RU2849194C1 - Modified mirna with reduced off-target activity - Google Patents

Abstract

FIELD: biotechnology; medicine.

SUBSTANCE: modified miRNA (small interfering RNA) with reduced off-target activity is proposed, containing a sense strand and an antisense strand, where the antisense strand has a chemical modification represented by formula (I) in at least one of the nucleotide positions 5, 6 or 7 of its 5'-region.

EFFECT: reduction in the number of toxic side effects resulting from off-target activity of miRNA; applicable for more effective inhibition of target gene expression.

13 cl, 9 dwg, 69 tbl, 32 ex

Description

This application claims priority to Chinese Patent Application CN202010772542.6 filed on August 4, 2020, Chinese Patent Application CN202110244977.8 filed on March 5, 2021, and Chinese Patent Application CN202110361502.7 filed on April 2, 2021, all of which are incorporated herein by reference in their entirety.

AREA OF TECHNOLOGY

The present invention relates to siRNA (small interfering RNA) that inhibits expression of a target gene, and in particular to modified siRNA with reduced off-target activity.

PRIOR ART

RNA interference (RNAi) is an effective method for silencing gene expression. Statistically, approximately 80% of proteins associated with human diseases are undetectable because they cannot be targeted by conventional small-molecule drugs and biomacromolecules. Using RNA interference technology, suitable siRNAs can be engineered to match the mRNA (messenger RNA) encoding these proteins to specifically target and degrade the target mRNAs, thereby inhibiting the production of related proteins. Therefore, siRNAs hold significant promise for drug development.

However, siRNAs often have varying degrees of off-target effects. One off-target effect is the siRNA-like off-target effect, i.e., inhibitory activity against an mRNA caused by complete or incomplete pairing of the seed region (positions 2–8 at the 5' end) of the siRNA antisense strand (also known as the AS strand) with the target mRNA. The off-target effect of a single siRNA molecule can affect multiple mRNAs. Thus, unpredictable toxic side effects may occur. This is a major cause of toxic side effects produced by siRNA-based drugs (Janas, M.M., Schlegel, M.K., Harbison, C.E. et al. Selection of GalNAc-conjugated siRNAs with limited off-target-driven rat hepatotoxicity. Nat Commun 9, 723 (2018)).

BRIEF DESCRIPTION OF THE INVENTION

miRNA

The present disclosure provides an siRNA that includes a chemical modification in its seed region to inhibit or reduce the off-target activity of the siRNA while maintaining (or even enhancing) the target activity of the siRNA.

The present invention relates to an siRNA that comprises a sense strand and an antisense strand, wherein each of the strands comprises from 15 to 35 nucleotides; the antisense strand comprises a chemical modification of formula (I) or a tautomeric modification thereof in at least one of nucleotide positions 2-8 (e.g. positions 2, 3, 4, 5, 6, 7 and 8) of its 5' region:

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

J ₂ is H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

Q ₁ is and Q ₂ is R ₂ ; or Q ₁ is R ₂ , and Q ₂ is

Where:

r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

J ₁ is H or C ₁ -C ₆ alkyl;

r ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base or base analogue;

where the chemical modification of formula (I) is not

In some embodiments, the antisense strand comprises a chemical modification of formula (I-1) or a tautomeric modification thereof at at least one of nucleotide positions 2-8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8) of its 5' region:

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR4(R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

each J ₁ and each J ₂ is independently H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

R ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly bonded to form a ring.

In some embodiments, the antisense strand comprises a chemical modification of formula (I-2) or a tautomeric modification thereof at at least one of nucleotide positions 2-8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8) of its 5' region:

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or C ₁ -C ₆ alkyl;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R, where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly bonded to form a ring.

In some embodiments, a nucleotide comprising a chemical modification of formula (I) or a tautomeric modification thereof is a nucleotide comprising a chemical modification of formula (I') or a tautomeric modification thereof,

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

J ₂ is H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

Q ₁ ' is and Q ₂ ' is R ₂ ; or Q ₁ ' is R ₂ , and Q ₂ ' is

where r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

J ₁ is H or C ₁ -C ₆ alkyl;

r ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where r ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

M represents O or S;

optionally, r ₁ and R ₂ are directly linked to form a ring;

B is a base or base analogue;

where the chemical modification of formula (I') is not

In some embodiments, the antisense strand comprises a chemical modification of formula (I-3) or a tautomeric modification thereof at at least one of nucleotide positions 2-8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8) of its 5' region:

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

each J ₁ and each J ₂ is independently H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where r ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

M represents O or S;

optionally, r ₁ and R ₂ are directly linked to form a ring.

In some embodiments, the antisense strand comprises a chemical modification of formula (I-4) or a tautomeric modification thereof at at least one of nucleotide positions 2-8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8) of its 5' region:

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or C ₁ -C ₆ alkyl;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

M represents O or S;

optionally, r ₁ and R ₂ are directly linked to form a ring.

In some embodiments, the chemical modification described above is not

In some embodiments, when X is NH-CO, R ₁ is not H.

In some embodiments, each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each of R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₃ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or C ₁ -C ₃ alkyl;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where r ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.

In some embodiments, each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H, methyl, ethyl, n-propyl or isopropyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or methyl;

r ₃ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and p is 1 or 2;

r ₁ is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and q is 1 or 2;

R ₂ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and r is 1 or 2;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2- modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _N6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

In some embodiments, each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H, methyl, ethyl, n-propyl or isopropyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or methyl;

r ₃ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and p is 1 or 2;

r ₁ is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and q is 1 or 2;

R ₂ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and r is 1 or 2;

optionally, r ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole and 3-nitropyrole.

In some embodiments, Y is O or NH; each X is independently selected from the group consisting of NH-CO, _CH2 , and NH;

n is 0 or 1; m is 0 or 1; s is 0 or 1;

each J ₁ and each J ₂ independently represents H;

r ₁ is selected from the group consisting of H, methyl and CH ₂ OH;

R ₂ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

r ₃ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

optionally, r ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, Y is O or NH; each X is independently selected from the group consisting of NH-CO, _CH2 , and NH;

n is 0 or 1; m is 0 or 1; s is 0 or 1;

each J ₁ and each J ₂ independently represents H;

r ₁ is selected from the group consisting of H, methyl and CH ₂ OH;

r ₂ is selected from the group consisting of H, methyl and CE ₂ OH;

R ₃ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

optionally, r ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, the chemical modification of formula (I) is selected from the group consisting of:

where B is a base or base analogue; for example, B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2 -modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _H6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is a base at a corresponding position among positions 2-8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8) of the 5' region of the antisense strand.

In some specific embodiments, B is a natural base at a corresponding position among positions 2-8 (e.g., positions 2, 3, 4, 5, 6, 7, and 8) of the 5' region of the antisense strand.

In some embodiments, the chemical modification of formula (I) is selected from the group consisting of:

where B is a base or base analogue; for example, B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2 -modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _N6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, the chemical modification of formula (I) is selected from the group consisting of:

where B is a base or base analogue; for example, B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2 -modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _N6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some other embodiments, B is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.

In some embodiments, the chemical modification of formula (I) is selected from the group consisting of:

where M is O or S;

where B is a base or base analogue; for example, B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2 -modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _N6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, the chemical modification of formula (I) is selected from the group consisting of:

where M is O or S;

B is a base or base analog; for example, B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2 -modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _N6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, the chemical modification of formula (I) is selected from the group consisting of:

where M is O or S;

B is a base or base analog; for example, B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2 -modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _N6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some other embodiments, B is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.

In some embodiments of the invention, the chemical modification of formula (I) includes, but is not limited to:

and those where adenine in the structure is replaced by guanine, cytosine, uracil or thymine.

In some embodiments, the antisense strand comprises a chemical modification of formula (I) or a tautomeric modification thereof described above at at least one of its nucleotide positions 2 to 8, 3 to 8, 4 to 8, 5 to 8, or 5 to 7 of the 5' region.

In some embodiments, the antisense strand comprises a chemical modification of formula (I) or a tautomeric modification thereof described above at nucleotide positions 5, 6, and 7 of its 5' region.

In some embodiments, the antisense strand comprises a chemical modification of formula (I) or a tautomeric modification thereof described above at nucleotide position 7 of its 5' region.

In some embodiments, the sense strand and the antisense strand independently comprise from 16 to 35, from 16 to 34, from 17 to 34, from 17 to 33, from 18 to 33, from 18 to 32, from 18 to 31, from 18 to 30, from 18 to 29, from 18 to 28, from 18 to 27, from 18 to 26, from 18 to 25, from 18 to 24, from 18 to 23, from 19 to 25, from 19 to 24, or from 19 to 23 nucleotides.

In some embodiments, the sense strand and the antisense strand are identical or different in length; the sense strand is 19-23 nucleotides in length and the antisense strand is 19-26 nucleotides in length. The ratio of the length of the sense strand to the length of the antisense strand of the siRNA according to the present invention may be 19/20, 19/21, 19/22, 19/23, 19/24, 19/25, 19/26, 20/20, 20/21, 20/22, 20/23, 20/24, 20/25, 20/26, 21/20, 21/21, 21/22, 21/23, 21/24, 21/25, 21/26, 22/20, 22/21, 22/22, 22/23, 22/24, 22/25, 22/26, 23/20, 23/21, 23/22, 23/23, 23/24, 23/25, or 23/26. In some embodiments, the ratio of the length of the sense strand to the length of the antisense strand of the siRNA is 19/21, 21/23, or 23/25. In some embodiments, the ratio of the length of the sense strand to the length of the antisense strand of the siRNA is 19/21.

In some embodiments, the antisense strand is at least partially reverse complementary to a target sequence to mediate RNA interference; in some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the antisense strand and the target sequence; in some embodiments, the antisense strand is completely reverse complementary to the target sequence.

In some embodiments, the sense strand is at least partially reverse complementary to the antisense strand such that they form a double-stranded region; in some embodiments, there are no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatch between the sense strand and the antisense strand; in some embodiments, the sense strand is completely reverse complementary to the antisense strand.

The present invention also relates to an siRNA, which is an siRNA as described above, with modifications, wherein in addition to a nucleotide modified by a chemical modification of formula (I) or its tautomerism by a modification described above, at least one nucleotide modified in a different manner is also contained in at least one of the sense strand and/or the antisense strand.

In some embodiments, in addition to the nucleotide modified by the chemical modification of formula (I) or its tautomerism modification described above, other nucleotides in the sense strand and/or antisense strand are nucleotides modified in a different manner.

In some embodiments, each of the modified nucleotides is independently selected from the group consisting of a deoxynucleotide, a 3'-terminal deoxythymine nucleotide, a 2'-O-methyl-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally constrained nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino-modified nucleotide, a 2'-O-allyl-modified nucleotide, a 2'-C-alkyl-modified nucleotide, a 2'-hydroxy-modified nucleotide, a 2'-methoxyethyl-modified nucleotide, a 2'-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a nucleotide containing a non-natural base, a tetrahydropyran-modified nucleotide, a 1,5-anhydrohexitol-modified nucleotide, a cyclohexenyl-modified nucleotide, a nucleotide containing a thiophosphate group, a nucleotide containing a methylphosphonate group, a nucleotide containing a 5'-phosphate, and a nucleotide containing a 5'-phosphate mimetic.

In some embodiments, each of the otherwise modified nucleotides is independently selected from the group consisting of a 2'-alkoxy-modified nucleotide, a 2'-substituted alkoxy-modified nucleotide, a 2'-alkyl-modified nucleotide, a 2'-substituted alkyl-modified nucleotide, a 2'-amino-modified nucleotide, a 2'-substituted amino-modified nucleotide, a 2'-fluoro-modified nucleotide, a 2'-deoxynucleotide, a 2'-deoxy-2'-fluoro-modified nucleotide, a 3'-deoxy-thymine nucleotide, an isonucleotide, LNA (locked nucleic acid), ENA (ethylene-bridged nucleic acids), cET (hindered ethyl), UNA (unlocked nucleic acid), and GNA ( glycerol nucleic acid).

In some embodiments, the otherwise modified nucleotides are independently selected from the group consisting of a 2'-methoxy-modified nucleotide, a 2'-fluoro-modified nucleotide, and a 2'-deoxy-modified nucleotide.

In the context of the present invention, a fluoro-modified nucleotide refers to a nucleotide in which the hydroxy group at the 2'-position of the ribosyl group of the nucleotide is substituted with fluorine. In some embodiments, the 2'-alkoxy-modified nucleotide is a methoxy-modified nucleotide (2'-OMe). In some embodiments, the 2'-substituted alkoxy-modified nucleotide can be, for example, a 2'-O-methoxyethyl-modified nucleotide (2'-MOE) or a 2'-amino-modified nucleotide (2'-NH2).

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 14, and 16 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 9, 12, and 14 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 9, 12, 14, and 18 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, at least one phosphoester group in the sense strand and/or antisense strand is a phosphoester group with a modification group. The modification group makes the siRNA more stable in a biological sample or environment.

In some embodiments, the phosphodiester moiety with the modification group is a phosphorothioate moiety. Specifically, a phosphorothioate moiety refers to a phosphodiester moiety modified by replacing one non-bridging oxygen atom with a sulfur atom.

In some embodiments of the invention, the phosphorothioate group is present at least in one position selected from the group consisting of:

position between the 1st and 2nd nucleotides of the 5'-end of the sense strand;

position between the 2nd and 3rd nucleotides of the 5' end of the sense strand;

position between the 1st and 2nd nucleotides of the 3' end of the sense strand;

position between the 2nd and 3rd nucleotides of the 3' end of the sense strand;

position between the 1st and 2nd nucleotides of the 5'-end of the antisense strand;

position between the 2nd and 3rd nucleotides of the 5' end of the antisense strand;

the position between the 1st and 2nd nucleotides of the 3' end of the antisense strand; and

position between the 2nd and 3rd nucleotides of the 3' end of the antisense strand.

In some embodiments, the sense strand has a nucleotide sequence of the formula shown below:

where each N _a and each N _b independently represents a modified nucleotide or an unmodified nucleotide, and the modifications on N _a and N _b are different;

and/or

The antisense strand has a nucleotide sequence of the formula shown below:

where each N _a ' and each N _b ' independently represents a modified nucleotide or an unmodified nucleotide, where the modifications on N _a ' and N _b ' are different;

each X' independently represents N _a ' or N _b ';Y' represents N _a ' or N _b ';

W' is a nucleotide containing a chemical modification of formula (I) or a tautomeric modification thereof as described above.

In some embodiments, the sense strand has a nucleotide sequence of the formula shown below:

where each N _a and each N _b independently represents a modified nucleotide or an unmodified nucleotide, and the modifications on N _a and N _b are different;

and/or

The antisense strand has a nucleotide sequence of the formula shown below:

where each N _a ' and each N _b ' independently represents a modified nucleotide or an unmodified nucleotide, where the modifications on N _a ' and N _b ' are different;

each X' independently represents N _a ' or N _b ';Y' represents N _a ' or N _b ';

W' is a nucleotide containing a chemical modification of formula (I) or a tautomeric modification thereof as described above.

In some embodiments, the sense strand has a nucleotide sequence of the formula shown below:

where each N _a and each N _b independently represents a modified nucleotide or an unmodified nucleotide, and the modifications on N _a and N _b are different;

and/or

The antisense strand has a nucleotide sequence of the formula shown below:

where each N _a ' and each N _b ' independently represents a modified nucleotide or an unmodified nucleotide, where the modifications on N _a ' and N _b ' are different;

each X' independently represents N _a ' or N _b ';Y' represents N _a ' or N _b ';

W' is a nucleotide containing a chemical modification of formula (I) or a tautomeric modification thereof as described above.

In some embodiments, N _a is a 2'-methoxy-modified nucleotide and N _b is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.

In some embodiments, N _a ' is a 2'-methoxy-modified nucleotide and N _b ' is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.

In some embodiments, at least one phosphoester group in the sense strand and/or antisense strand is a phosphoester group with a modification group that provides the siRNA with increased stability in a biological sample or environment.

In some embodiments, the phosphodiester moiety with the modification group is a phosphorothioate moiety. Specifically, a phosphorothioate moiety refers to a phosphodiester moiety modified by replacing one non-bridging oxygen atom with a sulfur atom.

In some embodiments of the invention, the phosphorothioate group is present at least in one position selected from the group consisting of:

position between the 1st and 2nd nucleotides of the 5'-end of the sense strand;

position between the 2nd and 3rd nucleotides of the 5' end of the sense strand;

position between the 1st and 2nd nucleotides of the 3' end of the sense strand;

position between the 2nd and 3rd nucleotides of the 3' end of the sense strand;

position between the 1st and 2nd nucleotides of the 5'-end of the antisense strand;

position between the 2nd and 3rd nucleotides of the 5' end of the antisense strand;

a position between the 1st and 2nd nucleotides of the 3' end of the antisense strand; and a position between the 2nd and 3rd nucleotides of the 3' end of the antisense strand. In some embodiments, the sense strand has a nucleotide sequence of the formula shown below:

where Nm is any methoxy-modified nucleotide, such as methoxy-modified C, G, U, A, or T; Nf is any fluoro-modified nucleotide, such as fluoro-modified C, G, U, A, or T;

lowercase letters indicate that the two nucleotides adjacent to the letters are linked by a phosphorothioate group; and/or

The antisense strand has a nucleotide sequence of the formula shown below:

where Nm' is any methoxy-modified nucleotide, such as methoxy-modified C, G, U, A, or T; Nf' is any fluoro-modified nucleotide, such as fluoro-modified C, G, U, A, or T;

lowercase letters indicate that the two nucleotides adjacent to the letters are linked by a phosphorothioate group; W' represents a nucleotide containing a chemical modification of formula (I) or its tautomeric modification described above.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, and 14 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 14, and 16 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 9, 12, and 14 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 10, 12, and 14 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 1, 4, 6, 9, 12, 14, and 18 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 10, 12, 14, and 18 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 9, 10, 12, 14, 16, and 18 of the antisense strand is independently a 2'-deoxynucleotide or a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, and 14 of the antisense strand is independently a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 14, and 16 of the antisense strand is independently a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 12, and 14 of the antisense strand is independently a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 12, 14, 16, and 18 of the antisense strand is independently a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand is independently a 2'-fluoro-modified nucleotide.

In some embodiments, in the 5' to 3' direction, the nucleotides at positions 2, 4, 6, 10, 12, 14, 16, and 18 of the antisense strand are each independently a 2'-fluoro-modified nucleotide.

In some embodiments of the invention, the sense strand of the siRNA of the present invention has a nucleotide sequence of the formula shown below:

where each N _a and each N _b independently represent a modified nucleotide or an unmodified nucleotide, and the modifications on N _a and N _b are different;

each X independently represents N _a or N _b .

In some embodiments, the antisense strand of the siRNA of the present invention has a nucleotide sequence of the formula shown below:

where each N _a ' and each N _b ' independently represents a modified nucleotide or an unmodified nucleotide, where the modifications on N _a ' and N _b ' are different;

each X' independently represents N _a ' or N _b ';Y' represents N _a ' or N _b ';

W' is a nucleotide comprising any of the chemical modifications of formula (I) or tautomeric modifications thereof according to the present invention.

In some embodiments of the invention, the modifications on X' and Y' are different.

In some embodiments, N _a is a 2'-methoxy-modified nucleotide and N _b is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.

In some embodiments, N _a ' is a 2'-methoxy-modified nucleotide and N _b ' is a 2'-fluoro-modified nucleotide or a 2'-deoxy-modified nucleotide.

In some embodiments, N _a is a 2'-methoxy-modified nucleotide and N _b is a 2'-fluoro-modified nucleotide.

In some embodiments, N _a ' is a 2'-methoxy-modified nucleotide and N _b ' is a 2'-fluoro-modified nucleotide.

In some embodiments, the antisense strand of the siRNA of the present invention has a nucleotide sequence of the formula shown below:

wherein each X' is independently _Na ' or _Nb ', Y' is _Na ' or _Nb ', and the modifications on X' and Y' are different; _Na ' is a 2'-methoxy-modified nucleotide, and _Nb ' is a 2'-fluoro-modified nucleotide; W' is a nucleotide containing any of the chemical modifications of formula (I) or tautomeric modifications thereof according to the present invention.

In some embodiments of the invention, the sense strand of the siRNA of the present invention has a nucleotide sequence of the formula shown below:

or

where N _a is a 2'-methoxy-modified nucleotide and N _b is a 2'-fluoro-modified nucleotide.

In some embodiments, the antisense strand of the siRNA of the present invention has a nucleotide sequence of the formula shown below:

or

where N _a is a 2'-methoxy-modified nucleotide and N _b is a 2'-fluoro-modified nucleotide; and/or N _a ' is a 2'-methoxy-modified nucleotide and N _b ' is a 2'-fluoro-modified nucleotide.

W' is a nucleotide comprising any of the chemical modifications of formula (I) or tautomeric modifications thereof according to the present invention.

In some specific embodiments, W' is a nucleotide comprising a chemical modification or a tautomeric modification thereof; the chemical modification is selected from the group consisting of:

wherein B is selected from the group consisting of guanine, adenine, cytosine, and uracil; in some specific embodiments, B is selected from a base corresponding to position 7 of the 5' region of the antisense strand.

In some specific embodiments, W' is a nucleotide comprising a chemical modification or a tautomeric modification thereof; the chemical modification is selected from the group consisting of:

wherein M is O or S; wherein B is selected from the group consisting of guanine, adenine, cytosine, and uracil; in some specific embodiments, B is selected from a base corresponding to position 7 of the 5' region of the antisense strand.

In some specific embodiments, M is S. In some specific embodiments, M is O.

In some embodiments, at least one phosphoester group in the sense strand and/or antisense strand is a phosphoester group with a modification group that provides siRNA with increased stability in a biological sample or environment; in some embodiments, the phosphoester group with a modification group is a phosphorothioate group. In particular, a phosphorothioate group refers to a phosphodiester group modified by replacing one non-bridging oxygen atom with a sulfur atom.

In some embodiments of the invention, the phosphorothioate group is present at least in one position selected from the group consisting of:

position between the 1st and 2nd nucleotides of the 5'-end of the sense strand;

position between the 2nd and 3rd nucleotides of the 5' end of the sense strand;

end of the 1st nucleotide of the 3'-end of the sense strand;

position between the 1st and 2nd nucleotides of the 3' end of the sense strand;

position between the 2nd and 3rd nucleotides of the 3' end of the sense strand;

position between the 1st and 2nd nucleotides of the 5'-end of the antisense strand;

position between the 2nd and 3rd nucleotides of the 5' end of the antisense strand;

end of the 1st nucleotide of the 3' end of the antisense strand;

the position between the 1st and 2nd nucleotides of the 3' end of the antisense strand; and

position between the 2nd and 3rd nucleotides of the 3' end of the antisense strand.

In some embodiments, the sense strand and/or antisense strand comprise a plurality of phosphorothioate groups that are present in:

position between the 1st and 2nd nucleotides of the 5'-end of the sense strand;

position between the 2nd and 3rd nucleotides of the 5' end of the sense strand;

position between the 1st and 2nd nucleotides of the 5'-end of the antisense strand;

position between the 2nd and 3rd nucleotides of the 5' end of the antisense strand;

position between the 1st and 2nd nucleotides of the 3' end of the antisense strand;

position between the 2nd and 3rd nucleotides of the 3' end of the antisense strand;

optionally the end of the 1st nucleotide of the 3'-end of the sense strand, and/or optionally the position between the 1st and 2nd nucleotides of the 3'-end of the sense strand.

In some embodiments, the sense strand is selected from a nucleotide sequence of the formula shown below:

where Nm is any 2'-methoxy-modified nucleotide, such as 2'-methoxy-modified C, G, U, A, or T; Nf is any 2'-fluoro-modified nucleotide, such as 2'-fluoro-modified C, G, U, A, or T;

lowercase s letters, when present between uppercase letters, indicate that the two nucleotides adjacent to the s letters are linked by a phosphorothioate group;

Lowercase s, when first at the 3' end, indicate that the left nucleotide adjacent to the s ends with a phosphorothioate group.

In some embodiments, the antisense strand has a nucleotide sequence of the formula shown below:

or

where Nm' is any 2'-methoxy-modified nucleotide, such as 2'-methoxy-modified C, G, U, A, or T; Nf' is any 2'-fluoro-modified nucleotide, such as 2'-fluoro-modified C, G, U, A, or T;

lowercase s, when present between uppercase letters, indicate that the two nucleotides adjacent to the s letters are linked by a phosphorothioate group, and lowercase s, when first at the 3' end, indicate that the left nucleotide adjacent to the s letter ends with a phosphorothioate group;

W' is a nucleotide containing a chemical modification or a tautomeric modification thereof; the chemical modification is selected from the group consisting of:

wherein B is selected from the group consisting of guanine, adenine, cytosine, and uracil; in some embodiments, B is selected from a base corresponding to position 7 of the 5' region of the antisense strand.

In some specific embodiments, W' is a nucleotide comprising a chemical modification or a tautomeric modification thereof; the chemical modification is selected from the group consisting of:

wherein M is O or S; wherein B is selected from the group consisting of guanine, adenine, cytosine, and uracil; in some specific embodiments, B is selected from a base corresponding to position 7 of the 5' region of the antisense strand.

In some specific embodiments, M is S. In some specific embodiments, M is O.

In some embodiments, the siRNA comprises a sense strand selected from Table 5.

In some embodiments, the siRNA comprises any antisense strand selected from Table 5.

In some embodiments, the siRNA comprises any sense strand selected from Table 8.

In some embodiments, the siRNA comprises any antisense strand selected from Table 8.

In some embodiments, the siRNA comprises any antisense strand selected from Table 9.

In some embodiments, the siRNA comprises any sense strand selected from Table 13.

In some embodiments, the siRNA comprises any antisense strand selected from Table 13.

In some embodiments, the siRNA comprises any sense strand selected from Table 15.

In some embodiments, the siRNA comprises any antisense strand selected from Table 15.

In some embodiments, the siRNA comprises any sense strand selected from Table 24.

In some embodiments, the siRNA comprises any antisense strand selected from Table 24.

In some embodiments, the siRNA comprises any sense strand selected from Table 25.

In some embodiments, the siRNA comprises any antisense strand selected from Table 25.

In some embodiments, the siRNA comprises any sense strand selected from Table 26.

In some embodiments, the siRNA comprises any antisense strand selected from Table 26.

In some embodiments, the siRNA comprises any sense strand selected from Table 66.

In some embodiments, the siRNA comprises any antisense strand selected from Table 66.

In some embodiments, the siRNA described above, when contacted with a cell expressing a target gene, inhibits expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as measured, for example, by psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR (polymerase chain reaction) or branched DNA (rDNA)-based methods, or protein-based methods such as immunofluorescence assay, for example, Western blotting or flow cytometry.

In some embodiments, the siRNA described above, when contacted with a cell expressing a target gene, results in a residual percentage expression of the target gene mRNA of no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%, as measured, for example, by psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR-based methods or branched DNA (rDNA)-based methods, or protein-based methods such as immunofluorescence analysis, such as Western blotting or flow cytometry.

In some embodiments, an siRNA comprising a chemical modification of the present invention, e.g., a chemical modification of Formula (I) or Formula (II), when contacted with a cell expressing a target gene, reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, while maintaining the target activity, as measured, e.g., by psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (rDNA)-based methods, or protein-based methods such as immunofluorescence assay, e.g., Western blot or flow cytometry.

In some embodiments, an siRNA comprising a chemical modification of the present invention, e.g., a chemical modification of Formula (I) or Formula (II), when contacted with a cell expressing a target gene, reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, reducing on-target activity by at least 20%, at least 19%, at least 15%, at least 10%, at least 5%, or more than 1%, as measured, for example, by a psiCHECK activity screen and reporter gene assay. luciferase, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence assay, eg, Western blot or flow cytometry.

In some embodiments, an siRNA comprising a chemical modification of the present invention, e.g., a chemical modification of Formula (I) or Formula (II), when contacted with a cell expressing a target gene, reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, while increasing target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, by at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, as measured, for example, by psiCHECK screening methods and luciferase assays, such as PCR, at least based on protein or based on protein, for example, obtained from a blank.

Conjugate

The present invention also relates to an siRNA conjugate that comprises any siRNA described above and a conjugate group linked to the siRNA.

In some embodiments, the conjugate moiety comprises a pharmaceutically acceptable targeting ligand and optionally a linker, and the siRNA, linker, and targeting ligand are covalently or non-covalently linked in sequence.

In some embodiments, the linker is linked to the 3' end of the sense strand of the miRNA.

The present invention also relates to an siRNA conjugate that comprises any siRNA described above and a targeting ligand linked to the siRNA.

In some embodiments, the siRNA and the targeting ligand are covalently or non-covalently linked.

In some embodiments, the targeting ligand is linked to the 3' end of the sense strand of the miRNA.

In some embodiments, the targeting ligand targets the liver.

In some embodiments, the targeting ligand binds to the asialoglycoprotein receptor (ASGPR).

In some embodiments, the targeting ligand is selected from the group consisting of a galactose cluster and a galactose derivative cluster, wherein the galactose derivative is selected from the group consisting of N-acetyl galactosamine, N-trifluoroacetyl galactosamine, N-propionyl galactosamine, N-n-butyryl galactosamine, and N-isobutyryl galactosamine.

In some embodiments, to facilitate siRNA entry into a cell, a lipophilic group such as cholesterol can be introduced at the end of the siRNA sense strand, and the lipophilic group is covalently linked to a small interfering nucleic acid (e.g., cholesterol, lipoprotein, vitamin E, etc.) to facilitate penetration through the cell membrane consisting of a lipid bilayer and interaction with mRNA in the cell. Meanwhile, siRNA can also be modified by non-covalent binding, such as binding to a phospholipid molecule, polypeptide, cationic polymer, etc., a hydrophobic bond, or an ionic bond to enhance stability and biological activity.

In some embodiments, the targeting ligand is linked to the end of the siRNA via a phosphoester group, a phosphorothioate group, or a phosphonic acid group.

In some embodiments, the targeting ligand is indirectly linked to the end of the siRNA via a phosphoester group, a phosphorothioate group, or a phosphonic acid group.

In some embodiments, the targeting ligand is directly linked to the end of the siRNA via a phosphoester group, a phosphorothioate group, or a phosphonic acid group.

In some embodiments, the targeting ligand is directly linked to the end of the siRNA via a phosphoester group or a phosphorothioate group.

In some embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the miRNA via a phosphoester group or a phosphorothioate group.

In some embodiments, the targeting ligand has a structure of formula (IV) shown below,

where T is a targeting moiety, E is a branching group, L ₁ is a linker moiety, and L ₂ is a connecting moiety between the targeting moiety and the branching group, where i is selected from an integer from 1 to 10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, i is selected from an integer from 2 to 8.

In some embodiments, i is selected from an integer from 3 to 5.

In some embodiments, L ₁ is

wherein each of R ⁹ and R ¹⁰ is independently selected from the group consisting of -S-, -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH ₂ -, -CH ₂ NH-, -CH ₂ O-, -NH-C(O)-CH ₂ -, -C(O)-CH ₂ -NH-, -NH(CO)NH- and from 3 to 12-membered heterocyclyl, wherein -CH _{2 -} is optionally substituted with a substituent selected from the group consisting of halogen, alkyl, alkoxy and alkylamino, and alkyl is optionally further substituted with a substituent selected from the group consisting of hydroxy, amino and halogen;

R ¹¹ is selected from the group consisting of deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxy, sulfhydryl, alkylsulfhydryl, alkoxy, alkylamino, -C(O)-alkyl, -C(O)-O-alkyl, -CONH ₂ , -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O-alkyl, -S(O)ONH ₂ , and -S(O)ONH-alkyl, where alkyl, alkenyl, alkynyl, alkylsulfhydryl, alkoxy, -C(O)-alkyl, -C(O)-O-alkyl, -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O-alkyl and -S(O)ONH-alkyl are optionally further substituted with a substituent selected from the group consisting of halogen, hydroxy, amino and sulfhydryl;

k is selected from the group consisting of 0, 1, 2, 3 and 4;

j is selected from an integer from 1 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

In some embodiments, L ₁ is

or

where R ¹¹ is selected from the group consisting of deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxy, sulfhydryl, alkylsulfhydryl, alkoxy, alkylamino, -C(O)-alkyl, -C(O)-O-alkyl, -CONH ₂ , -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O-alkyl, -S(O)ONH ₂ , and -S(O)ONH-alkyl, where alkyl, alkenyl, alkynyl, carboxy, alkylsulfhydryl, alkoxy, -C(O)-alkyl, -C(O)-O-alkyl, -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O-alkyl and -S(O)ONH-alkyl are optionally further substituted with a substituent selected from the group consisting of halogen, hydroxy, amino and sulfhydryl;

k is selected from the group consisting of 0, 1, 2, 3 and 4;

In some embodiments, L ₁ is

or

In some embodiments, L ₁ is

or

In some embodiments, L ₁ is

In some embodiments, E in the targeting ligand is

wherein R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ are each independently selected from the group consisting of -C(O)NH- and -C(O)-, wherein the carbonyl is optionally further substituted with alkyl, and the alkyl is optionally further substituted with a group selected from the group consisting of alkyl, hydroxy, -C(O)O-, -C(O)O-alkyl- and -C(O)NH-;

each of X ² , X ³ , X ⁴ and X ⁵ is independently selected from an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

In some embodiments, E in the targeting ligand is

wherein each of R** ¹² , R ¹³ , R ¹⁴ and R ¹⁵ is independently selected from the group consisting of -C(O)NH- and -C(O)-, where -C(O)NH- and -C(O)- are optionally further substituted with alkyl, and alkyl is optionally further substituted with a group selected from the group consisting of alkyl, hydroxy, -C(O)O-, -C(O)O-alkyl- and -C(O)NH-;

each of X ² , X ³ , X ⁴ and X ⁵ is independently selected from an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10).

In some embodiments, E in the targeting ligand is

where each of R ¹² , R ¹³ , R ¹⁴ and R ¹⁵ is independently selected from the group consisting of -C(O)NH-, -C(O)-,

And

each X ² , X ³ , X ⁴ , and X ⁵ is independently selected from an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10).

In some embodiments, E in the targeting ligand is

In some embodiments, E in the targeting ligand is selected from the group consisting of

And

In some embodiments, E in the targeting ligand is selected from the group consisting of

And

In some embodiments, E in the targeting ligand is selected from

In some embodiments, E in the targeting ligand is

and L ₁ is selected from the group consisting of the following structures:

wherein each of R ⁹ and R ¹⁰ is independently selected from the group consisting of -S-, -NH-, -O-, -C(O)-, -OC(O)-, -C(O)O-, -NHC(O)-, -C(O)NH-, -CH ₂ -, -CH ₂ NH-, -CH ₂ O-, -NH-C(O)-CH ₂ -, -C(O)-CH ₂ -NH-, -NH(CO)NH- and from 3 to 12-membered heterocyclyl, wherein -CH _{2 -} is optionally substituted with a substituent selected from the group consisting of halogen, alkyl, alkoxy and alkylamino, and alkyl is optionally further substituted with a substituent selected from the group consisting of hydroxy, amino and halogen;

R ¹¹ is selected from the group consisting of deuterium, halogen, alkyl, amino, cyano, nitro, alkenyl, alkynyl, carboxyl, hydroxy, sulfhydryl, alkylsulfhydryl, alkoxy, alkylamino, -C(O)-alkyl, -C(O)-O-alkyl, -CONH ₂ , -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O-alkyl, -S(O)ONH ₂ , and -S(O)ONH-alkyl, where alkyl, alkenyl, alkynyl, alkylsulfhydryl, alkoxy, -C(O)-alkyl, -C(O)-O-alkyl, -CONH-alkyl, -OC(O)-alkyl, -NH-C(O)-alkyl, -S(O)O-alkyl and -S(O)ONH-alkyl are optionally further substituted with a substituent selected from the group consisting of halogen, hydroxy, amino and sulfhydryl;

k is selected from the group consisting of 0, 1, 2, 3 and 4;

j is selected from the group consisting of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

In some embodiments, E in the targeting ligand is selected from the group consisting of:

And

and L ₁ is selected from the group consisting of:

And

In some embodiments, E in the targeting ligand is selected from the group consisting of:

and L ₁ is selected from the group consisting of: - that is, el ₁ represents

In some embodiments, E in the targeting ligand is selected from the group consisting of:

and L ₁ is selected from the group consisting of

And

that is, el ₁ represents

or

In some embodiments, E in the targeting ligand is selected from the group consisting of

And

and L ₁ is selected from

that is, el ₁ represents

or

In some embodiments, E in the targeting ligand is selected from the group consisting of

And

a L ₁ is selected from the group consisting of

And,

that is, el ₁ represents

or

In the present description, L ₂ is a linking moiety between the targeting moiety and the branching group, and L ₂ binds and distributes the targeting moiety and the branching group.

In some embodiments, one end of L ₂ is directly linked to the targeting ligand and the other end is directly linked to the branching group E.

In some embodiments, one end of L ₂ is directly linked to the targeting ligand and the other end is indirectly linked to the branching group E.

In some embodiments, one end of L ₂ is indirectly linked to the targeting ligand and the other end is indirectly linked to the branching group E.

In some embodiments, the targeting ligand disclosed herein comprises two L ₂ and two targeting moieties.

In some embodiments, the targeting ligand disclosed herein comprises three L ₂ and three targeting moieties.

In some embodiments, the targeting ligand disclosed herein comprises four L ₂ and four targeting moieties.

In some embodiments, the targeting ligand disclosed herein comprises a plurality of L ₂ and a plurality of targeting moieties.

In some embodiments, L ₂ herein is selected from one of or a combination of 2-20 of the following groups covalently linked (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20):

substituted or unsubstituted cycloalkyl (e.g., cyclohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cycloheptyl, or cyclooctyl), substituted or unsubstituted cycloalkenyl (e.g., cyclohexenyl, cyclobutenyl, cyclopentenyl, cycloheptenyl, cyclooctenyl, cyclohexadienyl, cyclopentadienyl, cycloheptadienyl, or cyclooctadienyl), substituted or unsubstituted aryl (e.g., phenyl, naphthyl, binaphthyl, or anthracenyl), substituted or unsubstituted heteroaryl (e.g., pyridyl, pyrimidinyl, pyrrole, imidazole, furan, benzofuran, or indole), and substituted or unsubstituted heterocyclyl (e.g., tetrahydrofuran, tetrahydropyran, piperidine, or pyrrolidine) covalently linked in combination.

In some embodiments, L ₂ herein is selected from one of or a combination of 2-20 of the following groups covalently linked (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20):

In some embodiments, the targeting ligand comprises L ₂ of the following structure,

where X ⁶ is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20).

In some embodiments, the targeting ligand comprises L ₂ of the following structure

In some embodiments, the targeting ligand comprises L ₂ of the following structure

In some embodiments, the targeting ligand comprises L ₂ of the following structure

In some embodiments, the targeting ligand comprises L ₂ of the following structure

In some embodiments, the targeting ligand comprises L ₂ of the following structure

In some embodiments, the targeting ligand comprises L ₂ of the following structure

where x ⁷ is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and Z is

In some embodiments, the targeting ligand comprises L ₂ of the following structure

In some embodiments, the targeting ligand comprises L ₂ of the following structure

where x ⁸ is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) and Z is

In some embodiments, the targeting ligand comprises L ₂ of the following structure

where x ⁹ and X ¹⁰ are each independently selected from an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and Z is

In some embodiments, the targeting ligand comprises L ₂ of the following structure

In some embodiments, the targeting ligand comprises L ₂ of the following structure

,

where each of x ⁷ and X ⁸ independently represents an integer selected from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and Z represents

.

In some specific embodiments, the targeting ligand has the structure shown below:

or

In some specific embodiments, the targeting ligand has the structure shown below:

or

In some specific embodiments, the targeting ligand has the structure shown below:

In some specific embodiments, the targeting ligand has the structure shown below:

or

In some embodiments, the targeting moiety of the targeting ligand consists of one or more targeting moieties, and the targeting ligand helps direct the delivery of the therapeutic agent bound to it to the desired target site. In some cases, the targeting moiety can bind to a cell or cell-specific receptor and initiate endocytosis to facilitate entry of the therapeutic agent into the cell. The targeting moiety can comprise a compound with affinity for a cell receptor or cell surface molecule or an antibody. Various targeting ligands containing targeting moieties can be linked to therapeutic agents and other compounds to target the agents to cells and specific cellular receptors.

In some embodiments, the types of targeting moieties include carbohydrates, cholesterol and cholesterol groups, or steroids. Targeting moieties that can bind to cellular receptors include saccharides such as galactose, galactose derivatives (e.g., N-acetyl-galactosamine, N-trifluoroacetylgalactosamine, N-propionylgalactosamine, N-n-butyrylgalactosamine, and N-isobutyrylgalactosamine), mannose, and mannose derivatives.

Targeting moieties that bind to asialoglycoprotein receptors (ASGPRs) are known to be used, in particular, to direct the delivery of oligomeric compounds to the liver. Asialoglycoprotein receptors are widely expressed on liver cells (hepatocytes). Targeting moieties of cellular receptors that target ASCPRs include galactose and galactose derivatives. In particular, galactose derivative clusters, including clusters consisting of 2, 3, 4, or more than 4 N-acetyl-galactosamines (GalNAc or NAG), can promote the uptake of certain compounds into hepatocytes. The GalNAc cluster linked to the oligomeric compound is used to direct the composition to the liver, where the N-acetyl-galactosamine saccharide can bind to asialoglycoprotein receptors on the liver cell surface. Binding to asialoglycoprotein receptors is thought to initiate receptor-mediated endocytosis, thereby facilitating entry of the compound into the cell.

In some embodiments, the targeting ligand may comprise 2, 3, 4, or more than 4 targeting moieties.

In some embodiments, the targeting ligand disclosed herein may comprise 1, 2, 3, 4, or more than 4 targeting moieties linked to the _L2 branching group.

In some embodiments, the targeting ligand is in the form of a galactose cluster.

In some embodiments, each targeting moiety comprises a galactosamine derivative that is N-acetyl-galactosamine. Other sugars that can be used as targeting moieties and that have affinity for the ac ialoglycoprotein receptors can be selected from the group consisting of galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, N-isobutyryl-galactosamine, etc.

In some embodiments, the targeting ligand herein comprises N-acetylgalactosamine as a targeting moiety.

In some embodiments, the targeting ligand comprises three terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for asialoglycoprotein receptors. In some embodiments, the targeting ligand comprises three terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

In some embodiments, the targeting ligand comprises four terminal galactosamines or galactosamine derivatives (such as N-acetyl-galactosamine), each of which has affinity for asialoglycoprotein receptors. In some embodiments, the targeting ligand comprises four terminal N-acetyl-galactosamines (GalNAc or NAG) as targeting moieties.

Terms commonly used in the art to refer to three terminal N-acetylgalactosamines include triantennary, trivalent, and trimeric.

Terms commonly used in the art to refer to the four terminal N-acetylgalactosamines include tetraantennary, tetravalent, and tetrameric.

In some specific embodiments, the targeting ligand of the present invention has the structure shown below,

or

In some specific embodiments, the targeting ligand provided herein has the structure shown below,

or

In some specific embodiments, the targeting ligand provided herein has the structure shown below,

In some specific embodiments, the targeting ligand provided herein has the structure shown below,

or

In some embodiments, an siRNA of the present invention is linked to a targeting ligand of the present invention to form an siRNA conjugate, as shown below,

where T is a targeting moiety, E is a branching group, L ₁ is a linker moiety, and L ₂ is a connecting moiety between the targeting moiety and the branching group, where x is selected from an integer from 1 to 10, and D is an siRNA according to any of the embodiments described above.

In some embodiments, D is an siRNA targeting ApoC3.

In some embodiments, D is an siRNA targeting HBV-X.

In some embodiments, D is an siRNA targeting F11.

In some embodiments, D is an siRNA that targets HBV-S.

In some embodiments, D is an siRNA targeting angiopoietin-like protein 3 (ANGPTL3).

In some embodiments, D is an siRNA targeting the transthyretin (TTR) gene.

In some embodiments, D is any siRNA of the present invention.

In some embodiments, L1 is linked to the 3' end of the sense strand of the miRNA.

In some embodiments, the targeting ligand is linked to the end of the siRNA via a phosphoester group, a phosphorothioate group, or a phosphonic acid group.

In some embodiments, the targeting ligand is indirectly linked to the end of the siRNA via a phosphoester group, a phosphorothioate group, or a phosphonic acid group.

In some embodiments, the targeting ligand is directly linked to the end of the siRNA via a phosphoester group, a phosphorothioate group, or a phosphonic acid group.

In some embodiments, the targeting ligand is directly linked to the end of the siRNA via a phosphoester group or a phosphorothioate group.

In some embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the miRNA via a phosphoester group or a phosphorothioate group.

In some specific embodiments, the siRNA conjugate described herein is shown below.

or

where D is an siRNA according to any of the embodiments described above.

In some embodiments, D is an siRNA targeting ApoC3.

In some embodiments, D is an siRNA targeting HBV-X.

In some embodiments, D is an siRNA targeting F11.

In some embodiments, D is an siRNA that targets HBV-S.

In some embodiments, D is an siRNA targeting angiopoietin-like protein 3 (ANGPTL3).

In some embodiments, D is an siRNA targeting the transthyretin (TTR) gene.

In some specific embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the miRNA via a phosphoester group or a phosphorothioate group.

In some specific embodiments, the siRNA conjugate described herein is shown below.

or

where D is an siRNA according to any of the embodiments described above.

In some embodiments, D is an siRNA targeting ApoC3.

In some embodiments, D is an siRNA targeting HBV-X.

In some embodiments, D is an siRNA targeting F11.

In some embodiments, D is an siRNA that targets HBV-S.

In some embodiments, D is an siRNA targeting angiopoietin-like protein 3 (ANGPTL3).

In some embodiments, D is an siRNA targeting the transthyretin (TTR) gene.

In some specific embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the miRNA via a phosphoester group or a phosphorothioate group.

In some specific embodiments, the siRNA conjugate described herein is shown below.

or

where D is an siRNA according to any of the embodiments described above.

In some embodiments, D is an siRNA targeting ApoC3.

In some embodiments, D is an siRNA targeting HBV-X.

In some embodiments, D is an siRNA targeting F11.

In some embodiments, D is an siRNA that targets HBV-S.

In some embodiments, D is an siRNA targeting angiopoietin-like protein 3 (ANGPTL3).

In some embodiments, D is an siRNA targeting the transthyretin (TTR) gene.

In some specific embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the miRNA via a phosphoester group or a phosphorothioate group.

In some specific embodiments, the siRNA conjugate described herein is shown below.

where D is an siRNA according to any of the embodiments described above.

In some embodiments, D is an siRNA targeting ApoC3.

In some embodiments, D is an siRNA targeting HBV-X.

In some embodiments, D is an siRNA targeting F11.

In some embodiments, D is an siRNA that targets HBV-S.

In some embodiments, D is an siRNA targeting angiopoietin-like protein 3 (ANGPTL3).

In some embodiments, D is an siRNA targeting the transthyretin (TTR) gene.

In some specific embodiments, the targeting ligand is directly linked to the 3' end of the sense strand of the miRNA via a phosphoester group or a phosphorothioate group.

In some specific embodiments, L ₁ is linked to D via a phosphoester group, a phosphorothioate group, or a phosphonic acid group.

In some specific embodiments, L ₁ is linked to the 3' end of the sense strand of D via a phosphoester group, a phosphorothioate group, or a phosphonic acid group.

In some specific embodiments, L ₁ is directly linked to the 3' end of the sense strand of D via a phosphoester group or a phosphorothioate group.

In some specific embodiments, L ₁ is indirectly linked to the 3' end of the sense strand of D via a phosphoester group, a phosphorothioate group. In some embodiments, to promote the penetration of siRNA into a cell, a lipophilic group such as cholesterol can be introduced at the end of the sense strand of siRNA, and the lipophilic group is covalently linked to a small interfering nucleic acid; for example, cholesterol, lipoprotein, vitamin E, etc., are introduced at the end to facilitate passage through the cell membrane consisting of a lipid bilayer and interaction with mRNA in the cell. Meanwhile, siRNA can also be modified by non-covalent binding, for example, by binding to a phospholipid molecule, a polypeptide, a cationic polymer, etc., a hydrophobic bond, or an ionic bond to enhance stability and biological activity.

Composition

In another aspect of the present invention, there is provided a composition that comprises a conjugate as described above and one or more pharmaceutically acceptable excipients, such as a carrier, filler, diluent and/or delivery polymer.

The present invention also relates to a pharmaceutical composition that comprises the siRNA or siRNA conjugate of the present invention.

In some embodiments, the pharmaceutical composition may further comprise a pharmaceutically acceptable excipient and/or adjuvant; the excipient may be one or more different formulations or compounds commonly used in the art. For example, a pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

Application and method

Another aspect of the present invention relates to the use of a conjugate or a composition comprising a conjugate as described above in the manufacture of a medicament for treating a disease in a subject; in some embodiments, the disease is selected from a liver disease.

Another aspect of the present invention relates to a method of treating a disease in a subject, which comprises administering to the subject a conjugate or composition as described above.

Another aspect of the present invention relates to a method of inhibiting mRNA expression in a subject, which comprises administering to the subject a conjugate or composition as described above.

Another aspect of the present invention relates to a method of delivering an expression-inhibiting oligomeric compound to the liver in vivo, which comprises administering to a subject the conjugate or composition described above.

The conjugate, composition, and methods described herein can reduce the level of a target mRNA in a cell, cell population, tissue, or subject, which comprises administering to the subject a therapeutically effective amount of an expression-inhibiting oligomer described herein. The expression-inhibiting oligomer is bound to a targeting ligand, thereby inhibiting the expression of the target mRNA in the subject.

In some embodiments, the subject has previously been identified as having pathogenic activation of a target gene in a target cell or target tissue.

The subject described herein refers to a subject having a disease or pathological condition that would benefit from a reduction or inhibition of the expression of the target mRNA.

Delivery may be by local administration (e.g., direct injection, implantation, or topical application), systemic administration, or via subcutaneous, intravenous, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intramuscular, transdermal, respiratory (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration.

In optional embodiments, the pharmaceutical composition of the present invention can be administered by injection, such as intravenous, intramuscular, intradermal, subcutaneous, intraduodenal, or intraperitoneal injection.

In optional embodiments, the conjugate may be assembled into a kit.

In some embodiments, the siRNA conjugate or pharmaceutical composition described above, upon contact with a cell expressing a target gene, inhibits expression of the target gene by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, as measured, for example, by psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR (polymerase chain reaction) or branched DNA (rDNA)-based methods, or protein-based methods such as immunofluorescence assay, for example, Western blotting or flow cytometry.

In some embodiments, the siRNA conjugate or pharmaceutical composition described above, when contacted with a cell expressing a target gene, results in a residual percentage expression of the target gene mRNA of no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%, as measured, for example, by psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR-based methods or branched DNA (rDNA)-based methods, or methods protein-based assays such as immunofluorescence assays, such as Western blotting or flow cytometry.

In some embodiments, when the siRNA conjugate or pharmaceutical composition is in contact with a cell expressing a target gene, the siRNA conjugate reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, while maintaining the target activity, as measured, for example, by psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (rDNA)-based methods, or protein-based methods such as immunofluorescence assay, for example, Western blot or flow cytometry.

In some embodiments, when the siRNA conjugate or pharmaceutical composition is in contact with a cell expressing a target gene, the siRNA conjugate reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, while reducing the target activity by no more than 20%, no more than 19%, no more than 15%, no more than 10%, no more than 5%, or more than 1%, as measured, for example, by a psiCHECK activity screen and a luciferase reporter gene assay, other methods such as PCR or branched DNA (bDNA)-based methods, or protein-based methods such as immunofluorescence analysis, for example, Western blotting or flow cytometry.

In some embodiments, when the siRNA conjugate or pharmaceutical composition is in contact with a cell expressing a target gene, the siRNA conjugate reduces off-target activity by at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75%, while enhancing on-target activity by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least by at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80%, as measured, for example, by psiCHECK activity screening and luciferase reporter gene assay, other methods such as PCR or branched DNA (rDNA)-based methods, or protein-based methods such as immunofluorescence assay, for example, Western blotting or flow cytometry.

The present invention also relates to a method for silencing a target gene or mRNA of a target gene in a cell, which comprises the step of introducing into the cell an siRNA, an siRNA conjugate and/or a pharmaceutical composition according to the present invention.

The present invention also relates to a method for silencing a target gene or mRNA of a target gene in a cell in vivo or in vitro, which comprises the step of introducing into the cell an siRNA, an siRNA conjugate and/or a pharmaceutical composition according to the present invention.

The present invention also relates to a method for inhibiting a target gene or the expression of mRNA of a target gene, which comprises administering to a subject in need thereof an effective amount or an effective dose of siRNA, siRNA conjugate and/or pharmaceutical composition according to the present invention.

In some embodiments, administration is carried out via routes of administration including intramuscular, intrabronchial, intrapleural, intraperitoneal, intraarterial, lymphatic, intravenous, subcutaneous, cerebrospinal, or combinations thereof.

In some embodiments, the effective amount or effective dose of siRNA, siRNA conjugate and/or pharmaceutical composition is from about 0.001 mg/kg body weight to about 200 mg/kg body weight, from about 0.01 mg/kg body weight to about 100 mg/kg body weight, or from about 0.5 mg/kg body weight to about 50 mg/kg body weight.

In some embodiments, the target gene is a hepatitis B virus (HBV) gene, angiopoietin-like protein-3 (ANGPTL3) gene, or a transthyretin (TTR) gene.

The present invention also relates to the use of the above-mentioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the manufacture of a medicament for the prevention and/or treatment of pathological conditions and diseases caused by the hepatitis B virus.

The present invention also relates to the use of the above-mentioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the manufacture of a medicament for the prevention and/or treatment of hepatitis B.

The present invention also relates to a method for treating hepatitis B, which comprises administering to a patient in need thereof the above-mentioned siRNA and/or pharmaceutical composition and/or siRNA conjugate.

The present invention also relates to a method for inhibiting the expression of an HBV gene in a cell infected with chronic hepatitis HBV, which comprises administering an effective amount or an effective dose of the above-mentioned siRNA and/or a pharmaceutical composition and/or a siRNA conjugate to a cell infected with chronic hepatitis HBV.

The present invention also relates to the use of the above-mentioned siRNA and/or pharmaceutical composition and/or siRNA conjugate in the manufacture of a medicament for the prevention and/or treatment of pathological conditions and diseases caused by aberrant expression of the ANGPTL3 gene or the TTR gene in mammals (e.g., humans).

The present invention also relates to a method for treating pathological conditions and diseases caused by aberrant expression of the ANGPTL3 gene or the TTR gene, which comprises administering an effective amount or dose of the above-mentioned siRNA and/or a pharmaceutical composition and/or siRNA conjugate.

Pathological conditions and diseases caused by aberrant expression of the ANGPTL3 gene include cardiovascular and/or metabolic diseases such as hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, obesity, diabetes and/or coronary heart disease.

Pathological conditions and diseases caused by aberrant TTR gene expression include sensory neuropathy (e.g., distal limb numbness or decreased sensation), autonomic neuropathy (e.g., gastrointestinal dysfunction such as gastric ulcer or orthostatic hypotension), motor neuropathy, seizures, dementia, myelopathy, polyneuropathy, carpal tunnel syndrome, autonomic defects, cardiomyopathy, vitreous opacities, renal failure, kidney disease, significant decrease in mBMI (change in body mass index), cranial nerve dysfunction, and lattice corneal dystrophy.

In some embodiments, the aforementioned siRNA and/or pharmaceutical composition and/or siRNA conjugate exhibit superior on-target activity and reduced off-target activity in regulating genes expressed in the liver or in treating pathological conditions or diseases caused by aberrant gene expression in liver cells. Genes expressed in the liver include, but are not limited to, the ApoB, ApoC, ANGPTL3, PCSK9, SCD1, TIMP-1, CollA1, FVII, STAT3, p53, HBV and HCV genes, etc. In some embodiments, said specific gene is selected from the group consisting of the hepatitis B virus gene, the angiopoietin-like protein 3 gene, or the apolipoprotein C3 gene. Accordingly, the disease is selected from the group consisting of chronic liver disease, hepatitis, liver fibrosis, proliferative liver disease, and dyslipidemia. In some embodiments, the dyslipidemia is hypercholesterolemia, hypertriglyceridemia, or atherosclerosis.

In some embodiments, the aforementioned siRNA, siRNA conjugate, and/or pharmaceutical composition can also be used to treat other liver diseases, including diseases characterized by unwanted cellular proliferation, hematological diseases, metabolic diseases, and diseases characterized by inflammation. A proliferative liver disease can be a benign or malignant disease, such as cancer, hepatocellular carcinoma (HCC), liver metastases, or hepatoblastoma. Hematological or inflammatory liver diseases can be diseases associated with coagulation factors and complement-mediated inflammation or fibrosis. Metabolic liver diseases include dyslipidemia and glucose regulation disorders.

host cell

The present invention also relates to a cell that comprises an siRNA or siRNA conjugate of the present invention.

Kit

The present invention also relates to a kit that contains the siRNA or siRNA conjugate of the present invention.

Intermediate product

The present invention also provides a compound of formula (II) or a tautomer thereof

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

J ₂ is H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

Q ₁ ' is and Q ₂ ' is R ₂ ; or Q ₁ ' is R ₂ , and Q ₂ ' is

where R ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

J ₁ is H or C ₁ -C ₆ alkyl;

R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ ; wherein R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base or base analogue; and

W is a leaving group and Z is a phosphorus-containing active reactive group.

In some embodiments, W is MMTr or DMTr.

In some embodiments, Z is .

In some embodiments, the compound described above is not

where W, B and Z are as defined above.

In some embodiments, when X is NH-CO, R1 is not H.

In some embodiments, the compound described above is not:

or

In some embodiments, the compound described above is not:

or

where Z is as defined above.

In some embodiments, the compound of formula (II) or tautomer thereof is, in particular, a compound of formula (II-1) or tautomer thereof

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

each J ₁ and each J ₂ is independently H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

R ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base or base analogue; and

W is a leaving group and Z is a phosphorus-containing reactive group.

In some embodiments, W is MMTr or DMTr.

In some embodiments, Z is .

In some embodiments, the compound described above is not

where W, B and Z are as defined above.

In some embodiments, when X is NH-CO, R1 is not H.

In some embodiments, the compound described above is not:

or

In some embodiments, the compound described above is not:

or

where Z is as defined above.

In some embodiments, the compound of formula (II) or tautomer thereof is, in particular, a compound of formula (II-2) or tautomer thereof

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or C ₁ -C ₆ alkyl;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base or base analogue; and

W is a leaving group and Z is a phosphorus-containing reactive group.

In some embodiments, W is MMTr or DMTr.

In some embodiments, Z is .

In some embodiments, the compound described above is not

where W, B and Z are as defined above.

In some embodiments, when X is NH-CO, R ₁ is not H.

In some embodiments, the compound described above is not:

or

In some embodiments, the compound described above is not:

or

where Z is as defined above.

In some embodiments, each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each of R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₃ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or C ₁ -C ₃ alkyl;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where r ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.

In some embodiments, each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H, methyl, ethyl, n-propyl or isopropyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or methyl;

r ₃ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and p is 1 or 2;

r ₁ is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and q is 1 or 2;

R ₂ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and r is 1 or 2;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2- modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _N6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

In some embodiments, each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , P ₄ ' and R ₅ is independently H, methyl, ethyl, n-propyl or isopropyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or methyl;

r ₃ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and p is 1 or 2;

r ₁ is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and q is 1 or 2;

R ₂ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and r is 1 or 2;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole and 3-nitropyrole.

In some embodiments, Y is O or NH; each X is independently selected from the group consisting of NH-CO, _CH2 , and NH;

n is 0 or 1; m is 0 or 1; s is 0 or 1;

each J ₁ and each J ₂ independently represents H;

r ₁ is selected from the group consisting of H, methyl and CH ₂ OH;

r ₂ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

r ₃ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

optionally, r ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, Y is O or NH; each X is independently selected from the group consisting of NH-CO, _CH2 , and NH;

n is 0 or 1; m is 0 or 1; s is 0 or 1;

each J ₁ and each J ₂ independently represents H;

r ₁ is selected from the group consisting of H, methyl and CH ₂ OH;

r ₂ is selected from the group consisting of H, methyl and CH ₂ OH;

r ₃ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

optionally, r ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some other embodiments, B is selected from the group consisting of adenine, guanine, cytosine, uracil, and thymine.

In some embodiments, the compound described above includes, but is not limited to:

and those compounds where adenine is replaced by guanine, cytosine, uracil or thymine.

The present invention also provides a compound of formula (III) or a tautomer thereof

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

J ₂ is H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₃ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

Q'' ₁ is and Q ₂ is R ₂ ; or Q ₁ is R ₂ , and Q" ₂ is

where r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

J ₁ is H or C ₁ -C ₆ alkyl;

R ₂ is selected from the group consisting of H, OH, halogen, NH2, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base or base analogue; and

W - leaving group.

In some embodiments, W is MMTr or DMTr.

In some embodiments, the compound described above is not

,

where W and B are as defined above.

In some embodiments, when X is NH-CO, R1 is not H.

In some embodiments, the compound described above is not one or more of the following compounds:

And

In some embodiments, the compound of formula (III) or tautomer thereof is, in particular, a compound of formula (III-1) or tautomer thereof,

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

each J ₁ and each J ₂ is independently H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

r ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

r ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base or base analogue; and

W - leaving group.

In some embodiments, W is MMTr or DMTr.

In some embodiments, the compound described above is not where W and B are as defined above.

In some embodiments, when X is NH-CO, R1 is not H.

In some embodiments, the compound of formula (III) is not one or more of the following compounds:

And

In some embodiments, the compound of formula (III) or tautomer thereof is, in particular, a compound of formula (III-2) or tautomer thereof,

where Y is selected from the group consisting of O, NH and S;

each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or C ₁ -C ₆ alkyl;

R ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

R ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is a base or base analogue; and

W - leaving group.

In some embodiments, W is MMTr or DMTr. In some embodiments, the compound described above is not

where W and B are as defined above.

In some embodiments, when X is NH-CO, R1 is not H.

In some embodiments, the compound described above is not one or more of the following compounds:

In some embodiments, each X is independently selected from the group consisting of CR ₄ (CR ₄ '), S, NR ₅ and NH-CO, wherein each of R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₃ alkyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or C ₁ -C ₃ alkyl;

R ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3;

R ₁ is selected from the group consisting of H, C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, and q is 1, 2 or 3;

R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₃ alkyl, C ₁ -C ₃ alkoxy, C ₂ -C ₄ alkenyl, C ₂ -C ₄ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₄ alkenyl and C ₂ -C ₄ alkynyl, and r is 1, 2 or 3;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine bases, pyrimidine bases, indole, 5-nitroindole and 3-nitropyrrole.

In some embodiments, each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H, methyl, ethyl, n-propyl or isopropyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or methyl;

R ₃ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and p is 1 or 2;

R ₁ is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and q is 1 or 2;

R ₂ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and r is 1 or 2;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, _C2- modified purine, _N8 -modified purine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, _N6 -alkyladenine, _O6 -alkylguanine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, _C5 -modified pyrimidine, thymine, indole, 5-nitroindole and 3-nitropyrrole.

In some embodiments, each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each R ₄ , R ₄ ' and R ₅ is independently H, methyl, ethyl, n-propyl or isopropyl;

n is 0, 1, or 2; m is 0, 1, or 2; s is 0 or 1;

each J ₁ and each J ₂ is independently H or methyl;

R ₃ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and p is 1 or 2;

R ₁ is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and q is 1 or 2;

R ₂ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and r is 1 or 2;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, isoguanine, hypoxanthine, xanthine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, 7-deazapurine, cytosine, 5-methylcytosine, isocytosine, pseudocytosine, uracil, pseudouracil, 2-thiouridine, 4-thiouridine, thymine, indole, 5-nitroindole and 3-nitropyrole.

In some embodiments, Y is O or NH; each X is independently selected from the group consisting of NH-CO, _CH2 , and NH;

n is 0 or 1; m is 0 or 1; s is 0 or 1;

each J ₁ and each J ₂ independently represents H;

R ₁ is selected from the group consisting of H, methyl and CH ₂ OH;

R ₂ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

R ₃ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In some embodiments, Y is O or NH; each X is independently selected from the group consisting of NH-CO, _CH2 , and NH;

n is 0 or 1; m is 0 or 1; s is 0 or 1;

each J ₁ and each J ₂ independently represents H;

R ₁ is selected from the group consisting of H, methyl and CH ₂ OH;

R ₂ is selected from the group consisting of H, methyl and CH ₂ OH;

R ₃ is selected from the group consisting of H, OH, NH ₂ , methyl and CH ₂ OH;

optionally, R ₁ and R ₂ are directly linked to form a ring;

B is selected from the group consisting of purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, and 3-nitropyrrole.

In other embodiments of the invention, B is selected from the group consisting of adenine, guanine, cytosine, uracil and thymine.

In some embodiments, the compound described above includes, but is not limited to:

and those connections

where adenine is replaced by guanine, cytosine, uracil, or thymine, and

and those compounds where the bases or base analogs are replaced by purine, adenine, guanine, 2,6-diaminopurine, 6-dimethylaminopurine, 2-aminopurine, cytosine, uracil, thymine, indole, 5-nitroindole, or 3-nitropyrrole.

The present invention also relates to an siRNA or siRNA conjugate, wherein the chemical modification of formula (I) or its tautomeric modification in the antisense strand of any siRNA or siRNA conjugate of the present invention is replaced by a 2'-methoxy modification.

The present invention also relates to an siRNA or siRNA conjugate, wherein the chemical modification of formula (I) contained in the antisense strand of any siRNA or siRNA conjugate of the present invention is a 2'-methoxy modification. The present invention also relates to an siRNA or siRNA conjugate, wherein the antisense strand comprises a modification in at least one of the nucleotide positions from 2 to 8 of its 5' region, and the modification is a chemical modification of formula (I) or a tautomer modification.

The present invention also provides an siRNA or siRNA conjugate wherein one or more U bases, such as 1, 2, 3, 3, 5, 6, 7, 8, 9 or 10 U bases of any siRNA or siRNA conjugate of the present invention, are replaced by T bases.

The present invention also relates to a method for producing the above-mentioned siRNA or siRNA conjugate, which comprises the following steps: (1) synthesizing a compound of formula (II) or a tautomer thereof; and (2) synthesizing siRNA or an siRNA conjugate using the compound or its tautomer from step (1).

In some embodiments, a compound of formula (II) or a tautomer thereof is synthesized using a compound of formula (III) or a tautomer thereof.

The present invention also relates to the use of a compound of formula (II) or a tautomer thereof as described above for inhibiting or reducing the off-target activity of miRNA.

The present invention also relates to the use of a compound of formula (II) or a tautomer thereof as described above in the production of siRNA. Terms

To facilitate understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which this invention pertains.

If no configuration is indicated, the compounds of the present invention may be present in specific geometric or stereoisomeric forms. The present invention contemplates all such compounds, including cis and trans isomers, (-) and (+) enantiomers, (R) and (S) enantiomers, diastereomers, the (D) isomer, the (L) isomer, and racemic mixtures and other mixtures thereof, such as enantiomerically or diastereomerically enriched mixtures, all of which are within the scope of the present invention. Additional asymmetric carbon atoms may be present in substituents, such as an alkyl group. All such isomers and mixtures thereof are included within the scope of the present invention.

The compounds and intermediates of the present invention may also exist in various tautomeric forms, and all such forms are included within the scope of the present invention.

The term "tautomer" or "tautomeric form" refers to structural isomers with different energies that can interconvert through a low energy barrier. For example, proton tautomers (also known as proton transfer tautomers) involve interconversion via proton migration, such as keto-enol and imine-enamine isomerization, or lactam-lactim isomerization. An example of a lactam-lactim equilibrium exists between A and B, as shown below.

All compounds in the present invention can be depicted as Form A or Form B. All tautomeric forms are within the scope of the present invention. The nomenclature of compounds does not exclude any tautomers.

The compounds of the present invention may be asymmetric; for example, the compounds have one or more stereoisomers. Unless a configuration is specified, all stereoisomers include, for example, enantiomers and diastereomers. Compounds of the present invention containing asymmetric carbon atoms may be isolated in optically active pure form or in racemic form. The optically active pure form may be isolated from a racemic mixture or synthesized using chiral starting materials or chiral reagents.

Optically active (R)- and (S)-enantiomers and D- and L-isomers can be prepared using chiral synthesis, chiral reagents, or other conventional methods. If a single enantiomer of a particular compound of the present invention is desired, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting mixture of diastereomers is separated and the auxiliary group is cleaved to yield the pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (e.g., amino) or an acidic functional group (e.g., carboxyl), salts of the diastereomers are formed with the appropriate optically active acid or base, followed by resolution of the diastereomers by conventional methods known in the art, and pure enantiomers are obtained by reduction. In addition, separation of enantiomers and diastereomers is usually accomplished by chromatography using a chiral stationary phase, optionally in combination with chemical derivatization (e.g., carbamate formation from amines).

The present invention also includes isotopically labeled compounds that are identical to those described herein, but have one or more atoms replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature. Examples of isotopes that may be included in a compound of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ^2H , ^3H , ^11C , ^13C , ^14C , ^13N , ^15N , ^15O , ^17O , 18O, ^31P , ^32P , ^35S , ^{18F , 123I} , ^125I , and ^36Cl .

Unless otherwise indicated, when a position is specifically designated as deuterium (D), this position is to be understood as deuterium having an abundance that is at least 1000 times greater than the natural abundance of deuterium (which is 0.015%) (i.e., at least 10% deuterium inclusion). The compounds of the examples contain deuterium having an abundance that is at least 1000 times greater than the natural abundance, at least 2000 times greater than the natural abundance, at least 3000 times greater than the natural abundance, at least 4000 times greater than the natural abundance, at least 5000 times greater than the natural abundance, at least 6000 times greater than the natural abundance, or more times greater than the natural abundance. The present invention also includes various deuterated forms of the compound of formula I. Each available hydrogen atom attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize deuterated forms of the compound of general formula I according to the relevant literature. Commercially available deuterated starting materials can be used to prepare deuterated forms of the compound of formula I, or they can be synthesized using conventional methods with deuterated reagents, including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane, and the like.

The term "optional" or "optionally" means that an event or circumstance described subsequently may, but need not, occur, and that such description includes instances in which the event or circumstance does or does not occur. For example, "C _1-6 alkyl optionally substituted with halogen or cyano" means that halogen or cyano may be present, but is not required, and the description includes the case in which the alkyl is substituted with halogen or cyano, and the case in which the alkyl is not substituted with halogen or cyano.

In the chemical structure of the compound of the present invention, the bond represents an indefinite configuration, namely, if chiral isomers exist in a chemical structure, the bond May be or , or contains both configurations And Although all the above structural formulas are presented as specific isomeric forms for simplicity, the present invention can include all isomers, such as tautomers, rotamers, geometric isomers, diastereomers, racemates and enantiomers. In the chemical structure of the compound of the present invention, the bond does not specify a configuration, that is, a configuration for communication may be an E configuration or a Z configuration, or include both an E configuration and a Z configuration.

Unless otherwise specified, the "chemical modification," "compound," "ligand," "conjugate," and "nucleic acid" of the present invention may each independently exist in the form of a salt, mixed salts, or a non-salt form (e.g., a free acid or a free base). If they exist in the form of a salt or mixed salts, they may be a pharmaceutically acceptable salt.

The term "pharmaceutically acceptable salt" includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

"Pharmaceutically acceptable acid addition salt" refers to salts that retain the biological effectiveness of the free base without any undesirable effects and that are formed with an inorganic acid or organic acids. Inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, phosphates, etc.; Salts of organic acids include, but are not limited to, formates, acetates, 2,2-dichloroacetates, trifluoroacetates, propionates, caproates, caprylates, caprates, undecenates, glycolates, gluconates, lactates, sebacates, adipates, glutarates, malonates, oxalates, maleates, succinates, fumarates, tartrates, citrates, palmitates, stearates, oleates, cinnamates, laurates, malates, glutamates, pyroglutamates, aspartates, benzoates, methanesulfonates, benzenesulfonates, p-toluenesulfonates, alginates, ascorbates, salicylates, 4-aminosalicylates, napadisylates, etc. These salts can be prepared using methods known in the art.

"Pharmaceutically acceptable base addition salt" refers to salts that are capable of maintaining the biological effectiveness of the free acids without any undesirable effects and that are formed with inorganic bases or organic bases. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, etc. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts, and magnesium salts; sodium salts are preferred. Salts derived from organic bases include, but are not limited to, salts of the following: primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, triethanolamine, dimethylethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. Preferred organic bases include isopropylamine, Diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine. These salts can be prepared using methods known in the art.

The term "bond" when referring to the relationship between two molecules means that the two molecules are linked by a covalent bond or that the two molecules are linked by a non-covalent bond (such as a hydrogen bond or an ionic bond) and includes direct bonding and indirect bonding.

The term "directly bonded" means that the first compound or group is bonded to the second compound or group without any atom or group of atoms located between them.

The term "indirectly linked" means that the first compound or group is linked to the second compound or group by an intervening group, compound, or molecule (e.g., a linking group).

In the context of RNA-mediated gene silencing, the sense strand (also referred to as the SS or SS strand) of an siRNA refers to the strand that contains a sequence identical or substantially identical to the sequence of the target mRNA; the antisense strand (also referred to as the AS or AS strand) of an siRNA refers to the strand that has a sequence complementary to the sequence of the target mRNA.

In this context, the terms "complementary" and "reverse complementary" are used interchangeably and have the meaning well known to those skilled in the art: that is, in a double-stranded nucleic acid molecule, the bases of one strand are complementarily paired with the bases of the other strand. In DNA, the purine base adenine (A) always pairs with the pyrimidine base thymine (T) (or uracil (U) in RNA), and the purine base guanine (C) always pairs with the pyrimidine base cytosine (G). Each base pair contains a purine and a pyrimidine. When adenines in one strand are always paired with thymines (or uracils) in the other strand, and guanines are always paired with cytosines, the two strands are considered complementary to each other, and the sequences of the strands can be deduced from the sequences of their complementary strands. Accordingly, a "mismatch" in this technical field means that in a double-stranded nucleic acid the bases at corresponding positions do not pair in a complementary manner.

The term "base" encompasses any known DNA and RNA bases and base analogues such as purines or pyrimidines, which also includes the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine and natural analogues.

In the present invention, base analogs are typically purine or pyrimidine bases, with the exception of the common bases: guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3-β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-β-D-ribofuranosyl-(1-methyl-pyrazolo[4,3-d]pyrimidine-5,7(4H,6H)-dione) (P), isocytosine (iso-C), isoguanine (iso-G), 1-β-D-ribofuranosyl-(5-nitroindole), 1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds), and pyrrole-2-carbaldehyde (Pa). 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methylhydroxyisoquinolyl, 5-methylhydroxyisoquinolyl and 3-methyl-7-propynyl hydroxyisoquinolyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidazopyridinyl, 9-methyl-imidazopyridinyl, pyrrolopyrazinyl, hydroxyisoquinolyl, 7-propynyl hydroxyisoquinolyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, naphthyl, anthracenyl, phenanthrenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl and their structural derivatives. Base analogs can also be universal bases.

In this context, a "universal base" refers to a heterocyclic moiety located at the 1'-position of a nucleotide sugar moiety in a modified nucleotide or an equivalent position in a nucleotide substitution of a sugar moiety, and a heterocyclic moiety present in a nucleic acid duplex may be located opposite more than one type of base without changing the double helical structure (e.g., the structure of the phosphate backbone). Furthermore, a universal base does not disrupt the ability of a single-stranded nucleic acid in which it is present to form a duplex with a target nucleic acid. The ability of a single-stranded nucleic acid containing a universal base to form a duplex with a target nucleic acid can be determined using methods obvious to those skilled in the art (e.g., UV absorption, circular dichroism, gel shift, and single-stranded nuclease sensitivity). Furthermore, the conditions under which duplex formation is observed can be altered to determine stability or duplex formation, for example, temperatures such as the melting temperature (Tm), which are associated with the stability of nucleic acid duplexes. Compared to a reference single-stranded nucleic acid that is precisely complementary to the target nucleic acid, a single-stranded nucleic acid containing a universal base forms a duplex with a target nucleic acid that has a lower Tm than the duplex formed by the complementary nucleic acid. However, compared with a reference single-stranded nucleic acid in which the universal base is replaced by a base to produce one mismatch, the single-stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than the duplex formed with the nucleic acid having a mismatch base.

Some universal bases are capable of base pairing by forming hydrogen bonds between the universal base and all of the guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) bases under base pairing conditions. A universal base is not a base that forms a base pair with only one complementary base. In a duplex, a universal base cannot form a hydrogen bond, one hydrogen bond, or more than one hydrogen bond with each of the G, C, A, T, and U opposite it on the opposite strand of the duplex. Preferably, a universal base does not interact with its opposite base on the opposite strand of the duplex. In a duplex, base pairing with a universal base will not alter the double helical structure of the phosphate backbone. A universal base can also interact with bases in adjacent nucleotides on the same strand of nucleic acid through stacking interactions. Such stacking interactions can stabilize the duplex, particularly in cases where the universal base does not form a hydrogen bond with the base opposite it on the opposite strand of the duplex. Non-limiting examples of universal stacking nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and/or 1-β-D-ribofuranosyl-3-nitropyrrole.

In this context, "chemical modification" or "modification" includes all changes made to a nucleotide by chemical means, such as the addition or deletion of a chemical moiety or the substitution of one chemical moiety for another.

In the chemical structural formulas according to the present invention, wavy lines represent binding sites, and asterisks "*" denote chiral centers.

In the context of the present invention

fragment in the group

can be replaced by any group capable of binding to an adjacent nucleotide.

The term "alkyl" refers to a saturated aliphatic hydrocarbyl group that is a straight or branched chain group containing from 1 to 20 carbon atoms, such as an alkyl group containing from 1 to 12 carbon atoms or an alkyl group containing from 1 to 6 carbon atoms. Non-limiting examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, or 2,3-dimethylbutyl.

The term "alkoxy" refers to the group -O-alkyl, wherein alkyl is as defined above. Non-limiting examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, and cyclohexyloxy. C ₁ -C ₆ alkoxy may be optionally substituted or unsubstituted, and when substituted, the substituent is preferably one or more of the following groups independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, carboxyl, or a carboxylate group.

The term "alkenyl" refers to a hydrocarbyl group containing at least one double bond. Non-limiting examples of alkenyl include, but are not limited to, ethenyl, 1-propenyl, 2-propenyl, 1-butenyl, or 2-butenyl, and their various branched-chain isomers.

The term "alkynyl" refers to a hydrocarbyl group containing at least one triple bond. Non-limiting examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, or 2-butynyl, and their various branched-chain isomers.

The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

In the present invention, the “ring” in “R ₁ and R ₂ are directly bonded to form a ring” may be “cycloalkyl” or “heterocycloalkyl”.

The term "cycloalkyl" may refer to "carbocycle" and refers to a saturated or partially unsaturated monocyclic or polycyclic substituent of a cyclohydrocarbon. A cycloalkyl ring contains from 3 to 20 carbon atoms, in some embodiments from 3 to 7 carbon atoms, and in some embodiments from 5 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, etc. Polycyclic cycloalkyl includes spirocycloalkyl, fused cycloalkyl, and bridged cycloalkyl. Cycloalkyl may be substituted or unsubstituted, and when substituted, the substituent may be substituted at any available bonding site; in some embodiments, the substituent is selected from the group consisting of one or more of the following groups independently selected from the group consisting of halogen, deuterium, hydroxy, oxo, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, and 5-6-membered aryl or heteroaryl, wherein the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, and 5-6-membered aryl or heteroaryl are optionally substituted with one or more groups selected from the group consisting of halogen, deuterium, hydroxy, oxo, nitro and cyano.

A cycloalkyl ring can be fused to an aryl or heteroaryl ring, where the ring attached to the parent structure is cycloalkyl. Non-limiting examples of cycloalkyl rings include indanyl, tetrahydronaphthyl, benzocycloheptyl, etc. Cycloalkyl may be optionally substituted or unsubstituted, and when substituted, the substituent in some embodiments is selected from the group consisting of one or more of the following groups independently selected from the group consisting of halogen, deuterium, hydroxy, oxo, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, and 5-6-membered aryl or heteroaryl, wherein C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy and The 5-6 membered aryl or heteroaryl is optionally substituted with one or more groups selected from the group consisting of halogen, deuterium, hydroxy, oxo, nitro and cyano.

The term "heterocycloalkyl," also known as "heterocycle" or "heterocyclyl," refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms, wherein one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen, and S(O)m (where m is an integer from 0 to 2), excluding the cyclic moiety -O-O-, -O-S-, or -S-S-, and the remaining ring atoms are carbon atoms. In some embodiments, heterocycloalkyl contains from 3 to 12 ring atoms, 1-4 of which are heteroatoms; in some embodiments, heterocycloalkyl contains from 3 to 7 ring atoms. Non-limiting examples of monocyclic heterocycloalkyl include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, dihydroimidazolyl, dihydrofuranyl, dihydropyrazolyl, dihydropyrrolyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocycloalkyl includes spiroheterocyclyl, fused heterocyclyl, and bridged heterocycloalkyl. Non-limiting examples of "heterocycloalkyl" include:

A heterocycloalkyl ring can be fused to an aryl or heteroaryl ring, wherein the ring attached to the parent structure is a heterocycloalkyl. Non-limiting examples of a heterocycloalkyl ring include, but are not limited to:

etc.

The heterocycloalkyl may be optionally substituted or unsubstituted, and when substituted, the substituent in some embodiments is selected from the group consisting of one or more of the following groups independently selected from the group consisting of halogen, deuterium, hydroxy, oxo, nitro, cyano, C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy, and 5-6-membered aryl or heteroaryl, wherein the C _1-6 alkyl, C _1-6 alkoxy, C _2-6 alkenyloxy, C _2-6 alkynyloxy, C _3-6 cycloalkoxy, 3-6-membered heterocycloalkoxy, C _3-8 cycloalkenyloxy and The 5-6 membered aryl or heteroaryl is optionally substituted with one or more groups selected from the group consisting of halogen, deuterium, hydroxy, oxo, nitro and cyano.

In the context of the present invention, Bz is a benzoyl protecting group; MMTr is methoxyphenylbenzhydryl; DMTr is a dimethoxytrityl protecting group.

Unless otherwise indicated, in the context of the present invention, the uppercase letters C, G, U, A, and T represent the basic components of a nucleotide; the lowercase letter d indicates that the right nucleotide adjacent to the letter d is a deoxyribonucleotide; the lowercase letter m indicates that the left nucleotide adjacent to the letter m is a methoxy-modified nucleotide; the lowercase letter f indicates that the left nucleotide adjacent to the letter f is a fluoro-modified nucleotide; the lowercase letter s indicates that the two nucleotides adjacent to the letter s are linked by a phosphorothioate group.

As used herein, the term "fluoro-modified nucleotide" refers to a nucleotide in which the hydroxy group at the 2' position of the ribosyl group of the nucleotide is replaced by fluorine, and a "non-fluoro-modified nucleotide" refers to a nucleotide or nucleotide analog in which the hydroxy group at the 2' position of the ribosyl group of the nucleotide is replaced by a non-fluorine-containing group. "Nucleotide analog" refers to a group that can replace a nucleotide in a nucleic acid but has a structure other than an adenine ribonucleotide, a guanine ribonucleotide, a cytosine ribonucleotide, a uracil ribonucleotide, or a thymine deoxyribonucleotide, such as an isonucleotide, a bridged nucleic acid (BNA), or an acyclic nucleotide. A methoxy-modified nucleotide refers to a nucleotide in which the 2'-hydroxy group of the ribosyl group is replaced by a methoxy group. An isonucleotide refers to a compound formed by changing the position of a base on the ribose ring of a nucleotide. In some embodiments, an isonucleotide may be a compound formed by moving a base from the 1' position to the 2' position or 3' position of the ribose ring. A MNC refers to a constrained or inaccessible nucleotide. An MNC may contain a five-membered, six-membered, or seven-membered ring bridge structure with a "fixed" C3'-end sugar wrinkle. The bridge is typically incorporated into the 2'-, 4'-position of the ribose to produce a 2',4'-MNC nucleotide. In some embodiments, an MNC may be a LNC, ENC, cET MNC, etc. Acyclic nucleotides are a class of nucleotides in which the sugar ring of the nucleotide is open. In some embodiments, the acyclic nucleotide may be an unblocked nucleic acid (RNA) or a glycerol nucleic acid (GNA).

As used herein, the term "inhibition" is used interchangeably with the terms "reduction," "silencing," "downregulation," "suppression," and other similar terms, and includes any level of inhibition. Inhibition can be assessed in terms of a reduction in the absolute or relative level of one or more of these variables relative to a control level. The control level can be any type of control level used in the art, such as a pre-dose baseline level or a level determined from a similar untreated or control (e.g., a buffer-only control or an inert agent control) treated subject, cell, or sample. For example, the remaining level of mRNA expression can be used to characterize the degree of inhibition of target gene expression by miRNA; for example, the residual mRNA expression level is no more than 99%, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10%. Inhibition of target gene expression can be measured using the Dual-Glo® Luciferase Assay System: Firefly chemiluminescence value and Renilla chemiluminescence value are read, and the relative value is calculated. Ratio = Ren/Fir and inhibition (%) = 1 - (ratio + siRNA / reporter only ratio) × 100%; in this description, the proportion of remaining mRNA expression (or remaining activity%) = 100% - inhibition (%).

"An effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results. For preventive use, beneficial or desired results include the elimination or reduction of the risk, a reduction in the severity, or a delay in the onset of a condition, including the biochemical, histological, and/or behavioral symptoms of the condition, their complications, and intermediate pathological phenotypes that appear during the progression of the condition. For therapeutic applications, beneficial or desired results include clinical results, such as a reduction in the incidence of various conditions associated with a target gene, target mRNA, or target protein of the present invention, or an alleviation of one or more symptoms of the condition, a reduction in the dose of other agents required to treat the condition, an enhancement of the therapeutic effect of another agent, and/or a delay in the progression of conditions associated with a target gene, target mRNA, or target protein of the present invention in a patient.

In this context, the term "angiopoietin-like protein 3" (also known as "ANGPTL3" or "ANGPTL3") can refer to any ANGPTL3 nucleic acid or protein. The human ANGPTL3 sequence is available under accession number NP_055310. "ANGPTL3 expression" refers to the level of mRNA transcribed from the gene encoding ANGPTL3 or the level of protein translated from the mRNA.

In this context, the term "transthyretin" ("TTR"), also known as ATTR, HsT2651, PALB, prealbumin, TVRA, and transthyretin (prealbumin, amyloidosis type I), can refer to any TTR nucleic acid or protein. The human TTR mRNA transcript sequence is available under accession number NM_000371. "TTR expression" refers to the level of mRNA transcribed from the gene encoding TTR or the level of protein translated from the mRNA.

A "pharmaceutical composition" comprises an siRNA or siRNA conjugate of the present invention and a pharmaceutically acceptable excipient and/or adjuvant; the excipient may be one or more different formulations or compounds commonly used in the art. For example, a pharmaceutically acceptable excipient may include at least one of a pH buffer, a protective agent, and an osmotic pressure regulator.

In this context, the terms "patient," "subject," and "individual" are used interchangeably and include humans or non-human animals, such as mammals such as humans or monkeys.

Various delivery systems are known that can be used for the siRNA or siRNA conjugate according to the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis, and construction of the nucleic acid as part of a retrovirus or other vectors.

The siRNA disclosed herein can be prepared using a production method known in the art (e.g., solid-phase synthesis and liquid-phase synthesis). Solid-phase synthesis is commercially available as a customization service. A modified nucleotide moiety can be introduced into the siRNA of the present invention using a nucleoside monomer with an appropriate modification. Methods for preparing a nucleoside monomer with an appropriate modification and introducing a modified nucleotide moiety into siRNA are also well known to those skilled in the art.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

Fig. 1A-1L show the experimental results of the off-target activity of siRNA containing various test compounds.

Fig. 2A-2G show the experimental results of the off-target activity of miRNA2 containing various test compounds.

Fig. 3A-3G show the experimental results of the off-target activity of miRNA3 containing the test compounds.

Figure 4 shows the inhibitory activity of siRNA conjugated to a cluster of galactose amine molecules against the mTTR gene in primary mouse hepatocytes.

Figure 5 shows the in vivo inhibitory activity of siRNA conjugated to a cluster of galactose amine molecules against the mouse mTTR gene.

Figure 6 shows the long-term inhibitory activity in vivo of siRNA conjugated to a cluster of galactosamine molecules against the mouse mTTR gene.

Fig. 7 shows the effect of siRNA agents on total cholesterol levels in Apos3 transgenic mice.

Fig. 8 shows the effect of siRNA agents on triglyceride levels in Apos3 transgenic mice.

Fig. 9 shows the effect of siRNA agents on the Apos3 protein level in Apos3 transgenic mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is further described below with reference to examples, which, however, are not intended to limit the scope of the present invention. The experimental procedures without conditions indicated in the examples of the present invention were generally carried out in accordance with conventional conditions or in accordance with the conditions recommended by the manufacturer of the starting materials or commercial products. If the source of a reagent is not specified, the reagent was obtained from any supplier of molecular biology reagents of quality/purity for use in molecular biology.

I. Obtaining chemical modifications and assessing activity

Example 1. Obtaining chemical modifications

1.1 Synthesis of compound 1-1a and compound 1-1b

Compound 1 (500 mg, 3.42 mmol) and triethylamine (Et3N, 692 mg, 6.84 mmol, 0.95 mL) were dissolved in dichloromethane (DCM, 10 mL). A solution of 4-toluenesulfonyl chloride (TsCl, 717 mg, 3.76 mmol) in dichloromethane (10 mL) was added dropwise in an ice bath. After complete dropwise addition, the reaction mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was quenched with water. The aqueous phase was extracted three times with dichloromethane (15 mL). The organic phases were combined, washed first with saturated aqueous sodium bicarbonate solution (10 mL) and then with saturated brine (20 mL), and then concentrated under reduced pressure to evaporate the solvent to give crude product 2 (820 mg, 80%), which was used directly in the next step. MS (mass spectrometry) m/z(mass/charge): C ₁₄ H ₂₁ O ₅ S, [M+H] ⁺ calculated: 301.10, found: 301.2.

Compound 3 (239 mg, 1.22 mmol) was dissolved in dimethylformamide (DMF, 10 mL). A solution of NaH (60% in mineral oil, 93 mg, 2.33 mmol) was added under ice bath conditions. The reaction mixture was stirred for 30 min, and then compound 2 (350 mg, 1.16 mmol) was added dropwise. After complete dropwise addition, the reaction mixture was stirred at 60°C for 5 h. After completion of the reaction, the mixture was quenched with water. The aqueous phase was extracted three times with ethyl acetate (15 mL). The combined organic phases were washed first with water (10 mL) three times and then with saturated brine (10 mL), then concentrated under reduced pressure to evaporate the solvent, purified by reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-50% (A: H2O, B: CH3CN), flow rate: 70 mL/min) and lyophilized to give compound 4 (220 mg). MS m/z: C ₁₉ H ₂₁ N ₅ O ₃ Na, [M+Na] ⁺ calculated: 390.16, found: 390.3.

Compound 4 (1.50 g, 4.08 mmol) was dissolved in 20 mL of a mixed solution of acetic acid and water (4:1) at room temperature. The mixture was stirred at 60°C for 30 minutes. After completion of the reaction, the mixture was concentrated under reduced pressure to evaporate the solvent and purified using

reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-25% (A: H2O, B: CH ₃ CN), flow rate: 70 mL/min) and lyophilized to give compound 5 (1.10 g). MS m/z: C ₁₆ H ₁₈ N ₅ O ₃ , [M+H] ⁺ calculated: 328.13, found: 328.4.

Compound 5 (1.00 g, 3.05 mmol) was dissolved in pyridine (Py, 10 mL). A solution of 4,4'-dimethoxytrityl chloride (DMTrCl, 1.50 g, 4.58 mmol) in pyridine (5 mL) was added dropwise under ice bath conditions. After completion of the dropwise addition, the reaction mixture was stirred at room temperature overnight. After completion of the reaction, the mixture was quenched with water, concentrated under reduced pressure to evaporate the solvent, purified by reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-80% (A: H ₂ O, B: CH ₃ CN), flow rate: 70 mL/min) and lyophilized to give compound 6 (1.00 g). MS m/z: C ₃₇ H ₃₅ N ₅ O ₅ , [M-H] ⁺ calculated: 630.26, found: 630.5. Racemate 6 was separated using a chiral column (Daicel CHIRALPAK® IE 250×4.6 mm, 5 μm, A: n-hexane, B: ethanol) into 6A(-) (410 mg) and 6B(+) (435 mg).

Compound 6A(-) (200 mg, 0.32 mmol), tetrazole (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), and molecular sieve 3A (500 mg) were dissolved in 10 mL of acetonitrile. Compound 7 (144 mg, 0.48 mmol) was added at room temperature. The mixture was stirred at room temperature overnight. After completion of the reaction, the molecular sieve was filtered off and dichloromethane (30 mL) was added. The mixture was washed three times with saturated aqueous sodium bicarbonate solution (10 mL) and then with saturated brine (20 mL). The filtrate was concentrated by rotary evaporation, purified by reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min) and lyophilized to afford compound 1-1a (200 mg). MS m/z: C ₄₀ H ₃₉ N ₆ O ₇ P, [N-diisopropyl+OH] ⁺ calculated: 747.26, found: 747.6. 1H NMR (400 MHz, acetonitrile-d3) δ 7.56, 7.54 (2s, 1H), 7.36-7.27 (m, 2H), 7.24-7.21 (m, 7H), 6.83-6.80 (m, 4H), 4.12-4.10 (m, 2H), 3.75-3.68 (m, 10H), 3.20-2.80 (m, 2H), 2.68-2.54 (m, 4H), 1.22-1.04 (m, 18H).

Compound 6B(+) (200 mg, 0.32 mmol), tetrazole (11 mg, 0.16 mmol), N-methylimidazole (5 mg, 0.06 mmol), and molecular sieve 3A (500 mg) were dissolved in 10 mL of acetonitrile. Compound 7 (144 mg, 0.48 mmol) was added at room temperature. The mixture was stirred at room temperature overnight. After completion of the reaction, the molecular sieve was filtered off and dichloromethane (30 mL) was added. The mixture was washed three times with a saturated aqueous solution of sodium bicarbonate (10 mL) and then with saturated brine (20 mL). The filtrate was concentrated by rotary evaporation, purified by reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min) and lyophilized to afford compound 1-1b (200 mg). MS m/z: C ₄₀ H ₃₉ N ₆ O ₇ P, [N-diisopropyl+OH] ⁺ calculated: 747.26, found: 747.5.

1.2 Synthesis of compound 1-2

Compound 1 (2 g, 8.36 mmol) was dissolved in DMF (20 mL). NaH (0.37 g, 9.2 mmol, 60% in mineral oil) was slowly added under argon atmosphere at room temperature. After stirring for 2 h at room temperature, compound 2 (3.3 g, 16.72 mmol) was added to the reaction mixture. After stirring for 12 h at room temperature, the reaction mixture was concentrated. The residue was recrystallized from ethanol (EtOH, 50 mL) to give the target product 3A (1.3 g, yield: 44.0%) (dichloromethane:ethyl acetate = 2:1, Rf = 0.2) and the target product 3B (0.6 g, mixture of compound 1) (dichloromethane:ethyl acetate = 2:1, Rf = 0.18).

Compound 3A (1.3 g, 3.68 mmol) was dissolved in a mixture of trifluoroacetic acid (TFA, 4 mL) and DCM (20 mL), and then the reaction mixture was stirred at room temperature for 12 h and concentrated. The resulting residue was purified using a reversed-phase column (C ¹⁸ , H ₂ O + acetonitrile) to give the target product 4 (1 g, yield: 91.44%). MS m/z: C ₃₉ H ₃₈ N ₆ O ₆ , [M + H] ⁺ : 687.5.

The compound (D-threonol 5, 1.2 g, 11.4 mmol) was dissolved in pyridine (10 mL), and then a solution of DMTrCl (4.64 g, 13.70 mmol) in pyridine (15 mL) was slowly added. After 16 h of stirring at room temperature, the reaction mixture was quenched with H ₂ O (10 mL) and concentrated. The reaction mixture was concentrated. The resulting residue was purified using a reversed phase column (C ¹⁸ , H ₂ O + acetonitrile) to give the target product 6 (4.0 g, yield: 86.0%). MS m/z: C ₂₅ H ₂₉ NO ₄ , [M + Na] ⁺ : 430.4.

Compound 6 (600 mg, 2.02 mmol), compound 4 (822.5 mg, 2.02 mmol), and dihydroquinoline (EEDQ, 998.2 mg, 4.04 mmol) were dissolved in DCM (10 mL) and methanol (MeOH, 5 mL). After the mixture was stirred at room temperature for 16 h, the solid was filtered off, and the filtrate was diluted with DCM (100 mL). The organic phase was washed three times with _H2O (30 mL), dried over _{anhydrous Na2SO4} , filtered, and concentrated. The resulting residue was purified using a reversed-phase column ( ^C18 , _H2O +acetonitrile) to afford the target product 7 (780 mg, yield: 56.3%). MS m/z: C ₃₉ H ₃₈ N ₆ O ₆ , [M+H] ⁺ : 687.5.

Compound 7 (780 mg, 1.13 mmol), tetrazole (39.8 mg, 0.57 mmol), and N-methylimidazole (18.7 mg, 0.23 mmol) were dissolved in CH ₃ CN (10 mL). Molecular sieve 3A (700 mg) was added. After stirring for 5 min at room temperature under argon, compound 8 (513.5 mg, 1.70 mmol) was added. After stirring for 1 h at room temperature, the molecular sieve was filtered off, and the solid was washed three times with DCM (30 mL). The filtrate was washed successively with saturated aqueous NaHCO ₃ (30 mL x 4) and H ₂ O (30 mL x 4). The organic phase was concentrated at 30 °C. The obtained residue was purified using a reverse phase column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 90%) and lyophilized to obtain the target compound 1-2 (700 mg, yield: 69.5%). MS m/z: C ₄₈ H ₅₅ N ₈ O ₇ P, [N-cyanoethyl-diisopropyl+OH] ^- : 749.3.

1.3 Synthesis of compound 1-3

Compound 1 (2 g, 8.36 mmol) was dissolved in DMF (20 mL). NaH (0.37 g, 9.2 mmol, 60% in mineral oil) was slowly added under argon atmosphere at room temperature. After stirring for 2 h at room temperature, compound 2 (3.3 g, 16.72 mmol) was added to the reaction mixture. After stirring for 12 h at room temperature, the reaction mixture was concentrated. The residue was recrystallized from EtOH (50 mL) to give the target product 3A (1.3 g, yield: 44.0%) (dichloromethane:ethyl acetate = 2:1, Rf = 0.2) and the target product 3B (0.6 g, mixture of compound 1) (dichloromethane:ethyl acetate = 2:1, Rf = 0.18).

Compound 3A (1.3 g, 3.68 mmol) was dissolved in a mixture of TFA (4 mL) and DCM (20 mL), and then the reaction mixture was stirred at room temperature for 12 h and concentrated. The resulting residue was purified using a reversed-phase column (C ¹⁸ , H ₂ O + acetonitrile) to give the target product 4 (1 g, yield: 91.44%). MS m/z: C ₃₉ H ₃₈ N ₆ O ₆ , [M + H] ⁺ : 687.5.

L-threoninol compound 5 (1.2 g, 11.4 mmol) was dissolved in pyridine (10 mL), and then a solution of DMTrCl (4.64 g, 13.70 mmol) in pyridine (15 mL) was slowly added. After stirring at room temperature for 16 h, the reaction mixture was quenched with H ₂ O (10 mL) and concentrated. The reaction mixture was concentrated. The resulting residue was purified using a reversed phase column (C ¹⁸ , H ₂ O + acetonitrile) to give the target product 6 (4.0 g, yield: 86.0%). MS m/z: C ₂₅ H ₂₉ NO ₄ , [M + Na] ⁺ : 430.4.

Compound 6 (600 mg, 2.02 mmol), compound 4 (822.5 mg, 2.02 mmol), tetramethyluronium hexafluorophosphate (HATU, 1.15 g, 3.03 mmol) and diisopropylethylamine (DIEA, 1 mL, 6.05 mmol) were dissolved in DMF (10 mL). After 16 h of stirring at room temperature, the reaction mixture was filtered and the filtrate was diluted with DCM (100 mL). The organic phase was washed three times with _H2O (30 mL), dried over _{anhydrous Na2SO4} , filtered and concentrated. The resulting residue was purified using a reversed-phase column ( ^C18 , _H2O + acetonitrile, acetonitrile 60%) and lyophilized to give the target compound 7 (1.0 g, yield: 72.1%). MS m/z: C ₃₉ H ₃₈ N ₆ O ₆ , [M+H] ⁺ : 687.5.

Compound 7 (1.2 g, 1.75 mmol), tetrazole (61.2 mg, 0.87 mmol), and N-methylimidazole (28.7 mg, 0.35 mmol) were dissolved in CH ₃ CN (10 mL). Molecular sieve 3A (700 mg) was added. After stirring for 5 min at room temperature under argon, compound 8 (0.79 g, 2.62 mmol) was added. After stirring for 1 h at room temperature, the molecular sieve was filtered off, and the solid was washed three times with DCM (30 mL). The filtrate was washed successively with saturated aqueous NaHCO ₃ (30 mL x 4) and H ₂ O (30 mL x 4). The organic phase was concentrated at 30 °C. The obtained residue was purified using a reversed phase column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 90%) and lyophilized to obtain the target compound 1-3 (1.2 mg, yield: 77.4%). MS m/z: C ₄₈ H ₅₅ N ₈ O ₇ P, [N-cyanoethyl-diisopropyl+OH] ^- : 749.3.

1.4 Synthesis of compound 1-4a and compound 1-4b

Compound 1A (6.73 g, 28.14 mmol) was dissolved in dry DMF (80 mL). NaH (60%, 1.24 g, 30.95 mmol) was slowly added under argon. After stirring the mixture at room temperature for 30 min, the reaction mixture was added to a solution of tetrakis(triphenylphosphine)palladium(0) (Pd( _PPh3 ) ₄ , 1.95 g, 1.69 mmol), triphenylphosphine ( _PPh3 , 0.74 g, 2.81 mmol), and compound 1 (4.0 g, 28.14 mmol) in tetrahydrofuran (THF, 60 mL). After the reaction mixture was stirred at 55 °C for 16 h, the solid was filtered and washed three times with DCM (60 mL). The filtrate was concentrated. The resulting residue was purified using a normal phase column (eluting first with ethyl acetate and then with ethyl acetate:methanol (12:1)) to give the desired product 2 (7 g, crude product).

Compound 2 (8 g, crude product) and DMTrCl (12.65 g, 37.34 mmol) were dissolved in pyridine (10 mL). The mixture was stirred at room temperature for 16 h, then quenched with water (80 mL) and concentrated. The resulting residue was purified using a reverse-phase column (C ¹⁸ , water + acetonitrile) and lyophilized to afford the target compound 3 (13 g, yield: 83.7%).

Compound 3 (5 g, 8.02 mmol) was dissolved in methanol (MeOH, 20 mL) and ammonia water (6 mL). After stirring at room temperature for 16 h, the reaction mixture was concentrated. The resulting residue was purified using a normal phase column (DCM:MeOH=20:1) to obtain the target compound 4 (4 g, yield: 96.0%).

A solution of borane ( _BH3 ) in tetrahydrofuran (1.0 M in THF, 38.54 mL, 38.54 mmol) was added dropwise to a solution of compound 4 (4.00 g, 7.71 mmol) in THF (12 mL) at 0 °C under argon. After the compound was stirred at 0 °C under argon for 6 h, _H2O (27 mL) was added dropwise. Then, after adding 3 M aqueous NaOH (52 mL, 156 mmol) dropwise to the reaction mixture at 0 _° C, 30% aqueous _H2O2 (106 mL) was added dropwise to the reaction mixture and EtOH (10 mL ₎ was added. After the reaction mixture was stirred at room temperature for 48 h, saturated _Na2S2O3 was slowly added at 0 °C until bubbling. _H2O (300 mL) was _added to the reaction mixture, and the mixture was extracted with DCM (4 times 200 mL). _The organic phase was dried over anhydrous _Na2SO4 , filtered and concentrated. The resulting residue was purified using a reverse phase column (C ¹⁸ , acetonitrile + H ₂ O, 50%) and lyophilized to obtain the target product 5a (730 mg, yield: 17.6%) and the target product 5b (1.1 g, 26.6%).

Compound 5a (730 mg, 1.36 mmol) was dissolved in pyridine (8 mL). TMSCl (0.67 g, 6.14 mmol) was added under argon at room temperature. After stirring for 1 h at room temperature, BzCl (0.29 mL, 2.46 mmol) was added to the reaction mixture. After stirring for 16 h at room temperature, the reaction mixture was quenched with H ₂ O (10 mL) and concentrated. The resulting residue was dissolved in THF (30 mL). Tetrabutylammonium fluoride (TBAF, 1 mL) was added. After stirring for 1 h at room temperature, ammonia water (0.5 mL) was added. The mixture was stirred at room temperature for 5 h. The reaction mixture was diluted with ethanol (EA, 100 mL) and washed five times with brine (30 mL). The organic phase was concentrated. The resulting residue was purified using a reversed phase column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 60%) and lyophilized to give the target product 6a (480 mg, yield: 74.8%). MS m/z: C ₃₈ H ₃₅ N ₅ O ₅ , [M+H] ⁺ : 642.6.

Compound 5b (1.1 g, 2.05 mmol) was dissolved in pyridine (20 mL). TMSCl (1.34 g, 1.28 mmol) was added under argon at room temperature. After stirring for 1 h at room temperature, benzoyl chloride (BzCl, 0.59 mL, 5.92 mmol) was added to the reaction mixture. After stirring for 16 h at room temperature, the reaction mixture was quenched with _H2O (10 mL) and concentrated. The resulting residue was dissolved in THF (30 mL). TBAF (2 mL) was added. After stirring for 1 h at room temperature, ammonia water (0.5 mL) was added. The mixture was stirred at room temperature for 5 h. The reaction mixture was diluted with EA (100 mL) and washed five times with brine (30 mL). The organic phase was concentrated. The resulting residue was purified using a reversed phase column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 60%) and lyophilized to give the target product 6b (1.4 mg, yield: 82.1). MS m/z: C ₃₈ H ₃₅ N ₅ O ₅ , [M+H] ⁺ : 642.5.

Compound 6a (700 mg, 1.04 mmol), tetrazole (26.2 mg, 0.37 mmol), and N-methylimidazole were dissolved in CH ₃ CN (10 mL). Molecular sieve 3A (500 mg) was added. After stirring for 5 min at room temperature under argon, compound 7 (470.4 mg, 1.56 mmol) was added. After stirring for 1 h at room temperature, the molecular sieve was filtered off, and the solid was washed three times with DCM (50 mL). The filtrate was washed successively with saturated aqueous NaHCO ₃ (50 mL, 4 times) and H ₂ O (50 mL, 4 times). The organic phase was concentrated at 30 °C. The obtained residue was purified using a reverse phase column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 90%) and lyophilized to obtain the target compound 1-4A (600 mg, yield: 66.1%). MS m/z: C ₄₇ H ₅₂ N ₇ O ₆ P, [N-cyanoethyl-diisopropyl+OH] ^- : 704.3.

Compound 6b (1.3 g, 2.03 mmol), tetrazole (71.0 mg, 1.01 mmol), and N-methylimidazole (33.3 mg, 0.41 mmol) were dissolved in CH ₃ CN (20 mL). Molecular sieve 3A (700 mg) was added. After stirring for 5 min at room temperature under argon, compound 7 (0.92 g, 3.04 mmol) was added. After stirring for 1 h at room temperature, the molecular sieve was filtered off, and the solid was washed three times with DCM (50 mL). The filtrate was washed successively with saturated aqueous NaHCO ₃ (50 mL x 4) and H ₂ O (50 mL x 4). The organic phase was concentrated at 30 °C. The obtained residue was purified using a reverse phase column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 90%) and lyophilized to give the target compound 1-4b (1.4 mg, yield: 82.1%). MS m/z: C ₄₇ H ₅₂ N ₇ O ₆ P, [N-cyanoethyl-diisopropyl] ^- : 704.3.

1.5. Synthesis of compound 1-5

Compound 1A (6.73 g, 28.14 mmol) was dissolved in dry DMF (80 mL). NaH (60%, 1.24 g, 30.95 mmol) was slowly added under argon. After stirring the mixture at room temperature for 30 min, the reaction mixture was added to a solution of Pd( _PPh3 ) ₄ (1.95 g, 1.69 mmol), _PPh3 (0.74 g, 2.81 mmol), and compound 1 (4.0 g, 28.14 mmol) in THF (60 mL). After the reaction mixture was stirred at 55 °C for 16 h, the solid was filtered and washed three times with DCM (60 mL). The filtrate was concentrated. The resulting residue was purified using a normal phase column (eluting first with ethyl acetate and then with ethyl acetate:methanol (12:1)) to give the desired solid 2 (7 g, crude product).

Compound 2 (8 g, crude product) and DMTrCl (12.65 g, 37.34 mmol) were dissolved in pyridine (10 mL). The mixture was stirred at room temperature for 16 h, then quenched with water (80 mL) and concentrated. The resulting residue was purified using a reverse-phase column (C ¹⁸ , water + acetonitrile) and lyophilized to afford the target compound 3 (13 g, yield: 83.7%).

Compound 3 (1 g, 1.60 mmol), _KHCO3 (0.48 g, 4.81 mmol), and ethylene glycol (0.40 g, 6.41 mmol) were dissolved in acetone (50 mL). _KMnO4 (40% in water, 0.67 g, 1.68 mmol) was slowly added at -30 °C. After 1 h of stirring at -30 °C, the reaction mixture was quenched with saturated aqueous sodium thiosulfate (30 mL). The mixture was extracted four times with DCM (30 mL). _The organic phase was dried over anhydrous _Na2SO4 , filtered, and concentrated. The residue was purified using a reversed phase column (C ¹⁸ , H2O + acetonitrile, acetonitrile 60%) and lyophilized to give the target product 4 (600 mg, yield: 56.9%). MS m/z: C ₃₈ H ₃₅ N ₅ O ₆ , [M + H] ⁺ : 658.5.

Reagent 4 (5.0 g, 7.601 mmol), _NaIO4, and 1,4-dioxane/water (50 mL/5 mL) were added to a 250 mL round-bottomed flask. The mixture was reacted at room temperature for 2 h and then concentrated under reduced pressure to remove the solvent to give a white solid (6.0 g). The solid was then dissolved in methanol (50 mL), and sodium borohydride (1.62 g, 38 mmol) was added.

After stirring the mixture at room temperature for 2 h, 10% ammonium chloride solution (10 mL) was added. After removing the solvent under reduced pressure, the residue was purified by ^C18 column chromatography (water/acetonitrile: 5-95%) to afford product P1 as a colorless oil 5 (2.0 g, 3.0315 mmol, 39%), LCMS (liquid chromatography-mass spectrometry), MS+, [M+H] ⁺ : 660.

Compound 5 (1.7 g, 2.58 mmol) and DBU (diazabicycloundecene) (0.77 mL, 5.15 mmol) were dissolved in DCM (20 mL). BzCl (0.5 M in DCM, 0.8 mL) was added dropwise to the reaction at -70 °C under argon atmosphere. The reaction mixture was left standing at -70 °C for 1 h and quenched with ethanol (5 mL). The quenched reaction mixture was diluted with DCM (100 mL) and washed three times with water (30 mL). The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The resulting residue was purified using a normal phase column (DCM:EA=1:1) to afford 6 as a white solid (80 mg, yield: 4.14%). MS m/z: C ₄₅ H ₄₁ N ₅ O ₇ , [M+H] ⁺ : 764.5.

Compound 4 (380 mg, 0.50 mmol), tetrazole (17.43 mg, 0.25 mmol), and N-methylimidazole (8.17 mg, 0.10 mmol) were dissolved in CH ₃ CN (10 mL). Molecular sieve 3A (500 mg) was added. After stirring for 5 min at room temperature under argon, compound 7 (224.95 mg, 0.75 mmol) was added. After stirring for 1 h at room temperature, the molecular sieve was filtered off, and the solid was washed three times with DCM (50 mL). The filtrate was washed successively with saturated aqueous NaHCO ₃ (50 mL x 4) and H ₂ O (50 mL x 4). The organic phase was concentrated at 30 °C. The obtained residue was purified using a reverse phase column (C ¹⁸ , H ₂ O + acetonitrile, acetonitrile 90%) and lyophilized to obtain the target product 1-5 (330 mg, yield: 68.8%). MS m/z: C ₅₄ H ₅₈ N ₇ O ₈ P, [N-cyanoethyl-diisopropyl] ^- : 826.3.

1.6 Synthesis of compound 1-6a

Compound 1 (10 g, 68.404 mmol), compound 2 (15 g, 62.186 mmol), and triphenylphosphine (32.62 g, 124.371 mmol) were dissolved in dry THF (30 mL). DIAD (24.656 mL, 124.371 mmol) was slowly added dropwise at 0 °C. The reaction mixture was reacted at 25 °C for 12 h, and LCMS showed that the reaction was complete. The reaction mixture was extracted with ethyl acetate (200 mL) and water (200 mL). The organic phase was dried. The filtrate was concentrated. The resulting residue was purified using a normal phase column (DCM/MeOH=10/1) to give the target product 3 (20 g).

Compound 3 (20 g, 28.585 mmol) was dissolved in acetic acid (24 mL, 426.016 mmol) and _H2O (12 mL). The mixture was stirred at 60 °C for 1 h. The reaction mixture was then concentrated to dryness by rotary evaporation. THF (12 mL) and _H2O (12 mL) were added. The mixture was stirred at 80 °C for 7 h. LCMS showed that the reaction was complete. The reaction mixture was extracted with ethyl acetate (200 mL) and water (100 mL). Solid sodium carbonate was added to the aqueous phase until a large amount of solid precipitated from the aqueous phase. The solid was collected by filtration and washed with water. The filter cake was dried with an oil pump to give the title compound 5 (9 g).

Compound 5 (6.8 g, 18.581 mmol) was dissolved in pyridine (80 mL) under nitrogen atmosphere. TMSCl (14.250 mL, 111.489 mmol) was slowly added at 0 °C. The mixture was stirred for 2 h. Then, isobutyryl chloride (2.044 mL, 19.511 mmol) was added at 0 °C. The mixture was stirred at 25 °C for 1 h, and LCMS showed that the reaction was complete. The mixture was extracted with dichloromethane (200 mL) and water (200 mL). After the organic phase was dried and concentrated to dryness by rotary evaporation, a sample for purification was obtained. The sample was purified using a normal phase column (eluting with DCM:MeOH=10:1, peak at 4.8%) to afford compound 6 as a yellow oil (12 g).

Compound 6 (5.5 g, 12.392 mmol) was dissolved in pyridine (30 mL) under nitrogen atmosphere. Molecular sieve 4A 1/16 (7 g, 12.392 mmol) was added, followed by solid DMTrCl (5.04 g, 14.870 mmol) added in portions at 0 °C. The mixture was reacted at 25 °C for 2 h, and TLC (PE:EtOAc=1:1, Rf=0.69) showed that the reaction was complete. The reaction mixture and TJN200879-040-P1 were combined and worked up together. The reaction mixture was extracted with ethyl acetate (200 mL) and water (200 mL). After the organic phase was dried and concentrated to dryness by rotary evaporation, a sample for purification was obtained. The sample was purified using a normal phase column (eluting with PE:EtOAc, peak at 84%) to afford compound 7 as a yellow oil (12 g).

Compound 7 (12 g, 15.389 mmol) was dissolved in EtOAc (140 mL). Wet palladium on carbon Pd/C (7 g, 15.389 mmol) was added. The reaction mixture was reacted at 25 °C under hydrogen (15 Psi) for 2 h. TLC (PE:EtOAc=0:1, Rf=0.09) showed that the reaction was complete. The reaction mixture was filtered. After the filter cake was washed three times with ethyl acetate (30 mL), the filtrate was collected. After the filtrate was concentrated to dryness by rotary evaporation, 50 mL of dichloromethane and 2 mL of triethylamine were added to obtain a sample for purification. The sample was purified using a normal phase column (eluting with DCM:MeOH=10:1, peak at 0.5%) to yield 9 g (yellow foamy solid). The resulting racemic compound was resolved by SFC (supercritical fluid chromatography) into the target compound 7A(-) (3.9 g) and the target compound 7B(+) (3.8 g).

Compound 7A(-) (3.30 g, 5.40 mmol), tetrazole (190 mg, 2.70 mmol), 1-methylimidazole (90 mg, 1.10 mmol), and molecular sieve 3A (500 mg) were dissolved in 30 mL of acetonitrile. Compound 8 (2.50 g, 8.10 mmol) was added at room temperature.

The mixture was stirred at room temperature for 2 h. After completion of the reaction, the molecular sieve was filtered off and DCM (150 mL) was added. The mixture was washed with saturated aqueous sodium bicarbonate solution (30 mL 3 times) and then with saturated brine (30 mL). The filtrate was concentrated by rotary evaporation, purified by reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min) and lyophilized to afford compound 1-6a (2.9 g, 66%). MS m/z: C ₄₃ H ₅₅ N ₇ O ₇ P [M+H] ⁺ , calculated: 812.38, found: 812.5. 1H NMR (400 MHz, acetonitrile-d3) δ 7.56, 7.54 (2s, 1H), 7.36-7.27 (m, 2H), 7.24-7.21 (m, 7H), 6.83-6.80 (m, 4H), 4.12-4.10 (m, 2H), 3.75-3.68 (m, 10H), 3.20-2.80 (m, 2H), 2.68-2.54 (m, 4H), 1.22-1.04 (m, 18H).

1.7. Synthesis of compound 1-7a

Compound 1 (5 g, 23.1272 mmol), compound 2 (6.76 g, 46.254 mmol), and triphenylphosphine (7.28 g, 27.753 mmol) were dissolved in 30 mL of dioxane under nitrogen atmosphere. DEAD (diethyl azodicarboxylate) (5.502 mL, 27.753 mmol) was slowly added dropwise at 0 °C. After complete dropwise addition, the reaction mixture was slowly warmed to 25 °C and reacted for another hour. The reaction mixture was extracted with 100 mL of _H2O and 100 mL of EtOAc. After the organic phases were combined, dried, filtered, and concentrated, a sample for purification was obtained. The sample was purified using a normal phase column (eluting with PE:EtOAc=1:1) to obtain the target product (4 g).

Compound 3 (3.3 g) was dissolved in HOAc (16 mL) and _H2O (4 mL). The reaction mixture was heated in an oil bath at 60°C for 0.5 h and concentrated to dryness by rotary evaporation. The resulting residue was purified using a normal phase column (elution with PE:EtOAc = 0:1) to afford the target product 4 (3 g).

Compound 4 (3 g, 8.873 mmol) was dissolved in 5 mL of pyridine. A solution of DMTrCl (3.91 g, 11.535 mmol) in 10 mL of pyridine was slowly added dropwise at 0 °C under a nitrogen atmosphere. After complete dropwise addition, the reaction mixture was warmed to 25 °C and reacted for another hour. The reaction mixture was extracted with 50 mL of water and 100 mL of ethyl acetate. The aqueous phase was extracted three more times with 100 mL of ethyl acetate. The organic phases were combined, dried, filtered, concentrated, and purified using a normal phase column (PE:EtOAc = 2:1) to obtain the target product 5 (4 g).

Compound 5 (4 g, 5.769 mmol) was dissolved in methanol (10 mL). A saturated solution of NH ₃ in methanol (40 mL) was added. The mixture was reacted at 0 °C for 6 h. The reaction mixture was concentrated to dryness by rotary evaporation and purified using a normal phase column (PE:EtOAc=0:1) to give the racemic compound (2.4 g). The compound was separated by SFC into the desired product 6A (750 mg, 100% purity) and the desired product 6B (400 mg, 99.16% purity).

Compound 6A(-) (700 mg, 1.40 mmol), tetrazole (50 mg, 0.70 mmol), 1-methylimidazole (23 mg, 0.28 mmol), and molecular sieve 3A (500 mg) were dissolved in 10 mL of acetonitrile. Compound 7 (630 mg, 2.10 mmol) was added at room temperature. The mixture was stirred at room temperature for 2 h. After completion of the reaction, the molecular sieve was filtered off and DCM (50 mL) was added. The mixture was washed with saturated aqueous sodium bicarbonate solution (10 mL 3 times) and then with saturated brine (20 mL). The filtrate was concentrated by rotary evaporation, purified by reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min) and lyophilized to afford compound 1-7a (700 g, 72%). MS m/z: C ₃₈ H ₄₇ N ₄ O ₇ PNa [M+Na] ⁺ , calculated: 725.32, found: 725.5.

1.8. Synthesis of compound 1-8a

Compound 1 (8.5 g, 76.508 mmol) and compound 2 (30.64 g, 91.809 mmol) were dissolved in DMF (150 mL). _CS2CO3 (29.91 g, 91.809 mmol) was added. _The reaction mixture was reacted under nitrogen atmosphere at 90 °C for 12 h. LCMS detection showed that the reaction was complete. The reaction mixture was filtered, concentrated to dryness by rotary evaporation using an oil pump, and separated and purified using a normal phase column (80 g, DCM/MeOH=10/1 to 5/1) to give the target product 3 (13.5 g, 80% purity).

Compound 3 (10.5 g, 35.105 mmol) was dissolved in pyridine (65 mL) and _CH3CN (65 mL). BzCl (4.894 mL, 42.126 mmol) was added dropwise to the solution. The mixture was reacted at 25°C for 2 h. LCMS detection showed that the starting materials were mainly reacted. The mixture was quenched with H2O (100 mL) and extracted with EtOAc (100 mL x 3). The extract was dried, concentrated to dryness by rotary evaporation and separated (in combination with TJN200872-101) and purified by column chromatography (80 g, PE/EtOAc=10/1-0/1, DCM/MeOH=10/1) to give the desired product 4 (14 g, 90% purity).

Compound 4 (14 g, 36.694 mmol) was dissolved in HOAc (56 mL, 314.796 mmol) and _H2O (14 mL). The mixture was reacted at 60°C for 2 h, and LCMS showed that the reaction was complete. The mixture was concentrated using an oil pump and separated using a normal phase column (40 g, DCM/MeOH = 1/0 to 5/1) to afford the target product 5 (8.4 g, 90% purity and 2.4 g, 80% purity).

Compound 5 (7.4 g, 21.957 mmol), DMAP (4-dimethylaminopyridine) (0.54 g, 4.391 mmol), and molecular sieve 4A (11.1 g, 2.967 mmol) were dissolved in pyridine (60 mL). The mixture was stirred under ice bath conditions for 10 min, and then DMTrCl (8.93 g, 26.348 mmol) was added. The reaction mixture was stirred for 1.8 h, and LCMS detection showed that about 19% of the starting material remained and about 60% was the target MS. The mixture was combined with TJN200872-105 and 106 and purified together. _H2O (50 mL) was added to the reaction mixture. The mixture was extracted with DCM (50 mL 3 times), dried, concentrated to dryness by rotary evaporation and separated by column chromatography (120 g, PE/(EA:DCM:TEA:TEA=1:1:0.05)=1/0 to 0/1 to DCM/MeOH=10/1) to give compound 6 as a yellow solid (11 g, 89% purity, TJN200872-105, 106 and 107). The starting material was recovered (3.0 g, 70% purity).

Compound 6 (15 g, 22.041 mmol) was cleaved using SFC (DAICEL CHIRALPAK AD (250 mm × 50 mm, 10 μm); 0.1% NH ₃ H ₂ O EtOH, B: 45% to 45%; 200 mL/min) to give the desired product 6A (5.33 g, 94.29% purity) and the desired product 6B (6.14 g, 97.91% purity). 1.0 g of compound 6 was isolated.

Compound 6B(-) (5.4 g, 8.92 mmol), tetrazole (312 mg, 4.46 mmol), 1-methylimidazole (146 mg, 1.78 mmol), and molecular sieve 3A (500 mg) were dissolved in 40 mL of acetonitrile. Compound 7 (4 g, 13.4 mmol) was added at room temperature. The mixture was stirred at room temperature for 2 h. After completion of the reaction, the molecular sieve was filtered off and DCM (200 mL) was added. The mixture was washed with saturated aqueous sodium bicarbonate solution (30 mL 3 times) and then with saturated brine (50 mL). The filtrate was concentrated by rotary evaporation, purified by reversed-phase preparative HPLC (C ¹⁸ , conditions: 5-100% (A: water, B: CH ₃ CN), flow rate: 70 mL/min) and lyophilized to afford compound 1-8a (5.8 g, 80%). MS m/z: C ₄₅ H ₅₁ N ₅ O ₇ P, [M+H] ⁺ , calculated: 804.36, found: 804.4.

Example 2. Synthesis of miRNA

The siRNA synthesis was the same as conventional solid-phase phosphoramidite synthesis. To synthesize the modified nucleotide at position 7 of the AS strand, the original nucleotide of the original sequence was replaced with the phosphoramidite monomer synthesized above.

The synthesis process is briefly described below: Nucleoside phosphoramidite monomers were coupled one after another according to the synthesis program on a Dr. Oligo48 synthesizer (Biolytic), starting from a universal CPG (controlled porosity glass) support. Except for the phosphoramidite monomer at the 5'-position 7 of the AS chain described above, other nucleoside monomer materials 2'-F RNA, 2'-O-methyl RNA, and other nucleoside phosphoramidite monomers were purchased from Hongene, Shanghai, or Genepharma, Suzhou. 5-ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M solution in acetonitrile), 0.22 M solution of PADS (phenylacetyl disulfide) in acetonitrile and collidine (1:1 by volume) (Kroma, Suzhou) as a sulfurizing agent, and iodopyridine/water solution (Kroma) as an oxidizing agent.

After completion of the solid-phase synthesis, the oligoribonucleotides were cleaved from the solid support and soaked in a solution of 28% ammonia water and ethanol (3:1) at 50°C for 16 h. The mixture was centrifuged, and the supernatant was transferred to another centrifuge tube. After the supernatant was concentrated to dryness by evaporation, the residue was purified by C ¹⁸ reversed-phase chromatography using 0.1 M TEAA (triethylammonium acetate) and acetonitrile as the mobile phase, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected, then lyophilized, identified as target products by LC-MS, and quantified by UV detection (260 nm).

The resulting single-stranded oligonucleotides were paired at equilibrium in a complementary manner and annealed. The final double-stranded siRNA was dissolved in 1× PBS (phosphate-buffered saline), and the solution was adjusted to the concentration required for the experiment so that it was ready for use.

Example 3. psiCHECK activity screening

3.1 Experimental materials and equipment

Synthesis of miRNA samples was as described above. Plasmids were obtained from Sangon Biotech (Shanghai) Co., Ltd. Consumables, reagents, and instruments for the psiCHECK assay are shown in Table 1 and Table 2.

3.2. psiCHECK Activity Screening Procedure

Cell seeding and transfection were performed. Specific quantities for obtaining the transfection complex are listed in Table 3.

Dilutions with different concentrations were prepared as working solutions for subsequent use to meet different experimental requirements according to Table 4.

At 24 h post-transfection, assays were performed according to the Dual-Glo® Luciferase Assay System kit instructions. Dual-Glo® Luciferase Assay System assays were performed using the Dual Luciferase Reporter Gene Assay Kit (Promega, Cat. No. E2940), and firefly chemiluminescence and Renilla chemiluminescence values were read. Relative values were calculated as Ren/FIR, and inhibition (%) was calculated as 1 - (ratio+siRNA/reporter-only ratio × 100%).

In this description, the proportion of remaining mRNA expression = 100% - inhibition (%).

Example 4. Experiments with targeted and non-targeted siRNA activity consisting of various chemical modifications

The following siRNAs were synthesized using the compounds of Example 1 and the method of Example 2, and the target activity and off-target activity of each siRNA were tested using the method of Example 3. The siRNAs had identical sense strands and contained the following modified nucleotides/chemical modifications, respectively, at position 7 of the 5' end of the antisense strand:

where the nucleotide synthesized using 2-hydroxymethyl-1,3-propanediol as a starting material was defined as hmpNA;

TJ-NA019(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-2 from the example in Section 1.1;

TJ-NA020(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-3 from the example in Section 1.1;

TJ-NA026(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-4a from the example in Section 1.1;

TJ-NA027(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-4b from the example in Section 1.1;

TJ-NA038(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-5 from the example in Section 1.1;

(+)hmpNA(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-1b from the example in Section 1.1, and its absolute configuration was (S)-hmpNA(A);

(-)hmpNA(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-1a from the example in Section 1.1, and its absolute configuration was (R)-hmpNA(A).

Similarly, the following structures were prepared by solid-phase synthesis and by varying the hmpNA base species, and their absolute configurations were determined:

(+)hmpNA(G), with absolute configuration (S)-hmpNA(G);

(-)hmpNA(G), with absolute configuration (R)-hmpNA(G), prepared by solid-phase synthesis using nucleoside phosphoramidite monomer 1-6a from Example Section 1.6;

(+)hmpNA(C), with absolute configuration (S)-hmpNA(C);

(-)hmpNA(C), with absolute configuration (R)-hmpNA(C), prepared by solid-phase synthesis using nucleoside phosphoramidite monomer 1-8a from Example Section 1.8;

(+)hmpNA(U), with absolute configuration (R)-hmpNA(U); and

(-)hmpNA(UU), with absolute configuration (S)-hmpNA(G), obtained by solid-phase synthesis using nucleoside phosphoramidite monomer 1-7a from Example Section 1.7;

The absolute configurations of (S)-hmpNA(G), (R)-hmpNA(G), (S)-hmpNA(C), (R)-hmpNA(C), (S)-hmpNA(U), and (R)-hmpNA(U) were determined from their intermediate or derivative by X-ray diffraction.

Structure of the intermediate or derivative:

TJ-NA067: is defined as a colorless massive crystal (0.30×0.10×0.04 mm3) belonging to the monoclinic crystal system with space group P21. Lattice parameter a=16.0496(5) Å, b=4.86260(10) Å, c=16.4686(5) Å, α=90°, β=118.015(4)°, γ=90°, V=1134.65(7) Å3, Z=4. Calculated density Dc=1.389 g/cm3; number of electrons in unit cell F(000)=504.0; linear absorption coefficient of unit cell μ (Cu Kα)=0.840 mm-1; temperature of the diffraction experiment T=150.00(11) K.

6A(+): is defined as a colorless massive crystal (0.30×0.20×0.10 mm3) belonging to the monoclinic crystal system with space group P21. Lattice parameter a=22.6688(7) Å, b=8.5595(2) Å, c=23.3578(5) Å, α=90°, β=113.876(3)°, γ=90°, V=4144.3(2) Å3, Z=2. Calculated density Dc=0.999 g/cm3; number of electrons in the unit cell F(000)=1318.0; linear absorption coefficient of the unit cell μ (Cu Kα)=0.570 mm-1; temperature of the diffraction experiment T=100.01(18) K.

TJ-NA048: is defined as a colorless needle-shaped crystal (0.30×0.04×0.04 mm3) belonging to the monoclinic crystal system with space group P1. Lattice parameter a=7.6165(4) Å, b=11.3423(5) Å, c=17.3991(8) Å, α=85.007(4)°, β=88.052(4)°, γ=70.532(4)°, V=1411.75(12) Å3, Z=2. Calculated density Dc=1.366 g/cm3; number of electrons in unit cell F(000)=620.0; linear absorption coefficient of unit cell μ (Cu Kα)=0.856 mm-1; temperature of the diffraction experiment T=150.00(13) K.

TJ-NA092: is defined as a colorless prismatic crystal (0.30×0.10×0.10 mm3) belonging to the triclinic crystal system with space group P1. The lattice parameter a=5.17960(10) Å, b=8.0667(2) Å, c=12.4077(2) Å, α=93.146(2)°, β=101.266(2)°, γ=96.134(2)°, V=503.993(18) Å3, Z=2. The calculated density Dc=1.412 g/cm3; the number of electrons in the unit cell F(000)=228.0; the linear absorption coefficient of the unit cell μ (Cu Kα)=0.945 mm-1; temperature of the diffraction experiment T=100.00(10) K.

The experimental results of the on-target activity are shown in Table 6, and the experimental results of the off-target activity are shown in Table 7 and Figs. 1A–1L. All the test sequences with the compounds of the current experiment showed the activity similar to or slightly better than that of the parent sequence, indicating that the modifications did not affect the on-target activity. The siRNAs containing GNA/Abasic/Id, TJ-NA019(A), TJ-NA020(A), TJ-NA026(A), (+)hmpNA(A), and (-)hmpNA(A), had the best activity. In addition, the parent sequence had significant off-target activity, and all the modifications showed significant inhibitory effects against the off-target activity. In particular, no off-target activity was observed in the siRNAs containing TJ-NA027(A), (+)hmpNA(A), and (-)hmpNA(A).

Table 6. Results of target activity of siRNA targeting HBV-S

Double-stranded
code Remaining percentage of expression (mean) of target gene mRNA (target activity) Meaning
IC ₅₀
(concentration
half-maximum
inhibition)
(nM) 40
nM 13.3
nM 4.44
nM 1.48
nM 0.493
nM 0.164 nM 0.054 nM 0.0182
nM 0.00609 nM 0.00203
nM 0.00067 nM TRD
4389 5.4% 4.1% 4.8% 4.8% 8.4% 21.7% 53.0% 82.5% 104.9% 99.4% 95.2% 0.0589 TRD
5252 3.4% 3.1% 3.1% 3.6% 5.7% 11.1% 22.1% 44.7% 72.8% 92.2% 86.6% 0.0162 TRD
5812 3.8% 3.0% 3.4% 3.6% 7.3% 9.8% 23.5% 44.8% 63.9% 90.4% 81.4% 0.0158 TRD
5813 5.1% 3.8% 4.4% 4.3% 6.1% 13.2% 33.5% 53.8% 74.5% 80.8% 96.4% 0.0214 TRD
5816 3.9% 3.8% 3.4% 4.9% 6.9% 16.1% 39.8% 71.8% 96.3% 92.9% 108.1% 0.0389 TRD
5817 4.8% 4.2% 4.5% 3.7% 6.6% 13.7% 31.0% 61.0% 81.8% 92.9% 103.7% 0.0251 TRD
5818 3.7% 3.3% 3.1% 3.7% 6.1% 10.9% 26.3% 55.8% 69.1% 87.4% 88.8% 0.0195 TRD
5820 4.4% 3.8% 3.5% 3.7% 4.2% 9.5% 22.8% 49.2% 79.4% 93.2% 91.7% 0.0191 TRD
5821 6.8% 5.2% 5.7% 6.1% 8.7% 19.7% 39.3% 69.9% 102.8% 92.9% 97.9% 0.0398 TRD
5822 4.4% 4.5% 4.1% 3.7% 5.3% 13.2% 24.6% 51.2% 82.3% 84.9% 101.9% 0.0200 TRD
5823 3.6% 3.8% 3.4% 3.4% 5.2% 11.0% 29.7% 58.3% 71.4% 84.7% 100.7% 0.0200 TRD
5825 4.3% 3.6% 3.4% 4.2% 7.1% 18.0% 32.7% 66.0% 88.7% 93.8% 103.2% 0.0302

Table 7. Results of off-target activity of HBV-S-targeted siRNA

Double-stranded code Remaining percentage of target gene mRNA expression (mean) (off-target activity) 40
nM 13.3
nM 4.44
nM 1.48
nM 0.493
nM 0.164
nM 0.054
nM 0.0182 nM 0.00609
nM 0.00203
nM 0.00067 nM TRD
4389 57.4
% 55.9
% 65.5
% 73.3
% 89.2
% 92.8
% 105.3
% 102.4
% 107.6
% 96.0
% 101.2
% TRD
5252 97.3
% 100.4
% 104.0
% 108.1
% 107.3
% 102.9
% 108.7
% 94.9
% 101.2
% 101.8
% 97.7% TRD
5812 98.2
% 107.0
% 99.1
% 100.7
% 110.1
% 125.2
% 113.7
% 105.3
% 105.5
% 99.5
% 93.8% TRD
5813 100.5
% 105.2
% 95.9
% 112.1
% 102.3
% 104.3
% 101.5
% 97.2
% 110.7
% 100.6
% 93.6% TRD
5816 108.3
% 101.5
% 97.2
% 109.5
% 116.7
% 122.8
% 108.5
% 113.2
% 121.6
% 112.9
% 106.8
% TRD
5817 104.5
% 106.7
% 110.0
% 109.3
% 119.4
% 120.9
% 127.3
% 113.6
% 117.7
% 112.2
% 105.0
% TRD
5818 83.7
% 89.7
% 83.0
% 91.0
% 117.5
% 79.4
% 99.1
% 103.4
% 89.2
% 92.9
% 98.7% TRD
5820 92.1
% 100.3
% 104.3
% 98.9
% 103.6
% 103.8
% 106.2
% 108.3
% 105.8
% 100.3
% 97.7% TRD
5821 102.9
% 99.3
% 98.3
% 99.6
% 106.8
% 106.4
% 108.7
% 108.1
% 104.5
% 95.4
% 107.8
% TRD
5822 106.1
% 93.8
% 81.6
% 100.4
% 100.4
% 96.9
% 105.3
% 101.9
% 94.6
% 101.4
% 94.0% TRD
5823 91.8
% 89.1
% 92.9
% 99.8
% 97.8
% 101.1
% 90.7
% 92.6
% 97.9
% 95.9
% 87.1% TRD
5825 84.9
% 89.7
% 97.7
% 106.7
% 103.9
% 104.7
% 100.0
% 100.9
% 90.2
% 112.7
% 98.3%

Example 5. Sequence dependence experiment of siRNAs containing various chemical modifications

It is known that abasic modification depends on the siRNA sequence, therefore, the inventors tested the test compounds of the present invention on several different sequences. siRNAs targeting mRNAs of four different genes (ANGPTL3, HBV-S, HBV-X and TTR) (their sequences are shown in Table 8) were used and modified at position 7 of the 5'-end of the AS chain with the compounds of Example 1: TJ-NA020(A), TJ-NA027(A), (+)hmpNA(A), (-)hmpNA(A), GNA(A) (as a control) and compound Id (sequences are shown in Table 9) and compared with the original sequences with respect to on-target and off-target activity.

The results of the target activity experiment are shown in Table 10. HNK(A) showed significant sequence dependence, and different sequences had significantly different target activities. The test compounds of the present invention did not show significant sequence dependence, indicating that they are more universally applicable. Furthermore, introducing only the 2'-F modification at position 9 of the 5'-end of the AS chain and only the 2'-OMe modification at position 10 resulted in similar activity, i.e., the test compounds of the present invention did not show significant sequence dependence.

The experimental results of the off-target activity of miRNA2 and miRNA3 are shown in Table 11, Figs. 2A-2G (targeting HBV-S) and Figs. 3A-3G (targeting HBV-X). It can be seen that the test compounds of the present invention significantly reduce the off-target activity of miRNA compared to the parent sequences. Furthermore, introducing only the 2'-F modification at position 9 of the 5' end of the AS chain and only the 2'-OMe modification at position 10 resulted in similar off-target activity, i.e., the modifications can similarly significantly reduce the off-target activity of miRNA.

II. Preparation and evaluation of the activity of targeting ligands

Example 6. Galactosamine compound 1-t bound to a solid phase support

The synthesis schemes look like this:

1) Scheme of synthesis of compound 1-g

2) Scheme of synthesis of compound 1-h

3) Scheme of synthesis of compound 1-1

4) Synthesis of compound 1-q

5) Synthesis of galactosamine 1-t compound bound to a solid phase support

Stage 1

Starting material 1-a (297 g, 763 mmol) and starting material 1-b (160 g, 636 mmol) were dissolved in 960 mL of DCE (1,2-dichloroethane). Sc(OTf)3 (15.6 g, 31.8 mmol) was added at 15 °C. Then, the reaction mixture was heated to 85 °C and stirred for 2 h. After completion of the reaction, 1.5 L of saturated _NaHCO3 was added to quench the reaction. The organic phase was separated, washed with 1.5 L of saturated brine, dried over anhydrous _{Na2SO4 ,} and filtered. The filtrate was distilled under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1 to 0:1) to give product 1-e as a light yellow oil (328 g, 544 mmol, yield: 85.5%, purity: 96.4%).

¹ HNMR: (400 MHz, CDCl3) δ 7.44-7.29 (m, 5H), 5.83 (d, J=8.8 Hz, 1H), 5.40-5.23 (m, 2H), 5.18-5.06 (m, 2H), 4.86 (s, 1H), 4.66 (d, J=8.4 Hz, 1H), 4.21-4.07 (m, 2H), 4.04-3.77 (m, 3H), 3.51-3.45 (m, 1H), 3.31-3.11 (m, 2H), 2.18 (d, J=2.0 Hz, 1H), 2.14 (s, 3H), 2.06 (s, 3H), 2.03-1.99 (m, 3H), 1.95 (s, 3H), 1.64-1.46 (m, 4H), 1.43-1.29 (m, 4H).

MS, C ₂₈ H ₄₀ N ₂ O ¹¹ , found: M ⁺ 581.3.

Stage 2

The compound obtained in step 1 was divided into two parts for parallel reactions, each of which was carried out as follows: Compound 1-c (72.0 g, 124 mmol) was added to 432 mL of THF. Pd/C (20.0 g, 10% purity) was added under argon, followed by the addition of TFA (14.1 g, 124 mmol, 9.18 mL). Hydrogen gas was introduced into the reaction solution and the gas pressure was maintained at 30 psi. The reaction solution was heated to 30 °C and stirred for 16 h.

After the reaction was complete, the two parallel reactions were combined. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure; the process was repeated three times. The residue was dried under reduced pressure to yield the target compound 1-d (139 g).

¹ HNMR(400 MHz, DMSO-d6) δ 7.85 (d, J=9.2 Hz, 1H), 7.74 (s, 3H), 5.21 (d, J=3.6 Hz, 1H), 4.97 (dd, J=2.8, 10.8 Hz, 1H), 4.48 (d, J=8.8 Hz, 1H), 4.06-3.98 (m, 3H), 3.93-3.82 (m, 1H), 3.73-3.68 (m, 1H), 3.63-3.56 (m, 1H), 3.43-3.38 (m, 1H), 2.82-2.71 (m, 2H), 2.13-2.09 (m, 3H), 2.01-1.97 (m, 3H), 1.91-1.87 (m, 3H), 1.77 (s, 3H), 1.76-1.73 (m, 1H), 1.52-1.44 (m, 4H), 1.28 (s, 4H).

Stage 3

Compound 1-d (139 g, 247 mmol) and compound 1-e (75.3 g, 223 mmol) were added to a solution of DMF (834 mL), and then DIPEA (diisopropylethylamine) (41.6 g, 322 mmol, 56.1 mL), HOBt (hydroxybenzotriazole) (36.8 g, 272 mmol) and EDCI (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (52.2 g, 272 mmol) were added at 0 °C. The reaction mixture was stirred at 15°C for 16 h. After completion of the reaction, the reaction mixture was diluted with dichloromethane (400 mL) and then washed successively with saturated ammonium chloride solution (1 L), saturated _NaHCO3 (1.00 L), and saturated brine. The organic phase was separated, dried over anhydrous sodium sulfate, filtered, and distilled under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1 to 0:1) to obtain the target compound 1-f (108 g, yield: 56.8%).

¹ HNMR(40 (400 MHz, DMSO-d6) δ 7.89-7.78 (m, 2H), 7.41-7.27 (m, 6H), 5.21 (d, J=3.2 Hz, 1H), 5.08-4.92 (m, 3H), 4.48 (d, J=8.4 Hz, 1H), 4.07-3.99 (m, 3H), 3.97-3.81 (m, 2H), 3.75-3.64 (m, 1H), 3.42-3.37 (m, 1H), 3.13-2.93 (m, 2H), 2.20 (t, J=8.0 Hz, 2H), 2.10 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.87-1.79 (m, 1H), 1.76 (s, 3H), 1.74-1.64 (m, 1H), 1.48-1.41 (m, 2H), 1.38 (s, 12H), 1.29-1.20 (m, 4H), 1.19-1.14 (m, 1H).

MS, C ₃₇ H ₅₅ N ₃ O ₁₄ , found: M ⁺ 766.4.

Stage 4

The compound 1-f obtained above was divided into two parts for parallel reactions, each of which was carried out as follows: Compound 6 (47.0 g, 61.3 mmol) was added to 280 mL of THE. Pd/C (15.0 g, 10% purity) was added under argon, followed by the addition of TFA (7.00 g, 61.3 mmol, 4.54 mL). Hydrogen gas was introduced into the reaction solution, and the gas pressure was maintained at 30 psi. The reaction solution was heated to 30 °C and stirred for 16 h. After the reaction was complete, the two reactions carried out in parallel were combined. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure; the process was repeated three times. The residue was dried under reduced pressure to give the title compound 1-g (94.0 g, crude product).

¹ HNMR(400 MHz, DMSO-d6) δ 8.38 (s, 1H), 8.10 (s, 3H), 7.83 (d, J=9.2 Hz, 1H), 5.21 (d, J=3.2 Hz, 1H), 4.96 (dd, J=3.6, 11.2 Hz, 1H), 4.47 (d, J=8.4 Hz, 1H), 4.06-3.98 (m, 3H), 3.92-3.82 (m, 1H), 3.75-3.67 (m, 2H), 3.60 (s, 1H), 3.43-3.37 (m, 1H), 3.18-3.04 (m, 2H), 2.30-2.24 (m, 2H), 2.10 (s, 3H), 2.00 (s, 3H), 1.95-1.90 (m, 2H), 1.89 (s, 3H), 1.78-1.75 (m, 3H), 1.49-1.41 (m, 3H), 1.40 (s, 9H), 1.26 (s, 4H).

Stage 5

The compound 1-f obtained above was divided into two parts for parallel reactions, each of which was carried out as follows: Compound 1-f (46.0 g, 60 mmol) was added to HCl-EtOAc (2.00 M, 276 mL), and the reaction mixture was stirred at 15 °C for 16 h. After completion of the reaction, the two reaction solutions were combined and concentrated by distillation under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure; the process was repeated three times. The residue was dried under reduced pressure to give light red compound 1h (91.0 g, crude product).

¹ HNMR(400 MHz, DMSO-d6) δ 7.91-7.80 (m, 2H), 7.42-7.26 (m, 6H), 5.21 (d, J=3.2 Hz, 1H), 5.07-4.92 (m, 4H), 4.48 (d, J=8.4 Hz, 1H), 4.06-3.98 (m, 3H), 3.98-3.82 (m, 3H), 3.73-3.65 (m, 1H), 3.44-3.35 (m, 1H), 3.12-2.94 (m, 2H), 2.22 (t, J=8.0 Hz, 2H), 2.10 (s, 3H), 2.01-1.97 (m, 4H), 1.94-1.90 (m, 1H), 1.89 (s, 3H), 1.87-1.79 (m, 2H), 1.76 (s, 3H), 1.74-1.67 (m, 1H), 1.49-1.40 (m, 2H), 1.40-1.32 (m, 2H), 1.24 (d, J=4.0 Hz, 4H), 1.19-1.13 (m, 1H).

MS, C ₃₃ H ₄₇ N ₃ O ₁₄ , found: M ⁺ 710.3.

Stage 6

Two reactions were carried out in parallel as follows: Compound 1-g (45.0 g, 60.3 mmol) and compound 1-h (38.5 g, 54.3 mmol) were added to 270 mL of DMF, then DIPEA (10.1 g, 78.4 mmol, 13.6 mL) was added at 0 °C, followed by the addition of HOBt (8.97 g, 66.3 mmol) and EDCI (12.7 g, 66.3 mmol). The reaction mixture was stirred at 15 °C for 16 h. After completion of the reaction, the two reaction solutions were combined, diluted with 300 mL of DCM and washed successively with saturated ammonium chloride (800 mL), saturated NaHCO ₃ (800 mL) and brine (800 mL).

The organic phase was dried over _{anhydrous Na2SO4} . After filtration, the filtrate was concentrated by evaporation under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate = 5:1 to 0:1) to give white compound 1-i (66.0 g, 47.4 mmol, yield: 39.3%, purity 95.1%).

¹ HNMR(400 MHz, DMSO-d6) δ 7.96-7.78 (m, 5H), 7.41-7.25 (m, 6H), 5.21 (d, J=3.6 Hz, 2H), 5.05-4.92 (m, 4H), 4.48 (d, J=8.8 Hz, 2H), 4.22-4.12 (m, 1H), 4.02 (s, 6H), 3.94-3.80 (m, 3H), 3.74-3.64 (m, 2H), 3.45-3.35 (m, 2H), 3.11-2.92 (m, 4H), 2.20-2.12 (m, 4H), 2.10 (s, 6H), 1.99 (s, 6H), 1.89 (s, 6H), 1.82-1.79 (m, 2H), 1.76 (s, 6H), 1.74-1.63 (m, 2H), 1.44 (d, J=6.0 Hz, 4H), 1.37 (s, 12H), 1.24 (s, 9H).

MS: C ₆₂ H ₉₄ N ₆ O ₂₅ , detected: m/z 1323.8.

Stage 7

This step was carried out in 11 reactions, each of which was carried out as follows: Compound 1-i (5.00 g, 3.78 mmol) and toluene (300 mL) were added, and silica gel (45.0 g) was added. The reaction mixture was stirred at 100 °C for 40 h. After completion of 11 reactions, the reaction mixtures were combined. After distillation of the solvent under reduced pressure, isopropanol and dichloromethane were added to the residue, and the mixture was stirred for 20 min. Insoluble materials were removed by filtration, and the filter cake was washed with isopropanol until the product was dissolved in isopropanol. The resulting solution was concentrated to remove the solvent and dried under reduced pressure to give light yellow compound 1-j (43.2 g, 34.0 mmol, yield: 82.0%).

¹ HNMR: (400 MHz, DMSO-d6) δ 8.01 (d, J=7.6 Hz, 1H), 7.93-7.79 (m, 2H), 7.39-7.27 (m, 3H), 5.21 (d, J=3.2 Hz, 1H), 5.06-4.91 (m, 2H), 4.48 (d, J=8.0 Hz, 1H), 4.07-3.97 (m, 3H), 3.94-3.82 (m, 2H), 3.73-3.65 (m, 1H), 3.45-3.36 (m, 2H), 3.10-2.94 (m, 2H), 2.15 (d, J=7.6 Hz, 2H), 2.10 (s, 3H), 1.99 (s, 3H), 1.89 (s, 3H), 1.86-1.79 (m, 1H), 1.77 (s, 3H), 1.74-1.65 (m, 1H), 1.44 (s, 2H), 1.37 (d, J=5.2 Hz, 2H), 1.24 (s, 4H).

MS: C ₅₈ H ₈₆ N ₆ O ₂₅ , found: m/z=1267.8.

Stage 8

This step was performed in two parallel reactions, each carried out as follows: Compound 1-d (11.8 g, 21.0 mmol) and compound 1-j (21.3 g, 16.8 mmol) were added to 70 mL of DMF, then DIPEA (3.54 g, 27.3 mmol, 4.77 mL) was added at 0 °C, followed by the addition of HOBt (3.13 g, 23.1 mmol) and EDCI (4.44 g, 23.1 mmol). The reaction mixture was stirred at 15 °C for 16 h. After completion of the reaction, the two reaction solutions were combined, diluted with 500 mL of DCM, and washed sequentially with saturated ammonium chloride (1.5 L), saturated NaHCO ₃ (1.5 L), and brine (1.5 L). The organic phase was dried over _{anhydrous Na2SO4} . After filtration, the filtrate was concentrated by evaporation under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol=50:1 to 10:1) to give light yellow compound 1-k (54.0 g, 31.8 mmol, yield: 75.6%).

¹ HNMR(400 MHz, DMSO-d6) δ 7.91 (d, J=7.6 Hz, 1H), 7.87-7.78 (m, 5H), 7.73 (t, J=5.2 Hz, 1H), 7.42-7.24 (m, 6H), 5.21 (d, J=3.6 Hz, 3H), 5.06-4.92 (m, 5H), 4.48 (d, J=8.4 Hz, 3H), 4.19-4.09 (m, 2H), 4.07-3.97 (m, 10H), 3.94-3.80 (m, 4H), 3.76-3.64 (m, 3H), 3.42-3.37 (m, 4H), 3.08-2.94 (m, 6H), 2.20-2.12 (m, 2H), 2.10 (s, 9H), 2.08-2.01 (m, 2H), 1.99 (s, 9H), 1.89 (s, 9H), 1.87-1.79 (m, 2H), 1.77 (s, 9H), 1.74-1.63 (m, 2H), 1.44 (d, J=5.6 Hz, 6H), 1.40-1.31 (m, 6H), 1.24 (s, 13H).

MS: C ₇₈ H ₁₁₈ N ₈ O ₃₃ , found: m/z=1696.1.

Stage 9

This step was performed in 3 parallel reactions, each carried out as follows: Compound 1-k (17.0 g, 10.0 mmol) and THF (100 mL) were added. Under argon atmosphere, Pd/C (5.0 g, 10% purity) was added, followed by TFA (1.14 g, 10.0 mmol, 742 μL). Hydrogen gas was introduced into the reaction solution and the gas pressure was maintained at 15 psi. The reaction solution was heated to 30 °C and stirred for 4 h. After the reaction was complete, the 3 parallel reactions were combined. The reaction mixture was filtered, and the filtrate was evaporated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure; the process was repeated three times. The residue was purified by preparative liquid chromatography (C ¹⁸ , mobile phase A 0.1% TFA-water, mobile phase B: 10-40% ACN (acetonitrile), 20 min) to give a white compound 1 L (17.3 g, 10.2 mmol, yield: 34.0%).

¹ HNMR: (400 MHz, DMSO-d6) δ 8.45 (t, J=5.2 Hz, 1H), 8.14 (d, J=5.2 Hz, 3H), 7.97 (t, J=5.2 Hz, 1H), 7.90-7.77 (m, 4H), 5.21 (d, J=2.8 Hz, 3H), 4.96 (dd, J=3.2, 11.6 Hz, 3H), 4.47 (d, J=8.4 Hz, 3H), 4.20-4.10 (m, 1H), 4.02 (s, 8H), 3.87 (q, J=9.6 Hz, 3H), 3.75-3.61 (m, 4H), 3.46-3.34 (m, 3H), 3.21-2.93 (m, 6H), 2.21 (s, 2H), 2.14-2.02 (m, 11H), 1.99 (s, 9H), 1.96-1.82 (m, 12H), 1.80-1.65 (m, 10H), 1.44 (d, J=5.6 Hz, 8H), 1.36 (d, J=6.4 Hz, 4H), 1.30-1.17 (m, 12H)

MS: C ₇₀ H ₁₁₂ N ₈ O ₃₁ , found: m/2z=781.8.

Stage 10

Compound 1-m (2 g, 12.64 mmol) was dissolved in pyridine (10 mL). A solution of DMTrCl (4.71 g, 13.90 mmol) in pyridine (10 mL) was added dropwise at room temperature. The reaction mixture was stirred at room temperature for 5 h. After completion of the reaction, the reaction mixture was quenched with methanol and concentrated under reduced pressure to obtain a crude product. The crude product was purified using silica gel (eluted with petroleum ether:ethyl acetate = 10:1). The product eluate was collected and concentrated under reduced pressure to evaporate the solvent to obtain compound 1-n (4 g).

MS m/z: C ₂₉ H ₃₂ O ₅ , [M+H] ⁺ found: 461.3.

Stage 11

Compound 1-n (2 g, 4.34 mmol), N,N-diisopropylethylamine (DIEA, 1.43 mL, 8.68 mmol), and HATU (2.47 g, 6.51 mmol) were dissolved in DMF (10 mL). A solution of compound 1-o in DMF (5 mL) was added at room temperature. The reaction mixture was stirred at room temperature for 8 h. After completion of the reaction, water was added to quench the reaction. The aqueous phase was extracted with ethyl acetate. The combined organic phases were first washed with water and then with saturated brine (20 mL), then concentrated under reduced pressure to evaporate the solvent, purified by reverse-phase preparative HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm, conditions: 25-80% (A: water 0.075% NH ₃ ⋅H ₂ O, B: CH ₃ CN), flow rate: 55 mL/min) and lyophilized to obtain compound 1-p (2.4 g).

MS m/z: C ₃₃ H ₃₉ NO ₇ , [M+H] ⁺ detected: 562.4.

Stage 12

Compound 1-p (2.4 g, 4.27 mmol) was dissolved in 15 mL of a mixed solution of methanol and water (2:1). LiOH (0.36 g, 8.54 mmol) was added at room temperature. The mixture was stirred overnight. After completion of the reaction, the mixture was concentrated under reduced pressure to evaporate the solvent, purified by reverse-phase preparative HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm, conditions: 25-75% (A: water 0.075% NH ₃ ⋅H ₂ O, B: CH ₃ CN), flow rate: 55 mL/min) and lyophilized to give compound 1-p (2 g).

MS m/z: C ₃₂ H ₃₇ NO ₇ , [M+H] ⁺ detected: 548.6.

Step 13

Compound 1-q (0.37 g, 0.69 mmol), DIEA (0.19 mL, 1.15 mmol), and HATU (0.32 g, 0.86 mmol) were dissolved in 2 mL of DMF. A solution of compound 1-l (0.9 g, 0.69 mmol) in DMF (2 mL) was added at room temperature. The mixture was stirred at room temperature overnight. After completion of the reaction, the reaction mixture was diluted with dichloromethane (10 mL) and washed successively with saturated NaHCO ₃ (20 mL) and brine (20 mL). The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered, and concentrated. The residue was purified by reversed-phase preparative HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm, conditions: 25-65% (A: water 0.075% NH ₃ ⋅H ₂ O, B: CH ₃ CN), flow rate: 45 mL/min) and lyophilized to obtain compound 1-r (0.5 g).

MS m/z: C ₁₀₂ H ₁₄₇ N ₉ O ₃₇ , [M-H] ⁺ detected: 2088.5.

Step 14

Compound 1-r (300 mg, 0.14 mmol) and succinic anhydride (28.70 mg, 0.28 mmol) were dissolved in tetrahydrofuran. DMAP (3.50 mg, 0.028 mmol) was added to the reaction mixture, and the mixture was stirred at 40°C overnight. After completion of the reaction, methanol (18.8 mg) was added. The reaction mixture was stirred for 10 min, then diluted with dichloromethane (3 mL) and washed twice with saturated NaHCO ₃ (5 mL). The organic phase was concentrated to dryness under reduced pressure and purified by reversed-phase preparative HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm, conditions: 25-65% (A: water 0.075% NH ₃ ⋅H ₂ O, B: CH ₃ CN), flow rate: 35 mL/min) and lyophilized to obtain compound 1-s (140 mg).

MS m/z: C ₁₀₆ H ₁₅₁ N ₉ O ₄₀ , [M-H] ⁺ detected: 2189.4.

Step 15

Compound 1-r (140 mg, 64 μmol), obtained in the previous step, was added to acetonitrile (5 mL). Then, HBTU (48.7 mg, 128 μmol) was added, an amine-modified surface solid phase support (CPG-NH ₂ , 2.3 g) was added, and DIEA (41.5 mg, 320 μmol, 55 μL) was added. The mixture was shaken at 30°C for 16 h. After completion of the reaction, the mixture was filtered and washed successively with methanol (8 mL 4 times) and dichloromethane (8 mL 4 times). The solid was added to pyridine:acetic anhydride (v:v=4:1, 10.0 mL), and the mixture was reacted with shaking at 30°C for another 16 h. After completion of the reaction, the mixture was filtered and washed successively with methanol (8 mL 4 times) and dichloromethane (8 mL 4 times) to obtain compound 1-t bound to a solid phase support (2.1 g).

Example 7. Galactosamine compound 2 bound to a solid phase support

The synthesis schemes look like this:

1) Synthesis of compound 2-b

2) Synthesis of compound 2-e

Stage 1

Compound 2-a (1.00 g, 2.37 mmol) was added to THF (7.5 mL) and _H2O (7.5 mL), followed by the addition of LiOH. _H2O (109 mg, 2.60 mmol) was added. The reaction mixture was stirred at 16 °C for 16 h. After completion of the reaction, the solvent was removed by evaporation under reduced pressure. The residue was further lyophilized to give white compound 2-b (960 mg, 2.32 mmol, yield: 97.8%).

¹ HNMR: (400 MHz, DMSO-d6) δ 7.44 (d, J=8.4 Hz, 2H), 7.34-7.23 (m, 6H), 7.22-7.15 (m, 1H), 6.86 (d, J=8.0 Hz, 4H), 3.73 (s, 6H), 3.66 (d, J=6.4 Hz, 1H), 3.32 (d, J=12.0 Hz, 1H), 3.11 (dd, J=2.0, 9.2 Hz, 1H), 2.85 (t, J=8.8 Hz, 1H).

MC m/z: C ₂₄ H ₂₄ O ₆ , detected: m/z: 407.2.

Stage 2

Compound 1-l (500 mg, 0.30 mmol) was added to dichloromethane (3 mL), then compound 2-b (0.14 g, 0.34 mmol) was added at 15 °C to the reaction, and HBTU (142 mg, 375 μmol) and DIEA (115 mg, 895 μmol) were added at 0 °C. The mixture was reacted at 15 °C for 16 h. After completion of the reaction, the reaction mixture was diluted with dichloromethane (10 mL) and washed successively with saturated NaHCO ₃ (20 mL) and saturated brine (20 mL). The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered, and then concentrated under reduced pressure. The residue was purified by preparative liquid chromatography (column: Welch Xtimate C ¹⁸ 250×70 mm #10 μm; mobile phase: [water-ACN]; B%: 40% to 66%, 18 min) to give compound 2-c.

MS m/z: C ₉₄ H ₁₃₄ N ₈ O ₃₆ , [M-H] ⁺ detected: 1952.1.

Stage 3

Compound 2-c (230 mg, 0.12 mmol) and succinic anhydride (23.5 mg, 0.26 mmol) were dissolved in dichloromethane solution (2 mL). DMAP (43.1 mg, 0.35 mmol) was added to the reaction mixture. The mixture was stirred at 15 °C for 16 h. After completion of the reaction, methanol (18.8 mg) was added. The reaction mixture was stirred for 10 min, then diluted with dichloromethane (3 mL) and washed twice with saturated NaHCO ₃ . The reaction mixture was concentrated to dryness under reduced pressure to give compound 2-d (240 mg, crude product).

MS m/z: C ₁₀₆ H ₁₅₁ N ₉ O ₄₀ , [M-H] ⁺ detected: m/2z: 2070.2

Stage 4

Compound 2-d (240 mg, 116 μmol), obtained in the previous step, was added to acetonitrile (8 mL). Then, HBTU (88.7 mg, 233 μmol) was added, an amine-modified surface solid phase support (CPG-NH ₂ , 4 g) was added, and DIEA (75.5 mg, 584 μmol, 101 μL) was added. The mixture was shaken at 30 °C for 16 h. After completion of the reaction, the mixture was filtered and washed sequentially with methanol (8 mL 4 times) and dichloromethane (8 mL 4 times). The solid was added to pyridine:acetic anhydride (v:v=4:1, 10.0 mL), and the mixture was reacted with shaking at 30°C for another 16 h. After completion of the reaction, the mixture was filtered and washed successively with methanol (8 mL 4 times) and dichloromethane (8 mL 4 times) to obtain the desired product 2-e bound to a solid phase support (3.7 g).

Example 8. Galactosamine compound 3-n bound to a solid phase support

The synthesis schemes look like this:

1) Synthesis of compound 3-d

2) Synthesis of compound 3-g

3) Synthesis of compound 3-n

Stage 1

Starting material 3-a (78.8 g, 202 mmol) and starting material 3-b (40 g, 168 mmol) were dissolved in DCE (250 mL). _CF3SO3H3 (4.15 g, 8.43 mmol) was _added at 15 °C. Then, the reaction mixture was heated to 75 °C and stirred for 2 h. After completion of the reaction _, 1 L of saturated _NaHCO3 was added to quench the reaction. The organic phase was separated, washed with 1 L of saturated brine, dried over anhydrous _{Na2SO4 ,} and filtered. The filtrate was distilled under reduced pressure and purified by silica gel column chromatography (petroleum ether:ethyl acetate=5:1 to 0:1) to obtain the target product 3-c (63.2 g, 107 mmol, yield: 63.5%).

¹ HNMR:(400 MHz, CDCl3) δ 7.35-7.26 (m, 5H), 5.88 (s, 1H), 5.34-5.25 (m, 2H), 4.65 (d, J=8.4 Hz, 1H), 4.16-4.13 (m, 2H), 3.92-3.87 (m, 3H), 3.18-3.17 (m, 1H), 3.15-3.14 (m, 2H), 2.16-1.91 (m, 15H), 1.58-1.50 (m, 5H), 1.49-1.36 (m, 2H).

MS m/z: C ₂₄ H ₄₀ N ₂ O ₁₁ , detected: m/z: 567.4.

Stage 2

The above-obtained compound 3-c (60.0 g, 106 mmol) was added to 360 mL of THF. Pd/C (15.0 g, 10% purity) was added under argon, followed by the addition of TFA (12.1 g, 106 mmol, 7.84 mL). Hydrogen gas was introduced into the reaction solution, and the gas pressure was maintained at 30 psi. The reaction solution was heated to 30 °C and stirred for 16 h. After completion of the reaction, the reaction mixture was filtered, and the filtrate was concentrated under reduced pressure. The residue was diluted with dichloromethane and concentrated under reduced pressure; the process was repeated three times (500 mL 3 times). The residue was dried under reduced pressure to give light yellow compound 3-d (44 g, 102 mmol, yield: 96.1%).

Stage 3

Compound 3-e (60.0 g, 447 mmol) was dissolved in DMF (300 mL). K ₂ CO ₃ (92.7 g, 671 mmol) was added, and BnBr (115 g, 671 mmol, 79.7 mL) was added dropwise at 0 °C. The reaction mixture was stirred at 25 °C for 6 h. The reaction mixture was poured into crushed ice and then extracted with ethyl acetate (100 mL 6 times). The organic phase was washed successively with water (100 mL 2 times) and saturated brine (100 mL 3 times). The organic phase was dried over anhydrous sodium sulfate and distilled under reduced pressure to remove the solvent. The residue was purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1 to 0:1) to give compound 3-f as a white solid (60.3 g, 269 mmol, yield: 60.1%).

¹ NMR: (400 MHz, CDCl ₃ ) δ 7.37-7.26 (m, 5H), 5.18 (d, J=4.4 Hz, 2H), 3.95-3.90 (m, 2H), 3.75-3.71 (m, 2H), 1.08 (s, 1H).

MS m/z: C ₁₂ H ₁₆ O ₄ , detected: m/z: 223.5.

Stage 4

Compound 3-f (50.0 g, 223 mmol) was dissolved in dichloromethane (300 mL). Pyridine (73.5 g, 929 mmol, 75 mL) and a solution of p-nitrophenyl chloroformate (180 g, 892 mmol) in dichloromethane (50 mL) were added. The reaction mixture was stirred at 25 °C under nitrogen atmosphere for 24 h. After completion of the reaction, the mixture was diluted with dichloromethane (250 mL) and washed successively with NaHSO ₄ solution (30 mL 3 times) and saturated brine (30 mL 2 times). The organic phase was dried over MgSO ₄ , filtered and concentrated under reduced pressure to evaporate the solvent. The obtained crude product was purified by silica gel column chromatography (petroleum ether:ethyl acetate=3:1) to give the target compound 3-g (37.0 g, 66.7 mmol, yield: 29.9%).

MS m/z: C ₂₆ H ₂₂ N ₂ O ₁₂ , detected: m/z: 553.4.

Stage 5

Compound 3-g (22.0 g, 39.7 mmol) was added to acetonitrile (120 mL), and triethylamine (24.1 g, 238 mmol, 33.1 mL) was added under a nitrogen atmosphere. The reaction mixture was cooled to 0 °C, and a solution of compound 3-d (42.1 g, 40 mmol) in acetonitrile (120 mL) was added dropwise. The reaction mixture was heated to 25 °C and stirred for 1 hour. After completion of the reaction, the mixture was concentrated under reduced pressure to remove the solvent and then purified by silica gel column chromatography (petroleum ether:ethyl acetate=2:1) to give the target compound 3-h (37.0 g, 12.0 mmol, yield: 30.2%).

MS m/z: C ₅₂ H ₇₆ N ₄ O ₂₄ , detected: m/z: 1141.8.

Stage 6

Compound 3-h (11.0 g, 9.64 mmol) was dissolved in ethyl acetate (60 mL). Pd/C (2.00 g, 10% purity) was added. Hydrogen gas was introduced into the reaction solution, and the gas pressure was maintained at 40 psi. The reaction solution was stirred at 25 °C for 8 h. After completion of the reaction, the mixture was filtered and concentrated to dryness by evaporation under reduced pressure to obtain the target compound 3-i (10.0 g, 9.42 mmol, yield: 97.7%).

¹ HNMR: (400 MHz, DMSO-d6) δ 7.79 (d, J=9.2 Hz, 2H), 7.10 (s, 2H), 5.74 (t, J=1.6 Hz, 2H), 5.21 (d, J=3.6 Hz, 2H), 4.98-4.95 (m, 2H), 4.48 (d, J=8.4 Hz, 2H), 4.02 (d, J=4.8 Hz, 11H), 3.87-3.84 (m, 2H), 3.69-3.67 (m, 2H), 3.41-3.39 (m, 2H), 2.94-2.90 (m, 4H), 2.10 (s, 5H), 1.99 (s, 7H), 1.89 (s, 6H), 1.77 (s, 6H), 1.47-1.35 (m, 8H), 1.26-1.24 (m, 4H), 1.23-1.08 (m, 3H).

MS m/z: C ₄₅ H ₇₀ N ₄ O ₂₄ , detected: m/z: 1051.4.

Stage 7

Compound 3-i (5.00 g, 4.76 mmol) was added to a mixed solvent of dichloromethane (30 ml) and DMF (30 ml), then compound 33 (312 mg, 2.38 mmol) was added, and HBTU (1.80 g, 4.76 mmol) and DIEA (615 mg, 4.76 mmol) were added. The reaction mixture was stirred at 25 °C for 12 h. After completion of the reaction, the reaction mixture was poured into ethyl acetate (100 ml), then washed with saturated brine, dried over _{anhydrous Na2SO4} , filtered, and concentrated under reduced pressure to evaporate the solvent. The residue was purified by preparative HPLC to give the target compound 3-k (2.1 g, 956 μmol, yield: 20.1%).

¹ HNMR: (400 MHz, DMSO-d6) δ 7.84-7.81 (m, 5H), 7.12-7.07 (m, 3H), 5.21 (d, J=3.6 Hz, 4H), 4.99-4.96 (m, 4H), 4.49 (d, J=8.4 Hz, 4H), 4.06-4.00 (m, 24H), 3.88-3.86 (m, 4H), 3.55-3.52 (m, 4H), 3.49-3.43 (m, 4H), 3.25-3.05 (m, 4H), 2.94-2.93 (m, 8H), 2.11 (s, 12H), 2.00 (s, 16H), 1.90 (s, 12H), 1.78 (s, 12H), 1.46-1.44 (m, 8H), 1.38-1.35 (m, 8H), 1.26-1.24 (m, 8H), 1.18-1.16 (m, 6H), 1.09-0.99 (m, 2H).

MS m/z: C ₉₆ H ₁₅₃ N ₁₁ O ₄₆ , detected: m/z: 2197.5.

Stage 8

Compound 3-k (100 mg, 45.5 μmol) was added to DMF (1 mL), then compound 2-b (21.1 mg, 54 μmol) was added to the reaction mixture, and HBTU (21.8 mg, 57.3 μmol) and DIEA (17.7 mg, 136 μmol) were added. The mixture was reacted at 15 °C for 16 h. After completion of the reaction, the reaction mixture was diluted with dichloromethane (10 mL) and washed successively with saturated NaHCO ₃ and saturated brine. The organic phase was dried over anhydrous Na ₂ SO ₄ , filtered and concentrated. The residue was purified by preparative liquid chromatography (column: Phenomenex Gemini-NX 150 × 30 mm × 5 μm; mobile phase: [water-ACN]; B%: 35% to 75%, 12 min) to give compound 3-1. MS m/z: C ₁₂₀ H ₁₇₅ N ₁₁ O ₅₁ , found: 2586.9.

Stage 9

Compound 3-1 (14 mg, 5.4 μmol) and succinic anhydride (1.08 mg, 10.8 μmol) were dissolved in dichloromethane solution (1 mL). DMAP (2.0 mg, 16 μmol) and TEA (1.1 mg, 10.8 μmol, 1.5 μL) were added to the reaction mixture. The mixture was stirred at 15 °C for 16 h. After completion of the reaction, methanol (0.9 mg) was added. The reaction mixture was stirred for 10 min, then diluted with dichloromethane and washed twice with saturated NaHCO ₃ . The reaction mixture was concentrated to dryness under reduced pressure to give compound 3-m (18 mg).

MS m/z: C ₁₂₄ H ₁₇₉ N ₁₁ O ⁵⁴ , detected: 2687.2.

Stage 10

Compound 3-m (18 mg, 6.7 μmol), obtained in the previous step, was added to acetonitrile (3 mL). Then, HBTU (5.1 mg, 13.4 μmol) was added, an amine-modified surface solid phase support (CPG-NH ₂ , 200 mg) was added, and DIEA (4.3 mg, 33.5 μmol, 5.8 μL) was added. The mixture was shaken at 30°C for 16 h. After completion of the reaction, the mixture was filtered and washed successively with methanol (2 mL 4 times) and dichloromethane (2 mL 4 times). The solid was added to pyridine:acetic anhydride (v:v=4:1, 2 mL), and the mixture was reacted with shaking at 30°C for another 16 h. After completion of the reaction, the mixture was filtered and washed successively with methanol and dichloromethane to obtain the target product compound 3-n bound to a solid phase support (200 mg).

Example 9. Galactosamine compound 4-c bound to a solid phase support

The synthesis schemes are as follows: 1) Synthesis of compound 4-c

Stage 1

Compound 3-k (149.5 mg, 68 μmol), DIEA (141.0 mg, 1.09 mmol), molecular sieve 3A (500 mg), and DEPBT (163.4 mg, 0.55 mmol) were dissolved in 5 mL of DCM. Compound 1-q (400 mg, 0.18 mmol) was added at room temperature. The mixture was stirred at room temperature overnight. After completion of the reaction, the molecular sieve was filtered off. The filtrate was concentrated to dryness by rotary evaporation, purified by reversed-phase preparative HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm, conditions: 5-50% (A: water, B: CH ₃ CN), flow rate: 45 mL/min) and lyophilized to obtain compound 4-a (118 mg, 32 μmol, yield: 62.6%).

MS m/z: C ₁₂₈ H ₁₈₈ N ₁₂ O ₅₂ , found: [M+HCOO ^- ]=2770.6.

Stage 2

Compound 4-a (110 mg, 4.0 μmol), DMAP (7.4 mg, 40 μmol), molecular sieve 3A (100 mg), and succinic anhydride (11.9 mg, 120 μmol) were dissolved in 5 mL of THF. The mixture was stirred at 40 °C under argon for 4 h. After completion of the reaction, the molecular sieve was filtered off. The filtrate was concentrated to dryness by rotary evaporation, purified by reverse-phase preparative HPLC (column: Boston Green ODS 150 × 30 mm × 5 μm, conditions: 5-50% (A: water, B: CH ₃ CN), flow rate: 45 mL/min), and lyophilized to obtain compound 4-b (80 mg, 28.3 μmol, yield: 70.8%).

MS m/z: C ₁₃₂ H ₁₉₂ N ₁₂ O ₅₅ , [M-H] ⁺ detected: 2824.6.

Stage 3

Compound 38 (71 mg, 25 μmol), obtained in the previous step, was added to acetonitrile (5 mL). Then HBTU (19.0 mg, 50 μmol) was added, an amine-modified surface solid phase support (CPG-NH ₂ , 0.86 g) was added, and DIEA (16.2 mg, 125 μmol, 21.6 μL) was added. The mixture was shaken at 30 °C for 16 h. After completion of the reaction, the mixture was filtered and washed sequentially with methanol (5 mL 4 times) and dichloromethane (5 mL 4 times). The solid was added to pyridine:acetic anhydride (v:v=4:1, 6.0 mL), and the mixture was reacted with shaking at 30°C for another 16 h. After completion of the reaction, the mixture was filtered and washed successively with methanol and dichloromethane to obtain the target product compound 4-c bound to a solid phase support (0.74 mg).

Example 10. Obtaining control compound L96

The control compound L96 was prepared using the method described in patent WO2014025805A1.

Example 11. Synthesis of siRNA Conjugated to a Galactosamine Cluster The siRNA used for testing, an siRNA targeting the mouse TTR gene mRNA (Molecular Therapy Vol. 26, No. 3, March 2018), is shown below. The galactosamine cluster M was covalently linked to the 3' end of the SS chain.

SS chain (5'-3'): CmsAmsGmUmGfUmUfCfUfUmGmCmUmCmUmAmUmAmAm-M

Chain AS (5'-3'): UmsUfsAmUmAmGfAmGmCmAmAmGmAmAfCmAfCmUmGmsUmsUm

Reference was made to the above-mentioned solid-phase phosphoramidite synthesis method, and the difference was that in the SS chain synthesis, instead of the universal CPG support, a CPG support to which a galactosamine cluster was linked was used.

The synthesis is briefly described below: Nucleoside phosphoramidite monomers were coupled one after another according to the synthesis program on a Dr. Oligo48 synthesizer (Biolytic), starting from the synthesized CPG support to which the galactosamine cluster described above was coupled. The nucleoside monomer materials 2'-F RNA, 2'-O-methyl RNA and other nucleoside phosphoramidite monomers were purchased from Hongene, Shanghai or Genepharma, Suzhou. 5-ethylthio-1H-tetrazole (ETT) was used as an activator (0.6 M solution in acetonitrile), 0.22 M PADS solution in acetonitrile and collidine (1:1 by volume) (Kroma, Suzhou) as a sulfurizing agent and iodopyridine/water solution (Kroma) as an oxidizing agent.

After completion of the solid-phase synthesis, the oligoribonucleotides were cleaved from the solid support and soaked in a solution of 28% ammonia water and ethanol (3:1) at 50°C for 16 h. The mixture was centrifuged, and the supernatant was transferred to another centrifuge tube. After the supernatant was concentrated to dryness by evaporation, the residue was purified by C ¹⁸ reversed-phase chromatography using 0.1 M TEAA (triethylammonium acetate) and acetonitrile as the mobile phase, and DMTr was removed using 3% trifluoroacetic acid solution. The target oligonucleotides were collected, then lyophilized, identified as target products by LC-MS, and quantified by UV detection (260 nm).

The resulting single-stranded oligonucleotides were paired in an equimolar ratio in a complementary manner and annealed with the AS strand. The final double-stranded siRNA was dissolved in 1× PBS (phosphate-buffered saline), and the solution was adjusted to the concentration required for the experiment.

We synthesized siRNAs conjugated to a galactosamine cluster. The siRNAs used in the experiment target mouse TTR mRNA. M=

Example 12. Inhibition of mRNA expression in primary hepatocytes using siRNA conjugated to a cluster of galactosamine molecules

Fresh primary hepatocytes were isolated from mice using the method described by Severgini et al. (Cytotechnology. 2012;64(2): 187-195).

After isolation, primary hepatocytes were inoculated into a 24-well plate at 100,000 cells per well. The tested siRNAs were added at final concentrations of 50 nM, 10 nM, 2 nM, 0.4 nM, 0.08 nM, 0.016 nM, 0.0032 nM, and 0.00064 nM. Subsequently, primary hepatocytes were cultured at 37°C with 5% _CO2 for 24 h. After 24 h, the mTTR mRNA expression level was determined using the qPCR (quantitative PCR) method.

As shown in Fig. 4, S-1, S-2, S-3, and S-4 exhibited excellent mTTR gene expression inhibition efficiency. The _IC50 values of S-1 and S-4 were lower than those of the other two groups. The _IC50 value of the control group S-L96 was 0.280 nM, while the _IC50 value of S-1 was 0.131 nM and that of S-4 was 0.135 nM, indicating that the siRNAs conjugated with compounds S-1 and S-4 had better in vitro uptake efficiency by primary hepatocytes than the control group, and that compounds S-1 and S-4 could more effectively mediate the entry of siRNA into primary hepatocytes.

Example 13. In vivo inhibition of mRNA expression using siRNA conjugated to a galactosamine cluster. Eight-week-old C57BL/6 mice (Joinnbio, SPF, female) were injected subcutaneously with the siRNAs described above. On day 1, 100 μl of a solution containing PBS or a dose (1 mg/kg body weight or 0.2 mg/kg body weight) of the corresponding siRNA (S-L96, S-3, S-2, S-4, or S-1) formulated in PBS were injected subcutaneously into the loose skin on the neck and shoulder of the mice. Six mice in each group received injections.

Three days after administration, mice were sacrificed by cervical dislocation, and mTTR mRNA expression levels in mouse liver tissues were determined by qPCR.

As shown in Fig. 5, S-1, S-2, S-3, and S-4 exhibited excellent potency in inhibiting mTTR gene expression. When administered at doses of 1 mg/kg body weight and 0.2 mg/kg body weight, S-2, S-3, S-4, and the control group S-L96 exhibited similar activity. S-1 showed better activity than the control group S-L96 when administered at 1 mg/kg body weight and 0.2 mg/kg body weight.

Example 14. Long-term efficacy experiment for inhibition of mRNA expression in vivo using siRNA conjugated to a cluster of galactosamine molecules

Two siRNA compounds, S-1-2 and S-L96-2 (see Table 13 for their SS and AS chains), were synthesized using the synthesis method in Example 11 and used for in vivo administration to mice. Eight-week-old C57BL/6 mice (Joinnbio, SPF, female) were subcutaneously injected with the galactosamine cluster-conjugated siRNAs described above. On day 0, 100 μl of a solution containing PBS (referred to as the sham group, i.e., control group) or a dose (1 mg/kg body weight) of the corresponding galactosamine cluster-conjugated siRNA (S-1 or S-L96) formulated in PBS were subcutaneously injected into the loose skin on the neck and shoulder of the mice. Nine mice in each group received injections.

Three mice were sacrificed by cervical dislocation at 7, 14, and 28 days post-injection. Two liver tissue samples were collected from each mouse, and mTTR mRNA expression levels in the mouse liver tissue were determined using qPCR.

At 7 days, 14 days, and 28 days after administration, the ratios of S-1-2 mRNA relative to the PBS group were 0.13, 0.12, and 0.21, respectively, and the ratios of S-L96-2 mRNA relative to the PBS group were 0.17, 0.13, and 0.29, respectively.

Figure 6 also shows the mRNA expression levels in mouse liver tissue 7 days, 14 days, and 28 days after administration of compounds S-1-2 and S-L96-2.

The results show that the administered siRNA still showed effective mRNA inhibition at day 28, and that S-1-2 had higher inhibition than the control S-L96-2 group.

III. Verification of activities

Example 15. Synthesis of siRNA conjugates

Nucleoside monomers were linked one after another in the 3'-5' direction, following the order in which the nucleotides were arranged, using the solid-phase phosphoramidite method. Each time a nucleoside monomer was linked, four reactions occurred: protection, coupling, capping, oxidation, and sulfonation. The sense strand and antisense strand were synthesized under identical conditions.

Oligonucleotide synthesis instrument models: Biolytic Dr. Oligo 48 solid-phase oligonucleotide synthesizer and GE oligo pilot100 solid-phase oligonucleotide synthesizer.

Detection method: The purity of the sense and antisense strands described above was determined, and the molecular weights were analyzed using a Waters Acquity UPLC-SQD2 LCMS (column: ACQUITY UPLC BEH C ¹⁸ ). The values found were consistent with the calculated values, indicating that the synthesized sense strands were conjugated to molecules at the 3' end and antisense strands. The siRNAs had sense and antisense strands as shown in Table 15.

where the nucleotide synthesized using 2-hydroxymethyl-1,3-propanediol as a starting material was defined as hmpNA;

(-)hmpNA(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-1a from the example in section 1.1;

(-)hmpNA(G) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-6a from the example in section 1.6;

(-)hmpNA(C) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-8a from the example in section 1.8;

(-)hmpNA(U) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-7a from the example in section 1.7.

In Table 15, the structure of NAG1 is as shown in Example 11.

Example 16. Screening for siRNA Activity Level and Off-Target Activity In HEK293A cells, the compounds of the present invention were screened for the level of on-target and off-target activity using in vitro molecular modeling.

Target sequences and non-target sequences corresponding to siRNA sequences were constructed and inserted into psiCHECK-2 plasmids. The plasmids contained the renilla luciferase gene and the firefly luciferase gene. The plasmids were dual reporter gene systems. The target siRNA sequence was inserted into the 3' UTR (3'-untranslated region) of the renilla luciferase gene. The siRNA activity for the target sequence was reflected by measuring renilla luciferase expression after calibration with firefly luciferase. The dual luciferase reporter assay system (Promega, E2940) was used for the measurement.

HEK293A cells were cultured at 37°C with 5% _CO2 in high-glucose DMEM containing 10% fetal bovine serum. Twenty-four hours before transfection, HEK293A cells were inoculated into 96-well plates at a density of 10,000 cells per well. Each well contained 100 µl of medium.

Cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine2000 (Thermo Fisher, 11668019) according to the instructions. 0.2 μl of Lipofectamine2000 was used for each well. The amount of plasmid transfection was 10 ng per well. A total of 5 concentration points or 11 siRNA concentration points were set for the target sequence plasmids and non-target sequence plasmids. When 5 concentration points were set, the highest transfection concentration point was 10 nM, and a 10-fold serial dilution was performed. When 11 concentration points were set, the highest final transfection concentration point was 20 nM, and a 3-fold serial dilution was performed. At 24 h post-transfection, non-target levels were determined using a dual-luciferase reporter assay system (Promega, E2940).

The results shown in Table 16 - Table 19 show that the compounds TRD006890 and TRD006924 of the present invention have low off-target activity with high on-target activity, and both are significantly better than the positive control AD81890.

The results presented in Table 20 show that HNA modification was significantly dependent on the sequence site. When the HNA modification site was located at position 5'7 (TRD006912) or position 6 (AD81890) of the AS chain, off-target activity was higher, indicating greater toxicity. Furthermore, TRD006912 demonstrated a further decrease in on-target activity (from 0.68 to 0.96) compared with AD81890.

Example 17. In vitro testing of anti-HBV activity of siRNA compounds using HepG2.2.15 cells

On day 1, HepG2.2.15 cells were inoculated into a 96-well plate at 20,000 cells per well. While the cells were inoculated, HepG2.2.15 cells were transfected with varying concentrations of siRNA using RNAiMax. On day 4, the cell culture supernatant was collected and tested for HBsAg using ELISA (enzyme-linked immunosorbent assay) (the remaining supernatant was frozen for later use). Finally, the cells were harvested, and RNA was extracted from the cells. Total HBV RNA (including 3.5kb + 2.4kb + 2.1kb + 0.7kb RNA (kb - kilobase pairs)) and 3.5kb HBV RNA (including pgRNA (pregenomic RNA) + prenuclear RNA) were measured by RT-PCR (reverse transcription PCR), while GAPDH gene RNA was measured as an internal standard. Five concentration points were set for the test compounds, and two wells were analyzed in parallel with duplicates. The final DMSO concentration in the culture was 0.5%.

The percentage of inhibition was calculated using the formulas below:

% HBsAg inhibition = (1 - HBsAg content in sample / HBsAg content in DMSO control group) × 100

% HBV RNA inhibition = (1 - HBV RNA content in sample / HBV RNA content in DMSO control group) × 100

% cell viability = (sample absorbance - control culture absorbance) / (DMSO control absorbance - control culture absorbance) × 100.

EC ₅₀ values (half-maximal effective concentration) were calculated by analysis using Graphpad Prism software (four-parameter logistic equation).

As shown in Table 21 regarding the control compounds AD66810 and AD81890 and the test indicators of antiviral activity, the test compounds TRD006890, TRD006894, TRD006895, TRD006896, TRD006897, TRD006899, TRD006900, TRD006905, TRD006906, TRD0069 07 and TRD006908 showed excellent antiviral activity on HepG2.2.15 cells.

Example 18. Evaluation of in vitro activity against HBV infection of siRNA compounds using primary human hepatocytes

On day 0, primary human hepatocytes were inoculated into a 48-well plate at 120,000 cells per well. While the cells were inoculated, test compounds were added. siRNA was transferred into primary human hepatocytes by free uptake. siRNA was diluted 5-fold from a starting concentration of 200 nM to seven concentrations. On day 1, HBV type D was added to infect primary human hepatocytes. On days 2, 4, and 6, the medium was replaced with fresh medium. The final DMSO concentration in the cultures was 2%. On day 8, the cell culture supernatant was collected and tested for HBV DNA by qPCR and for HBeAg and HBsAg by ELISA. Seven concentration points were set for the test compounds and the control compound, and two wells were analyzed in parallel for replicates.

As shown in Table 22 regarding the control compound AD81890 and the antiviral activity test indicators, the test compound TRD006894 demonstrated significantly better antiviral activity on primary human hepatocytes.

Example 19. In vivo activity against HBV of siRNA compounds

On day 28, mice (C57BL/6 males) were injected with rAAV8-1.3HBV via the tail vein. On days 14 and 21 post-virus injection, blood was collected from the submandibular veins of all experimental mice to collect plasma. Plasma HBV DNA, HBeAg, and HBsAg levels were measured.

On day 28 post-virus injection, mice were randomized into groups based on the results of plasma samples tested on day 14 and day 21 post-virus injection.

All mice were injected subcutaneously once at a dose of 3 mg/kg on day 28 post-injection. Before injection, blood was collected from the submandibular vein of all mice to collect plasma, which was then tested for HBV DNA, HBeAg, HBsAg, and ALT. The day of injection was day 0. On days 7, 14, and 21 post-injection, blood was collected from the submandibular vein of all mice to collect plasma for analysis.

Plasma HBV DNA was quantified using qPCR. Plasma HBeAg and HBsAg were quantified using ELISA.

On day 7, compared to the control compound AD81890, the test compound TRD006894 demonstrated superior antiviral activity in mice. The test compound TRD006894 can maintain activity in vivo for a long time: it can effectively inhibit viral activity on both days 14 and 21.

Example 20. Design and Synthesis of Human ApoC3 siRNA 1) Design of siRNA: The human ApoC3 gene (NM_000040.3) was used as the target gene to satisfy the general rules for active siRNA for the design of 19/21 nucleotide siRNA. The sequences of the unmodified sense strand and antisense strand are detailed in Table 14, where the SS strand and AS strand of the unmodified siRNA are unmodified.

2) siRNA synthesis: siRNA was synthesized on a Dr.Oligo48 synthesizer (Biolytic) at a specification of 200 nmol using a universal solid support (Biocomma, Shenzhen) mediated by phosphoramidite chemistry. Target oligonucleotides were collected, then lyophilized, identified as target products by LC-MS, and quantified by UV detection (260 nm).

In synthesizing the modified nucleotide at position 5'7 of the AS strand, the original nucleotide of the starting sequence was replaced by the phosphoramidite monomers synthesized in Example 1. The sequences of the antisense strands modified at position 5'7 are detailed in Table 14, where W' is selected from the group consisting of

where M is O or S; where B is selected from the natural base at the corresponding position in Table 24.

The sequences of the sense strands and antisense strands of ApoC3 siRNA modified with 2'-fluoro, 2'-methoxy, etc. are detailed in Table 25, and the sequences of the sense strands and antisense strands of ApoC3 siRNA conjugates are detailed in Table 26.

The sensor and antisense strands were synthesized following the steps described above and annealed at equilibrium to form double-stranded structures via hydrogen bonding. Finally, the resulting double-stranded siRNAs were dissolved in 1× PBS, and the solutions were adjusted to the concentrations required for the experiment.

Table 24. Sense strands and antisense strands of human ApoC3 miRNA

SEQ ID NO SS chain (5'-3') SEQ ID NO Unmodified chain AS (5'-3') SEQ ID NO AS chain with chemical modification in
position 7 (5'-3') SEQ ID NO:
115 GCCUCUGCCC GAGCUUCAA SEQ ID NO: 116 UUGAAGCUCGG GCAGAGGCCA SEQ ID NO: 117 UUGAAGW'UCG GGCAGAGGCCA SEQ ID NO:
118 GCUUCAUGCA GGGUUACAU SEQ ID NO: 119 AUGUAACCCUG CAUGAAGCUG SEQ ID NO: 120 AUGUAAW'CCUG CAUGAAGCUG SEQ ID NO:
121 UGAGCAGCGU GCAGGAGUU SEQ ID NO: 122 AACUCCUGCAC GCUGCUCAGU SEQ ID NO:123 AACUCCW'GCAC GCUGCUCAGU SEQ ID NO:
124 CAGUUCCCUG AAAGACUAU SEQ ID NO: 125 AUAGUCUUUCA GGGAACUGAA SEQ ID NO: 126 AUAGUCW'UUC AGGGAACUGAA SEQ ID NO:
127 AAGUCCACCU GCCUAUCCA SEQ ID NO: 128 UGGAUAGGCAG GUGGACUUGG SEQ ID NO: 129 UGGAUAW'GCA GGUGGACUUGG SEQ ID NO:
130 UCUCAGUGCU CUCCUACCU SEQ ID NO: 131 AGGUAGGAGAG CACUGAGAAU SEQ ID NO: 132 AGGUAGW'AGA GCACUGAGAAU SEQ
ID NO:
133 GGCAUGCUGG
CCUCCCAAU SEQ ID
NO: 134 AUUGGGAGGCC
AGCAUGCCUG SEQ ID
NO: 135 AUUGGGW'GGC
CAGCAUGCCUG SEQ ID NO:
136 GCAUGCUGGC CUCCCAAUA SEQ ID NO: 137 UAUUGGGAGGC CAGCAUGCCU SEQ ID NO: 138 UAUUGGW'AGG CCAGCAUGCCU SEQ ID NO:
139 CUGGCCUCCC AAUAAAGCU SEQ ID NO: 140 AGCUUUAUUGG GAGGCCAGCA SEQ ID NO: 141 AGCUUUW'UUG GGAGGCCAGCA SEQ ID NO:
142 GGCCUCCCAA UAAAGCUGA SEQ ID NO: 143 UCAGCUUUAUU GGGAGGCCAG SEQ ID NO: 144 UCAGCUW'UAU UGGGAGGCCAG SEQ ID NO:
145 UAAAGCUGGA CAAGAAGCU SEQ ID NO: 146 AGCUUCUUGUC CAGCUUUAUU SEQ ID NO: 147 AGCUUCW'UGUC CAGCUUUAUU SEQ ID NO:
148 UAUUCUCAGU GCUCUCCUA SEQ ID NO: 149 UAGGAGAGCAC UGAGAAUACU SEQ ID NO: 150 UAGGAGW'GCA CUGAGAAUACU SEQ ID NO:
151 CCGUUAAGGA CAAGUUCUU SEQ ID NO: 152 AAGAACUUGUC CUUAACGGUG SEQ ID NO: 153 AAGAACW'UGU CCUUAACGGUG SEQ ID NO:
154 CUGCGAGCUC CUUGGGUCU SEQ ID NO: 155 AGACCCAAGGA GCUCGCAGGA SEQ ID NO: 156 AGACCCW'AGGA GCUCGCAGGA SEQ
ID NO:
157 ACAGUAUUCU CAGUGCUCU SEQ ID NO: 158 AGAGCACUGAG AAUACUGUCC SEQ ID NO: 159 AGAGCAW'UGA GAAUACUGUCC SEQ ID NO:
160 UUCUCAGUGC UCUCCUACU SEQ ID NO: 161 AGUAGGAGAGC ACUGAGAAUA SEQ ID NO: 162 AGUAGGW'GAG CACUGAGAAUA SEQ ID NO:
163 AAGGGACAGU AUUCUCAGU SEQ ID NO: 164 ACUGAGAAUAC UGUCCCUUUU SEQ ID NO: 165 ACUGAGW'AUA CUGUCCCUUUU SEQ ID NO:
166 AAUAAAGCUG GACAAGAAA SEQ ID NO: 167 UUUCUUGUCCA GCUUUAUUGG SEQ ID NO: 168 UUUCUUW'UCCA GCUUUAUUGG SEQ ID NO:
169 GACAAGUUCU CUGAGUUCU SEQ ID NO: 170 AGAACUCAGAG AACUUGUCCU SEQ ID NO: 171 AGAACUW'AGA GAACUUGUCCU SEQ ID NO:
172 CGAGGAUGCC UCCCUUCUU SEQ ID NO: 173 AAGAAGGGAGG CAUCCUCGGC SEQ ID NO: 174 AAGAAGW'GAG GCAUCCUCGGC SEQ ID NO:
175 ACUACUGGAG CACCGUUAA SEQ ID NO: 176 UUAACGGUGCU CCAGUAGUCU SEQ ID NO: 177 UUAACGW'UGC UCCAGUAGUCU SEQ ID NO:
178 AUAAAGCUGG ACAAGAAGU SEQ ID NO: 179 ACUUCUUGUCC AGCUUUAUUG SEQ ID NO: 180 ACUUCUW'GUCC AGCUUUAUUG SEQ ID NO:
181 AGGGACAGUA UUCUCAGUA SEQ ID NO: 182 UACUGAGAAUA CUGUCCCUUU SEQ ID NO: 183 UACUGAW'AAU ACUGUCCCUUU SEQ
ID NO:
184 GCCUCCCAAU AAAGCUGGA SEQ ID
NO: 185 UCCAGCUUUAU UGGGAGGCCA SEQ ID
NO: 186 UCCAGCW'UUAU GGGAGGCCA SEQ ID NO:
187 UGCUGGCCUC CCAAUAAAA SEQ ID NO: 188 UUUUAUUGGGA GGCCAGCAUG SEQ ID NO: 189 UUUUAUW'GGG AGGCCAGCAUG SEQ ID NO:
190 AUUCUCAGUG CUCUCCUAU SEQ ID NO: 191 AUAGGAGAGCA CUGAGAAUAC SEQ ID NO: 192 AUAGGAW'AGC ACUGAGAAUAC SEQ ID NO:
193 UUCAGUUCCC UGAAAGACU SEQ ID NO: 194 AGUCUUUCAGG GAACUGAAGC SEQ ID NO: 195 AGUCUUW'CAG GGAACUGAAGC SEQ ID NO:
196 CAUGCUGGCC UCCCAAUAA SEQ ID NO:197 UUAUUGGGAGG CCAGCAUGCC SEQ ID NO: 198 UUAUUGW'GAG GCCAGCAUGCC SEQ ID NO:
199 UAUUCUCAGU GCUCUCCUU SEQ ID NO: 200 UAGGAGAGCAC UGAGAAUACU SEQ ID NO: 201 UAGGAGW'GCA CUGAGAAUACU SEQ ID NO:
202 UAUUCUCAGU GCUCUCCUC SEQ ID NO: 203 UAGGAGAGCAC UGAGAAUACU SEQ ID NO: 204 UAGGAGW'GCA CUGAGAAUACU SEQ ID NO:
205 UAUUCUCAGU GCUCUCCUG SEQ ID NO: 206 UAGGAGAGCAC UGAGAAUACU SEQ ID NO: 207 UAGGAGW'GCA CUGAGAAUACU SEQ
ID NO:
208 CCGUUAAGGA CAAGUUCUA SEQ ID NO: 209 AAGAACUUGUC CUUAACGGUG SEQ ID NO: 210 AAGAACW'UGU CCUUAACGGUG SEQ ID NO:
211 CCGUUAAGGA CAAGUUCUC SEQ ID NO: 212 AAGAACUUGUC CUUAACGGUG SEQ ID NO: 213 AAGAACW'UGU CCUUAACGGUG SEQ ID NO:
214 CCGUUAAGGA CAAGUUCUG SEQ ID NO: 215 AAGAACUUGUC CUUAACGGUG SEQ ID NO: 216 AAGAACW'UGU CCUUAACGGUG SEQ ID NO:
217 AAUAAAGCUG GACAAGAAU SEQ ID NO: 218 UUUCUUGUCCA GCUUUAUUGG SEQ ID NO: 219 UUUCUUW'UCCA GCUUUAUUGG SEQ ID NO:
220 AAUAAAGCUG GACAAGAAC SEQ ID NO: 221 UUUCUUGUCCA GCUUUAUUGG SEQ ID NO: 222 UUUCUUW'UCCA GCUUUAUUGG SEQ ID NO:
223 AAUAAAGCUG GACAAGAAG SEQ ID NO: 224 UUUCUUGUCCA GCUUUAUUGG SEQ ID NO: 225 UUUCUUW'UCCA GCUUUAUUGG SEQ ID NO:
226 GCACCGUUAA GGACAAGUA SEQ ID NO: 227 AACUUGUCCUU AACGGUGCUC SEQ ID NO: 228 AACUUGW'CCUU AACGGUGCUC SEQ ID NO:
229 GCACCGUUAA GGACAAGUC SEQ ID NO: 230 AACUUGUCCUU AACGGUGCUC SEQ ID NO: 231 AACUUGW'CCUU AACGGUGCUC SEQ ID NO:
232 GCACCGUUAA GGACAAGUG SEQ ID NO: 233 AACUUGUCCUU AACGGUGCUC SEQ ID NO: 234 AACUUGW'CCUU AACGGUGCUC SEQ ID NO:
235 GACAAGUUCU CUGAGUUCA SEQ ID NO:236 AGAACUCAGAG AACUUGUCCU SEQ ID NO: 237 AGAACUW'AGA GAACUUGUCCU SEQ ID NO:
238 GACAAGUUCU CUGAGUUCC SEQ ID NO: 239 AGAACUCAGAG AACUUGUCCU SEQ ID NO: 240 AGAACUW'AGA GAACUUGUCCU SEQ ID NO:
241 GACAAGUUCU CUGAGUUCG SEQ ID NO: 242 AGAACUCAGAG AACUUGUCCU SEQ ID NO: 243 AGAACUW'AGA GAACUUGUCCU SEQ ID NO:
244 AUUCUCAGUG CUCUCCUAA SEQ ID NO: 245 AUAGGAGAGCA CUGAGAAUAC SEQ ID NO: 246 AUAGGAW'AGC ACUGAGAAUAC SEQ ID NO:
247 AUUCUCAGUG CUCUCCUAC SEQ ID NO: 248 AUAGGAGAGCA CUGAGAAUAC SEQ ID NO: 249 AUAGGAW'AGC ACUGAGAAUAC SEQ ID NO:
250 AUUCUCAGUG CUCUCCUAG SEQ ID NO: 251 AUAGGAGAGCA CUGAGAAUAC SEQ ID NO: 252 AUAGGAW'AGC ACUGAGAAUAC SEQ ID NO:
253 GCACCGUUAA GGACAAGUU SEQ ID NO: 254 AACUUGUCCUU AACGGUGCUC SEQ ID NO: 255 AACUUGW'CCUU AACGGUGCUC SEQ ID NO:
256 CCGUUAAGGA CAAGUUCUU SEQ ID NO: 257 AAGAACUUGUC CUUAACGGUG SEQ ID NO: 258 AAGAACW'UGU CCUUAACGGUG SEQ
ID NO: 259 AUUCUCAGUG CUCUCCUAU SEQ ID NO: 260 AUAGGAGAGCA CUGAGAAUAC SEQ ID NO: 261 AUAGGAW'AGC ACUGAGAAUAC SEQ ID NO:
262 AAUAAAGCUG GACAAGAAA SEQ ID NO: 263 UUUCUUGUCCA GCUUUAUUGG SEQ ID NO: 264 UUUCUUW'UCCA GCUUUAUUGG SEQ ID NO:
265 GACAAGUUCU CUGAGUUCU SEQ ID NO: 266 AGAACUCAGAG AACUUGUCCU SEQ ID NO: 267 AGAACUW'AGA GAACUUGUCCU SEQ ID NO:
268 UAUUCUCAGU GCUCUCCUA SEQ ID NO: 269 UAGGAGAGCAC UGAGAAUACU SEQ ID NO: 270 UAGGAGW'GCA CUGAGAAUACU SEQ ID NO:
271 AUUCUCAGUG CUCUCCUAU SEQ ID NO: 272 AUAGGAGAGCA CUGAGAAUAC SEQ ID NO: 273 AUAGGAW'AGC ACUGAGAAUAC SEQ ID NO:
274 UAUUCUCAGU GCUCUCCUG SEQ ID NO: 275 UAGGAGAGCAC UGAGAAUACU SEQ ID NO: 276 UAGGAGW'GCA CUGAGAAUACU SEQ ID NO:
277 GACAAGUUCU CUGAGUUCC SEQ ID NO: 278 AGAACUCAGAG AACUUGUCCU SEQ ID NO: 279 AGAACUW'AGA GAACUUGUCCU SEQ ID NO:
280 GCACCGUUAA GGACAAGUC SEQ ID NO: 281 AACUUGUCCUU AACGGUGCUC SEQ ID NO: 282 AACUUGW'CCUU AACGGUGCUC

In Table 25-26, the nucleotide synthesized using 2-hydroxymethyl-1,3-propanediol as the starting material is identified as hmpNA; hmpNA is a racemic structure;

(-)hmpNA(A) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-1a from the example in section 1.1;

(+)hmpNA(A) is an optical isomer;

(-)hmpNA(G) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-3a of type section 1.6;

(+)hmpNA(G) is an optical isomer;

(-)hmpNA(C) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-8a of type section 1.8;

(+)hmpNA(C) is an optical isomer;

(-)hmpNA(U) was prepared by solid-phase synthesis using the nucleoside phosphoramidite monomer 1-7a of type section 1.7;

(+)hmpNA(U) is an optical isomer;

The lowercase letter m indicates that the left nucleotide adjacent to the letter m is a 2'-methoxy-modified nucleotide; the lowercase letter f indicates that the left nucleotide adjacent to the letter f is a 2'-fluoro-modified nucleotide;

lowercase s letters, when present between uppercase letters, indicate that the two nucleotides adjacent to the s letters are linked by a thiophosphate group;

The lowercase s, being first at the 3' end, indicates that the left nucleotide adjacent to the s ends with a phosphorothioate group.

In Table 26, the structure of NAG1 is as shown in Example 11.

Example 21. Inhibition of human ApoC3 in Huh7 cells by siRNA - screening for inhibitory activity at a single concentration point

The effect of siRNAs targeting human ApoC3 on the expression level of human ApoC3 mRNA was tested in vitro. Huh7 cells were cultured at 37°C with 5% _CO2 in high-glucose DMEM containing 10% fetal bovine serum. Twenty-four hours prior to transfection, Huh7 cells were inoculated into 96-well plates at a density of 10,000 cells per well. Each well contained 100 µl of medium.

Cells were transfected with siRNA at a final concentration of 10 nM using Lipofectamine RNAiMAX (ThermoFisher, 13778150) according to the product instructions. Twenty-four hours after treatment, cells were lysed using the TaqMan™ Fast Advanced Cells-to-CT™ Kit (ThermoFisher, A35378), and one-step reverse transcription and quantitative real-time PCR detection were performed. Human ApoC3 mRNA levels were measured and corrected based on the level of the internal reference gene ACTIN.

The experimental materials and tools for cell viability (CV) screening in 96-well plate are shown in Table 1 and Table 2 in the example of Section 3.1.

Experimental method for cell viability screening (Cells-to-CT) in a 96-well plate:

(I) Cell transfection. The quantities of the components of the transfection complex are given in Table 27:

(II) Extraction of cellular RNA by the Cell-to-CT method and reverse transcription of cellular RNA. The reverse transcription reaction system is shown in Table 29, and the reaction conditions are shown in Table 30.

After completion of reverse transcription, samples could be stored in a refrigerator at 4°C before use in Taqman Q-PCR or stored in a freezer at -40°C (6 months).

(III) Detection of Q-PCR by Taqman probe

1. Reaction kit (ThermoFisher TaqMan Fast Advanced Master Mix (4444964); the kit expiration date was checked; the kit components were stored in a freezer at -40°C and in a refrigerator at 4°C after reconstitution and use);

2. The following reaction mixtures (Table 32) were prepared in microtubes. The working concentration of primers was 10 μM.

The samples were placed into the RT-PCR apparatus and the reaction was carried out according to the reaction program given in Table 33 (40 reaction cycles).

3. Method of results analysis

After the Q-PCR detection by the Taqman probe was completed, the corresponding Ct values were obtained according to the threshold value automatically set by the system, and the expression of a certain gene was relatively quantified by comparing the Ct values: Ct comparison refers to calculating the differences in gene expression according to the differences from the Ct value of an internal reference gene, and is also referred to as ΔΔCt=[(target gene of experimental group Ct - internal standard of experimental group Ct) - (target gene of control group Ct - internal standard of control group Ct)]. Inhibition (%)=(1 - remaining amount of target gene expression) × 100%.

Experimental results are expressed relative to the remaining percentage of human ApoC3 mRNA expression in cells treated with control siRNA. The results are presented in Table 34.

Example 22. Inhibition of human ApoC3 in Huh7 cells by siRNA - inhibitory activity at five concentration points

Screening was performed in Huh7 cells using siRNA in five concentration gradients. Each siRNA sample for transfection was serially diluted 10-fold from an initial final concentration of 10 nM to five concentration points.

Huh7 cells were cultured at 37°C with 5% _CO2 in high-glucose DMEM containing 10% fetal bovine serum. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at a density of 10,000 cells per well. Each well contained 100 µl of medium.

Cells were transfected with siRNA at final concentrations of 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM using Lipofectamine RNAiMAX (ThermoFisher, 13778150) according to the product instructions. Twenty-four hours after treatment, cells were lysed using the TaqMan™ Fast Advanced Cells-to-CT™ Kit (Thermo Fisher, A35378), and one-step reverse transcription and quantitative real-time PCR detection were performed. Human ApoC3 mRNA levels were measured and corrected based on the level of the internal reference gene ACTIN.

Results are expressed relative to the remaining percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC ₅₀ inhibition results are presented in Table 35.

The experiment was performed with reference to the cell viability screening (Cells-to-CT) in a 96-well plate in Example 21.

Example 23. Checking the level of target activity and non-target activity of miRNA using psiCHECK

Screening of target activity and off-target activity was performed in vitro at the molecular level using 11 concentration gradients in Huh 7 cells. The results show that although the siRNAs of the present invention have high activity, they also have low off-target activity.

Huh7 cells were cultured at 37°C with 5% _CO2 in high-glucose DMEM containing 10% fetal bovine serum. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at a density of 10,000 cells per well. Each well contained 100 µl of medium.

Cells were co-transfected with siRNA and the corresponding plasmid using Lipofectamine2000 (Thermo Fisher, 11668019) according to the instructions. 0.2 µl of Lipofectamine2000 was used for each well. The plasmid transfection amount was 10 ng per well. A total of 11 siRNA concentration points were determined for targeting and non-targeting plasmids. The highest final concentration at the concentration point was 40 nM, and a 3-fold serial dilution was performed (40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.494 nM, 0.165 nM, 0.0549 nM, 0.0183 nM, 0.00609 nM, 0.00203 nM, and 0.000677 nM). At 24 h posttransfection, non-target levels were determined using a dual-luciferase reporter assay system (Promega, E2940). The results are shown in Tables 37 through 40.

In the Huh7 cell line, the target/off-target activity of the miRNAs in Table 35 with good activity was determined by performing psi-CHECK screening.

Psi-CHECK plasmids were purchased from Synbio Technologies (Suzhou) Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd.

The experimental materials and instruments are detailed in Table 1 and Table 2 in Example 3.1, and the experimental results are detailed in Tables 37–40. See Example 3.2 for the psiCHECK activity screening experimental procedure, in which the multi-concentration dilution protocol for miRNA samples is shown in Table 36. The results are presented in Tables 37–40.

Table 36. Multiple concentration dilution protocol for miRNA samples

miRNA concentration
(μM) Final concentration
(nm) Addition of water and siRNA 20 / / 4 40 4 µl siRNA + 16 µl H ₂ O 1.33333 13,33333 20 µl siRNA + 40 µl H ₂ O 0.444444 4,444444 20 µl siRNA + 40 µl H ₂ O 0.148148 1.481481 20 µl siRNA + 40 µl H ₂ O 0.049383 0.493827 20 µl siRNA + 40 µl H ₂ O 0.016461 0.164609 20 µl siRNA + 40 µl H ₂ O 0.005487 0.05487 20 µl siRNA + 40 µl H ₂ O 0.001829 0.01829 20 µl siRNA + 40 µl H ₂ O 0.00061 0.006097 20 µl siRNA + 40 µl H ₂ O 0.000203 0.002032 20 µl siRNA + 40 µl H ₂ O 6.77E-05 0.000677 20 µl siRNA + 40 µl H ₂ O

Table 37. Results of screening of target activity of psiCHECK miRNA (GSCM)

Double chain number 40 Nm 13.3 nM 4.44 nM 1.48 nM 0.494 nM 0.165 nM 0.0549 nM 0.0183 nM 0.00609 nM 0.00203 nM 0.000677 nM IC ₅₀ value (nM) TRD005088 0.21 0.19 0.20 0.25 0.38 0.53 0.76 0.84 0.85 0.93 1.04 0.1950 TRD005092 0.28 0.25 0.24 0.28 0.34 0.44 0.73 0.84 0.96 1.07 1.09 0.1349 TRD005126 0.36 0.29 0.24 0.28 0.34 0.48 0.68 0.79 0.83 0.96 0.95 0.1349 TRD005131 0.25 0.25 0.28 0.29 0.38 0.55 0.72 0.83 0.98 0.96 0.94 0.1995 TRD005140 0.19 0.16 0.16 0.24 0.32 0.52 0.69 0.84 0.98 0.95 1.00 0.1660 TRD005143 0.13 0.13 0.13 0.13 0.18 0.26 0.38 0.56 0.75 0.88 0.94 0.0263 TRD005151 0.11 0.11 0.11 0.20 0.33 0.52 0.69 0.75 0.85 0.94 0.90 0.1738 TRD005181 0.23 0.25 0.23 0.23 0.22 0.32 0.49 0.66 0.85 0.91 0.92 0.0447 TRD005197 0.06 0.07 0.08 0.12 0.20 0.34 0.60 0.68 0.85 0.94 1.00 0.0692 TRD005198 0.31 0.33 0.27 0.33 0.42 0.61 0.67 0.79 0.90 0.85 0.91 0.2630 TRD005202 0.11 0.10 0.12 0.13 0.21 0.35 0.49 0.73 0.95 0.97 0.98 0.0603 TRD005204 0.06 0.05 0.05 0.06 0.09 0.14 0.18 0.31 0.55 0.77 0.93 0.0074 TRD005206 0.06 0.05 0.05 0.08 0.13 0.28 0.54 0.78 0.90 0.88 0.94 0.0676 TRD005219 0.13 0.12 0.12 0.15 0.27 0.45 0.73 0.85 0.90 0.97 0.92 0.1413 TRD005220 0.16 0.15 0.15 0.23 0.39 0.61 0.72 0.88 0.92 0.88 0.92 0.2570

Table 38. Results of psiCHECK off-target activity screening of AS miRNA (GSSM) chain seed regions

Double chain number 40 Nm 13.3 nM 4.44 nM 1.48 nM 0.494 nM 0.165 nM 0.0549 nM 0.0183 nM 0.00609 nM 0.00203 nM 0.000677 nM IC ₅₀ value (nM) TRD005077 0.98 0.94 0.94 0.99 0.95 1.03 1.01 0.93 1.08 1.04 0.96 more than 40 nM TRD005088 1.17 1.14 1.13 1.12 1.19 1.15 1.20 1.08 1.25 1.00 1.15 more than 40 nM TRD005092 0.89 0.96 0.94 1.05 1.04 1.03 0.94 1.10 1.05 1.10 1.21 more than 40 nM TRD005112 0.72 0.69 0.77 0.85 0.92 0.89 1.06 0.96 1.12 1.02 0.98 more than 40 nM TRD005124 0.99 0.95 0.96 1.02 1.08 0.97 0.99 1.04 1.03 0.95 1.12 more than 40 nM TRD005126 0.88 1.01 1.03 0.96 0.90 0.96 1.05 0.98 0.98 1.03 1.04 more than 40 nM TRD005140 0.95 0.99 0.92 0.93 0.94 0.92 0.99 1.01 1.01 1.01 0.95 more than 40 nM TRD005143 1.08 1.21 1.00 1.11 1.00 0.89 1.06 1.00 0.90 1.08 1.13 more than 40 nM TRD005151 0.94 0.95 0.96 0.89 0.93 0.92 0.86 0.89 0.89 0.90 0.77 more than 40 nM TRD005171 0.94 1.16 1.01 1.00 1.18 0.94 1.10 1.17 1.07 1.03 1.03 more than 40 nM TRD005181 1.01 0.99 0.98 1.04 1.09 1.07 1.25 1.02 1.07 0.98 1.09 more than 40 nM TRD005197 0.70 0.87 0.89 1.05 0.93 0.94 0.98 0.89 0.90 0.88 0.85 more than 40 nM TRD005198 0.94 0.91 0.92 0.99 1.04 1.01 1.21 0.86 0.97 1.02 1.01 more than 40 nM TRD005202 0.53 0.44 0.50 0.67 0.75 0.81 0.85 0.83 0.87 0.93 0.99 39.81 TRD005204 0.75 0.73 0.83 0.89 0.85 1.03 0.95 0.85 0.78 1.01 0.99 more than 40 nM TRD005205 0.89 0.88 0.75 0.98 0.83 0.91 0.88 0.94 0.83 0.82 0.83 more than 40 nM TRD005206 0.64 0.64 0.64 0.92 0.86 0.98 0.86 1.06 1.12 0.97 1.02 more than 40 nM TRD005207 0.67 0.89 0.84 0.97 1.01 1.04 0.99 0.99 1.01 0.97 1.09 more than 40 nM TRD005208 0.72 0.71 0.69 0.83 0.95 0.84 0.97 1.01 1.04 0.94 1.04 more than 40 nM TRD005209 0.73 0.72 0.79 0.96 1.02 1.01 0.99 1.11 1.06 1.05 1.23 more than 40 nM TRD005210 0.76 0.85 0.77 0.86 1.04 0.99 0.99 1.07 1.04 1.01 1.09 more than 40 nM TRD005212 0.79 0.89 0.91 0.96 0.93 0.96 0.95 1.04 0.98 0.88 1.01 more than 40 nM TRD005214 0.92 0.89 0.99 1.00 0.92 1.06 1.02 0.98 1.11 0.99 1.04 more than 40 nM TRD005216 0.88 0.87 0.83 0.92 0.91 0.83 0.86 0.81 0.84 0.92 0.98 more than 40 nM TRD005217 0.91 0.93 0.96 0.85 0.92 0.85 0.82 0.84 0.90 0.80 0.83 more than 40 nM TRD005219 0.70 0.68 0.80 0.84 0.76 0.93 0.88 0.81 0.87 0.89 0.90 more than 40 nM TRD005220 0.98 1.03 0.93 1.08 1.02 1.05 0.98 1.15 1.13 1.04 1.01 more than 40 nM TRD005221 0.74 0.74 0.87 0.99 1.02 0.90 0.99 0.97 1.01 0.88 1.09 more than 40 nM

Table 39. Off-target activity screening results (PSCM)

Double chain number 40 nM 13.3 nM 4.44 nM 1.48 nM 0.494 nM 0.165 nM 0.0549 nM 0.0183 nM 0.00609 nM 0.00203 nM 0.000677 nM IC ₅₀ value (nM) TRD005088 0.98 1.00 1.00 0.93 0.95 0.97 0.98 0.94 1.01 1.00 0.97 more than 40 nM TRD005092 1.01 1.03 0.98 1.05 1.03 1.08 1.08 0.96 1.09 1.02 0.92 more than 40 nM TRD005126 0.99 0.93 1.03 0.98 1.00 1.01 0.95 0.99 0.97 1.04 1.03 more than 40 nM TRD005140 1.02 0.97 1.03 1.12 1.07 1.07 1.05 1.08 1.16 0.98 0.98 more than 40 nM TRD005143 1.01 0.90 0.97 0.96 1.04 0.98 1.01 0.95 0.91 1.03 0.95 more than 40 nM TRD005151 0.96 0.94 0.90 0.92 0.90 0.99 0.95 0.97 0.93 0.88 0.96 more than 40 nM TRD005181 0.96 0.99 0.84 0.91 0.84 0.86 0.84 0.86 0.89 0.93 0.95 more than 40 nM TRD005197 0.84 0.78 0.83 0.91 0.92 0.87 0.83 0.88 0.84 0.88 1.01 more than 40 nM TRD005198 0.84 0.89 0.99 0.90 0.99 0.98 1.02 1.02 1.01 0.91 0.92 more than 40 nM TRD005202 0.95 0.83 0.85 0.94 0.99 0.93 0.91 0.95 0.98 0.74 0.84 more than 40 nM TRD005204 0.92 0.85 0.86 0.99 1.01 1.00 1.02 1.03 0.92 1.13 0.95 more than 40 nM TRD005206 1.03 1.02 1.03 1.07 1.17 1.14 1.19 1.14 0.98 1.17 1.07 more than 40 nM TRD005219 1.15 1.09 0.99 0.97 0.96 1.05 1.00 1.06 1.02 0.89 1.12 more than 40 nM TRD005220 0.90 1.04 0.94 0.93 1.09 0.94 0.90 0.95 1.04 0.86 1.02 more than 40 nM

Example 24. Inhibition of Human ApoC3 in Huh7 Cells. The inhibitory activities of siRNAs at 11 concentration points, which showed 80% or more inhibition in vitro (20% or less remaining mRNA expression level) in Table 17, were subjected to off-target modification (modifications at position 7 of the AS chain) in Huh7 cells using 11 concentration gradients, and then Huh7 cell viability was screened. Each siRNA sample for transfection was serially diluted 3-fold from an initial final concentration of 40 nM to 11 concentration points.

Huh7 cells were cultured at 37°C with 5% _CO2 in high-glucose DMEM containing 10% fetal bovine serum. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at a density of 10,000 cells per well. Each well contained 100 µl of medium.

Cells were transfected with siRNA at final concentrations of 40 nM, 13.3 nM, 4.44 nM, 1.48 nM, 0.494 nM, 0.165 nM, 0.0549 nM, 0.0183 nM, 0.00609 nM, 0.00203 nM, and 0.000677 nM using Lipofectamine RNAiMAX (ThermoFisher, 13778150) according to the product instructions. After 24 h of treatment, cells were lysed using the TaqMan™ Fast Advanced Cells-to-CT™ Kit (ThermoFisher, A35378), and one-step reverse transcription and quantitative real-time PCR detection were performed. Human ApoC3 mRNA levels were measured and corrected based on the level of the internal reference gene ACTIN.

Results are expressed relative to the remaining percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC ₅₀ inhibition results are presented in Table 41.

The experiment was performed with reference to the cell viability screening (Cells-to-CT) in a 96-well plate in Example 21.

Table 41. Multidose inhibitory activity of siRNA against human ApoC3 in Huh7 cells

Double chain number 40 nM 13.3 nM 4.44 nM 1.48 nM 0.494 nM 0.165 nM TRD005874 3.5% 3.8% 6.2% 9.1% 12.8% 22.8% TRD005875 4.7% 6.2% 11.4% 13.6% 26.0% 47.2% TRD005878 5.8% 12.3% 10.1% 11.8% 23.4% 38.1% TRD005879 4.2% 7.8% 12.5% 17.0% 35.0% 39.7% TRD005882 7.7% 8.6% 12.8% 14.6% 22.2% 37.8% TRD005885 12.9% 22.1% 35.5% 28.6% 30.0% 63.6% TRD005891 6.7% 10.5% 15.3% 21.6% 28.9% 49.3%

Double chain number 0.0549 nM 0.0183 nM 0.00609 nM 0.00203 nM 0.000677 nM IC ₅₀ value for Huh7 cells (nM) TRD005874 45.4% 76.1% 116.3% 122.8% 118.9% 0.0457 TRD005875 86.1% 122.0% 135.6% 106.8% 106.2% 0.1585 TRD005878 106.6% 131.2% 148.7% 149.6% 119.6% 0.1349 TRD005879 78.5% 132.2% 140.2% 142.9% 140.5% 0.1318 TRD005882 78.5% 114.3% 165.6% 145.6% 109.9% 0.1072 TRD005885 100.7% 130.5% 155.8% 117.4% 105.0% 0.2239 TRD005891 95.8% 109.4% 122.0% 128.9% 105.3% 0.1862

Example 25. Checking the level of target activity and non-target activity of miRNA using psiCHECK

After modification of the siRNA (at position 7 of the AS chain), in vitro molecular screening for on-target and off-target activity was performed in HEK293A cells using 11 concentration gradients. The results demonstrate that although the siRNAs of the present invention exhibit high activity, they also exhibit low off-target activity. See Example 16 for the experimental procedure. To improve detection sensitivity, the off-target plasmid GSSM-5hits was constructed, containing five identical GSSM sequences linked by TTCC for the antisense strands of the siRNA.

The results are shown in Tables 43 through 46. The results show that all siRNAs had high in vitro inhibitory activity against the target (IC ₅₀ GSCM value less than 0.3 nM) and had no significant off-target effect. psiCHECK Activity Screening Procedure

In the HEK293A cell line, miRNA activity was determined using psi-CHECK activity assays. Experimental materials and instruments are detailed in Table 1 and Table 2 in Example 3.1, and experimental results are detailed in Tables 43–46.

See Example 3.2 for the psiCHECK activity screening experimental procedure, in which the multi-concentration dilution protocol for miRNA samples is shown in Table 42.

Table 42. Multiple concentration dilution protocol for siRNA

miRNA concentration (μM) Final concentration (nm) Addition of water and siRNA 20 / / 4 40 4 µl siRNA + 16 µl H ₂ O 1.33333 13,33333 20 µl siRNA + 40 µl H ₂ O 0.444444 4,444444 20 µl siRNA + 40 µl H ₂ O 0.148148 1.481481 20 µl siRNA + 40 µl H ₂ O 0.049383 0.493827 20 µl siRNA + 40 µl H ₂ O 0.016461 0.164609 20 µl siRNA + 40 µl H ₂ O 0.005487 0.05487 20 µl siRNA + 40 µl H ₂ O 0.001829 0.01829 20 µl siRNA + 40 µl H ₂ O 0.00061 0.006097 20 µl siRNA + 40 µl H ₂ O 0.000203 0.002032 20 µl siRNA + 40 µl H ₂ O 6.77E-05 0.000677 20 µl siRNA + 40 µl H ₂ O

Table 43. Results of screening of target activity of psiCHECK miRNA (GSCM)

Double chain number 40 nM 13.3 nM 4.44 nM 1.48 nM 0.494 nM 0.165 nM TRD005874 7.0% 4.9% 4.7% 4.6% 5.3% 8.5% TRD005875 27.1% 19.8% 14.0% 12.4% 12.6% 18.7% TRD005876 49.9% 24.8% 16.9% 14.5% 15.2% 18.6% TRD005877 16.6% 8.2% 6.0% 6.5% 9.5% 19.8% TRD005878 21.9% 12.9% 9.2% 7.1% 7.8% 10.9% TRD005879 15.1% 8.0% 4.6% 4.8% 4.7% 6.5% TRD005882 18.4% 9.7% 9.3% 9.0% 8.8% 10.4% TRD005883 38.3% 22.4% 13.0% 13.4% 19.3% 38.6% TRD005884 27.1% 18.0% 13.6% 14.2% 18.7% 33.9% TRD005886 49.5% 30.0% 19.5% 17.0% 18.0% 30.6% TRD005887 21.0% 12.0% 10.3% 9.6% 11.3% 19.4% TRD005889 50.7% 32.9% 25.8% 28.5% 39.2% 58.7% TRD005890 77.9% 48.0% 33.0% 27.7% 27.6% 37.9% TRD005891 27.0% 16.6% 12.2% 10.1% 8.9% 12.0% TRD005893 84.6% 58.2% 40.8% 30.6% 29.5% 42.6%

Double chain number 0.0549 nM 0.0183 nM 0.00609 nM 0.00203 nM 0.000677 nM _IC50 value (nM) TRD005874 16.6% 35.4% 70.5% 85.8% 97.8% 0.0115 TRD005875 34.0% 55.5% 80.1% 95.7% 99.9% 0.0229 TRD005876 31.2% 53.9% 82.8% 91.6% 99.5% 0.0214 TRD005877 43.7% 72.5% 90.6% 97.6% 98.1% 0.041 TRD005878 21.5% 51.9% 74.9% 86.5% 98.0% 0.017 TRD005879 12.6% 30.3% 55.7% 79.3% 91.8% 0.0076 TRD005882 20.3% 45.7% 75.5% 92.6% 99.6% 0.0148 TRD005883 69.4% 85.9% 95.3% 96.6% 101.4% 0.1023 TRD005884 59.3% 81.4% 93.2% 97.4% 101.3% 0.0759 TRD005886 57.8% 81.1% 89.5% 101.8% 99.0% 0.0646 TRD005887 45.5% 71.4% 85.4% 95.2% 94.3% 0.0417 TRD005889 75.6% 85.2% 91.0% 96.2% 95.6% 0.2399 TRD005890 60.9% 86.1% 94.5% 102.2% 104.4% 0.0813 TRD005891 25.9% 52.6% 75.5% 90.3% 98.8% 0.0186 TRD005893 61.0% 75.5% 89.2% 92.1% 96.8% 0.0851 Note: Since the transfection efficiency was low at the highest concentration (40 nM) due to the internal synthesis process, the experimental data corresponding to the highest concentration (40 nM) were discarded during data processing.

Table 44. Results of psiCHECK off-target activity screening of AS miRNA chain seed regions (GSSM-5hits)

Double chain number 40 Nm 13.3 Nm 4.44 Nm 1.48 Nm 0.494
Nm 0.165
Nm 0.0549 Nm 0.0183 Nm 0.00609
Nm 0.002 03
Nm 0.000677 nM More importantly
IC ₅₀
(nM) TRD005875 1.00 0.97 0.98 1.00 1.04 1.06 1.00 1.07 1.01 1.16 1.03 ND TRD005882 0.72 0.75 0.89 0.92 1.01 0.96 0.92 1.01 0.90 1.00 0.91 80.4 TRD005891 0.78 0.76 0.84 0.90 0.98 0.98 1.00 0.98 1.01 0.97 0.95 84.4 TRD005874 0.72 0.69 0.74 0.93 0.98 1.00 0.99 0.98 0.95 1.12 0.93 59.0 TRD005887 0.75 0.74 0.82 0.85 0.91 0.92 0.94 1.02 1.03 0.98 1.08 101.0

Note: ND = not detectable.

Example 26. Inhibition of human ApoC3 in Huh7 cells by siRNA - inhibitory activity at 11 concentration points

Following modification of siRNA (at position 7 of the AS chain), Huh7 cells were screened for viability using 11 concentration gradients. Each siRNA sample for transfection was serially diluted 3-fold from an initial final concentration of 20 nM to 11 concentration points.

Huh7 cells were cultured at 37°C with 5% _CO2 in high-glucose DMEM containing 10% fetal bovine serum. Twenty-four hours before transfection, Huh7 cells were inoculated into 96-well plates at a density of 10,000 cells per well. Each well contained 100 µl of medium.

Cells were transfected with siRNA at final concentrations of 20 nM, 6.67 nM, 2.22 nM, 0.741 nM, 0.247 nM, 0.0823 nM, 0.0274 nM, 0.00914 nM, 0.00305 nM, 0.00102 nM, and 0.000339 nM using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product instructions. Total cellular RNA was extracted from cells 24 h after treatment using a high-throughput cellular RNA extraction kit, and RNA reverse transcription and quantitative real-time PCR detection were performed. Human ApoC3 mRNA levels were measured and corrected based on the level of the internal reference gene ACTIN.

Results are expressed relative to the remaining percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC ₅₀ inhibition results are presented in Table 53.

The experimental materials for cell viability screening (nucleic acid extractor) in 96-well plate are shown in Table 1 and Table 2.

Experimental method for cell viability screening (nucleic acid extractor) in a 96-well plate:

(I) Transfection of cells

Reference was made to the cell transfection procedure in Example 21. The quantities of the components of the transfection complex are given in Table 47:

II. Extraction of cellular RNA using a nucleic acid extractor (magnetic particle method)

1. Preparation: High-throughput cellular RNA extraction kit (FG0417-L/FG0418-XL, magnetic particle method).

2. The following reagents were added to 6 deep-well plates.

Cell lysate preparation: 200 μl LB lysis solution + 3.5 μl 1 M DTT solution; the culture supernatant in the 96-well plate was completely aspirated, the solution mixture was added at 200 μl/well and lysis was performed for 5 min.

Deoxyribonuclease I dilution solution: 3.4 µl of Deoxyribonuclease I dilution solution + 5 µl + 41.6 µl of 0.1% DEPC water (50 µl per well, well mixed). The resulting Deoxyribonuclease I mixture was placed on ice.

Selecting the device program: cellular RNA 96.

3. Six deep-well plates were placed into six corresponding nucleic acid extractor cartridges and labeled, and the tip combs were placed into 96-well plate 3. The instrument was started, and the cellular RNA extraction program was initiated. After 35 minutes, the program was paused. 96-well plate 2 was removed and 220 µl of WB1 buffer was added. The cellular RNA extraction program was then resumed.

4. After nucleic acid extraction and concentration measurements, the 96-well plates were sealed with aluminum foil and fully labeled. Plates can be stored in a refrigerator at 4°C before use in reverse transcription or stored in a freezer at -40°C.

III. Reverse transcription of cellular RNA

1. Preparation: (1) Reverse transcription kit (Takara PrimeScript™ II 1st Strand cDNA Synthesis Kit) (6210A); the expiration date was checked and all kit components were stored in a freezer at -40°C).

2. The following reaction mixture (Mix1) was prepared in a microtube.

After 5 min of incubation at 65°C, the mixture was rapidly cooled on ice for 2 min. (Note: The above treatment may denature the messenger RNA, increasing the efficiency of reverse transcription.)

3. The following reaction mixture for reverse transcription (Mix2) was prepared in a microtube.

10 µl of Mix2 were added to Mix1 to give a total volume of 20 µl. Inversion was performed as follows: 42°C for 45 min, 95°C for 5 min, 4°C forever.

4. After completion of the inversion, 80 μL DEPC water (final concentration: 10 ng/μL) was added to each tube, and the samples could be stored in a refrigerator at 4°C before use in Taqman Q-PCR or stored in a freezer at -40°C.

IV. Q-PCR analysis with Taqman probe. See Example 16 for the experimental procedure and Table 53 for the results.

Table 53. Multidose inhibitory activity of siRNA against human ApoC3 in Huh7 cells

Double chain number 20 nM 6.67 nM 2.22 nM 0.741 nM 0.247 nM 0.0823 nM TRD006884 1.7% 2.6% 4.1% 7.4% 25.1% 55.2% TRD006885 4.1% 2.4% 3.2% 5.7% 11.9% 30.0% TRD006886 2.8% 3.0% 4.5% 7.6% 17.4% 45.8% TRD006887 1.1% 1.3% 2.5% 6.0% 17.3% 51.6% TRD006888 1.1% 2.4% 1.8% 2.7% 4.9% 12.4% TRD006925 1.7% 1.2% 2.4% 2.8% 6.5% 21.3% TRD006928 0.9% 0.9% 1.2% 1.8% 2.6% 5.2% TRD006937 3.1% 2.1% 3.5% 6.9% 7.8% 22.5% TRD006964 3.2% 3.9% 2.4% 6.2% 8.8% 23.0%

Double chain number 0.0274 nM 0.00914 nM 0.00305 nM 0.00102 nM 0.000339 nM IC ₅₀ value for Huh7 cells (nM) TRD006884 104.3% 168.0% 162.9% 129.1% 111.7% 0.0933 TRD006885 65.1% 107.4% 102.2% 112.7% 92.3% 0.0912 TRD006886 94.5% 110.2% 101.7% 115.6% 99.9% 0.138 TRD006887 65.9% 171.1% 135.3% 135.9% 105.2% 0.1096 TRD006888 32.3% 66.7% 116.0% 109.9% 101.0% 0.0288 TRD006925 54.8% 103.0% 116.4% 118.5% 104.3% 0.0871 TRD006928 11.1% 26.2% 47.0% 72.1% 64.9% 0.0031 TRD006937 38.9% 58.4% 102.3% 82.3% 78.7% 0.0195 TRD006964 53.0% 65.8% 102.7% 73.7% 106.9% 0.024

Example 27. Checking the level of target activity and non-target activity of miRNA using psiCHECK

In vitro screening of target and off-target activity was performed on test compounds in HEK293A cells at the molecular level using 11 concentration gradients. The results show that although the siRNAs of the present invention have high activity, they also have low off-target activity. Psi-CHECK plasmids were purchased from Synbio Technologies (Suzhou) Co., Ltd. and Sangon Biotech (Shanghai) Co., Ltd. See Example 16 for the experimental procedure. To improve detection sensitivity, the off-target plasmid GSSM-5hits was constructed, i.e., five identical GSSM sequences linked by TTCC, for the antisense strands of the siRNA.

The results show that all six miRNAs had high in vitro inhibitory activity against the target (GSCM _IC50 value less than 0.3 nM). The results of the off-target activity analysis (GSSM-5hits, PSCM, PSSM) of the miRNAs show that five miRNAs did not exhibit significant off-target effects.

In the HEK293A cell line, the activity of 6 miRNAs was determined using psi-CHECK activity assays. The experimental results are detailed in Tables 54–57.

Table 54. Results of screening of target activity of psiCHECK miRNA (GSCM)

Double chain number 20 nM 6.67 nM 2.22 nM 0.741 nM 0.247 nM 0.0823 nM TRD006884 15.9% 12.6% 12.7% 14.6% 21.4% 42.7% TRD006885 11.5% 8.4% 7.2% 6.7% 9.3% 23.7% TRD006886 10.3% 9.7% 9.5% 9.1% 10.5% 20.4% TRD006887 15.8% 13.2% 13.2% 18.3% 33.5% 60.9% TRD006888 5.8% 5.1% 4.9% 5.5% 8.1% 18.1% TRD005205 25.1% 18.8% 19.6% 24.8% 52.0% 75.8% TRD006925 6.8% 5.6% 5.5% 6.3% 10.9% 34.6% TRD006971 4.8% 4.5% 4.5% 4.3% 5.6% 11.7% TRD006973 14.2% 13.3% 12.4% 14.5% 24.6% 51.4% TRD006975 12.5% 11.5% 11.6% 11.2% 17.5% 35.9% TRD006926 6.1% 5.5% 5.3% 5.9% 8.2% 14.7% TRD006927 5.7% 5.5% 5.4% 5.7% 7.2% 11.8% TRD006928 5.1% 5.3% 5.2% 5.2% 7.0% 11.6% TRD006929 14.9% 13.2% 12.7% 14.7% 28.3% 53.0% TRD006930 14.5% 12.6% 13.1% 15.8% 28.4% 53.6% TRD006931 16.8% 14.1% 13.5% 15.2% 24.2% 44.3% TRD006932 10.7% 11.2% 11.4% 11.2% 13.7% 25.9% TRD006933 11.1% 10.5% 11.1% 10.6% 13.6% 21.2% TRD006934 11.3% 11.5% 12.0% 11.6% 12.7% 22.0% TRD006935 18.7% 15.2% 14.1% 15.9% 27.6% 52.5% TRD006936 18.0% 14.6% 13.5% 14.4% 22.1% 45.9% TRD006937 17.5% 13.9% 13.0% 13.4% 21.4% 41.5% TRD006938 12.3% 9.3% 8.3% 7.7% 10.7% 21.8% TRD006939 17.8% 11.2% 8.8% 8.0% 10.6% 23.9% TRD006940 9.9% 8.8% 8.4% 7.8% 11.0% 22.3% TRD006963 12.6% 10.1% 9.8% 11.4% 20.9% 46.7% TRD006964 11.8% 10.1% 10.5% 11.5% 20.1% 39.9% TRD006965 11.6% 10.3% 10.2% 11.7% 20.4% 40.9% TRD006966 13.5% 10.6% 11.8% 17.5% 39.2% 74.5% TRD005883 13.4% 10.8% 11.3% 18.9% 43.9% 77.6%

Double chain number 0.0274 nM 0.00914 nM 0.00305 nM 0.00102 nM 0.000339 nM _IC50 value (nM) TRD006884 71.7% 97.5% 99.7% 100.3% 101.6% 0.0753 TRD006885 58.5% 83.0% 94.0% 96.9% 97.4% 0.0349 TRD006886 46.2% 79.6% 91.0% 102.6% 101.0% 0.0271 TRD006887 86.9% 93.0% 96.8% 99.6% 103.5% 0.1433 TRD006888 49.6% 81.3% 95.1% 100.4% 108.9% 0.0282 TRD005205 87.4% 97.2% 99.2% 103.3% 106.3% 0.2847 TRD006925 69.7% 90.8% 100.5% 103.7% 102.1% 0.0531 TRD006971 26.1% 60.0% 92.8% 99.6% 104.7% 0.014 TRD006973 75.9% 95.3% 100.6% 98.4% 97.4% 0.094 TRD006975 67.5% 88.2% 97.8% 98.9% 99.0% 0.0555 TRD006926 37.3% 78.0% 91.4% 100.4% 97.5% 0.0198 TRD006927 25.5% 63.2% 86.5% 95.5% 99.0% 0.0129 TRD006928 27.6% 72.2% 86.0% 101.4% 99.8% 0.0151 TRD006929 81.6% 113.2% 111.4% 108.1% 102.2% 0.0905 TRD006930 80.4% 95.9% 102.3% 97.9% 101.0% 0.0912 TRD006931 75.7% 95.7% 102.2% 105.3% 106.1% 0.0656 TRD006932 55.0% 92.9% 104.8% 109.0% 101.1% 0.0326 TRD006933 54.6% 83.1% 98.1% 101.1% 92.9% 0.03 TRD006934 48.2% 88.6% 97.3% 119.5% 105.7% 0.0261 TRD006935 81.8% 96.9% 97.9% 104.7% 102.6% 0.0891 TRD006936 76.5% 96.4% 100.2% 99.5% 90.3% 0.0687 TRD006937 70.0% 91.3% 93.3% 95.1% 92.3% 0.0589 TRD006938 58.1% 93.1% 96.3% 100.4% 94.1% 0.0336 TRD006939 61.5% 92.2% 97.3% 100.7% 98.4% 0.036 TRD006940 62.9% 88.7% 99.2% 87.3% 92.1% 0.0366 TRD006963 74.5% 83.7% 97.3% 98.6% 92.7% 0.0676 TRD006964 73.7% 94.1% 97.4% 105.6% 97.8% 0.0593 TRD006965 69.8% 90.4% 94.4% 98.5% 92.8% 0.0584 TRD006966 88.8% 101.9% 100.4% 102.5% 98.9% 0.1711 TRD005883 88.9% 101.2% 99.2% 98.6% 92.7% 0.1995

Table 55. Results of psiCHECK off-target activity screening of AS miRNA chain seed regions (GSSM-5hits)

Double chain number 20 nM 6.67 nM 2.22 nM 0.741 nM 0.247 nM 0.0823 nM TRD006884 92.8% 95.2% 108.1% 118.2% 112.6% 114.2% TRD006885 85.1% 93.6% 106.3% 115.6% 117.8% 107.1% TRD006886 89.9% 93.4% 102.7% 109.5% 112.2% 113.3% TRD006887 63.2% 63.6% 89.5% 106.7% 113.7% 106.5% TRD006888 71.9% 80.9% 95.5% 107.9% 110.9% 108.0% TRD005205 48.2% 42.9% 55.7% 78.9% 96.3% 104.2% TRD006971 72.8% 78.0% 89.4% 94.4% 97.3% 108.2% TRD006973 41.5% 44.2% 73.6% 93.8% 104.1% 122.2% TRD006975 37.2% 47.7% 75.0% 102.0% 104.1% 108.7%

Double chain number 0.0274 nM 0.00914 nM 0.00305 nM 0.00102 nM 0.000339 nM IC ₅₀ value fit for GSSM-5hits (nM) TRD006884 115.8% 111.6% 107.9% 115.5% 105.0% 309 TRD006885 113.1% 106.2% 110.9% 111.9% 108.6% 131 TRD006886 114.1% 113.4% 106.7% 107.1% 108.4% 180 TRD006887 107.0% 105.0% 105.0% 114.5% 113.3% 24 TRD006888 107.5% 101.4% 100.1% 104.4% 106.1% 46 TRD005205 103.8% 105.0% 112.0% 102.5% 106.4% 6 TRD006971 106.9% 102.9% 102.0% 96.2% 93.1% 41 TRD006973 111.2% 108.6% 110.8% 104.0% 97.8% 8 TRD006975 101.8% 103.5% 104.0% 103.9% 93.3% 8

Table 56. Results of psiCHECK off-target activity screening of SS miRNA chains (PSCM)

Double chain number 20 nM 6.67 nM 2.22 nM 0.741 nM 0.247 nM 0.0823 nM TRD006884 81.7% 87.8% 102.8% 118.3% 116.1% 113.8% TRD006885 87.0% 106.7% 121.9% 127.4% 126.7% 111.9% TRD006886 104.0% 103.4% 99.3% 95.1% 105.1% 109.6% TRD006887 116.9% 117.1% 112.6% 111.7% 112.9% 104.0% TRD006888 79.0% 89.0% 95.1% 105.6% 110.0% 111.9% TRD005205 83.2% 98.0% 101.9% 110.4% 108.9% 107.9% TRD006925 76.7% 89.6% 103.4% 104.6% 110.0% 120.0% TRD006971 76.5% 86.2% 94.5% 99.8% 103.8% 119.7% TRD006973 111.6% 115.5% 109.3% 109.1% 102.4% 125.1% TRD006975 119.4% 118.6% 130.2% 107.8% 98.3% 104.0%

Double chain number 0.0274 nM 0.00914 nM 0.00305 nM 0.00102 nM 0.000339 nM Fitting the IC ₅₀ value for PSCM(nM) TRD006884 121.3% 105.8% 108.8% 110.9% 104.9% 90 TRD006885 114.1% 111.2% 112.8% 112.2% 108.5% 342 TRD006886 116.2% 107.2% 100.8% 107.9% 105.6% ND TRD006887 104.1% 102.0% 104.6% 108.1% 107.2% ND TRD006888 116.9% 104.8% 99.0% 105.5% 105.4% 72 TRD005205 113.2% 110.7% 120.9% 101.6% 118.9% 121 TRD006925 117.9% 107.5% 106.3% 97.9% 94.3% 70 TRD006971 113.0% 110.6% 112.3% 99.8% 102.4% 59 TRD006973 111.6% 104.2% 101.6% 98.4% 92.0% ND TRD006975 97.1% 98.8% 104.0% 97.8% 95.6% ND

Note: ND = not detectable.

Table 57. Results of psiCHECK off-target activity screening of SS miRNA seed5 regions (PSSM)

Double chain number 20 nM 6.67 nM 2.22 nM 0.741 nM 0.247 nM 0.0823 nM TRD006884 108.3% 103.8% 77.5% 111.3% 105.7% 109.0% TRD006885 87.1% 102.6% 131.1% 133.4% 126.8% 113.6% TRD006886 105.4% 109.7% 110.3% 110.1% 107.9% 108.1% TRD006887 120.1% 123.7% 130.2% 118.6% 120.5% 109.6% TRD006888 69.5% 87.3% 94.1% 103.7% 108.1% 107.4% TRD005205 89.9% 97.9% 112.5% 113.1% 108.0% 115.1% TRD006925 81.3% 92.4% 102.2% 107.8% 109.2% 126.1% TRD006971 80.0% 83.9% 96.6% 94.8% 97.3% 125.7% TRD006973 109.0% 110.4% 109.1% 103.1% 102.5% 116.0% TRD006975 127.0% 128.9% 133.1% 117.0% 107.7% 121.5%

Double chain number 0.0274 nM 0.00914 nM 0.00305 nM 0.00102 nM 0.000339 nM Fitting the IC ₅₀ value for PSSM(nM) TRD006884 108.9% 98.8% 102.1% 104.9% 104.8% ND TRD006885 116.0% 114.0% 116.4% 112.9% 113.6% 340 TRD006886 110.5% 110.5% 105.6% 103.5% 98.7% ND TRD006887 107.5% 105.5% 108.9% 109.0% 109.8% ND TRD006888 110.4% 103.1% 99.8% 103.3% 105.2% 46 TRD005205 114.1% 112.3% 126.3% 114.3% 124.3% 251 TRD006925 111.4% 103.3% 104.9% 105.2% 91.6% 94 TRD006971 102.1% 101.0% 99.5% 89.8% 92.6% 66 TRD006973 108.6% 104.1% 101.3% 98.3% 95.7% ND TRD006975 106.9% 108.2% 116.3% 110.3% 97.0% ND

Example 28. Inhibition of human ApoC3 in Huh7 cells by siRNA - inhibitory activity at 11 concentration points

Hep3B cell viability screening was performed using test compounds in Hep3B cells using 11 concentration gradients. Each siRNA sample for transfection was serially diluted 3-fold from an initial final concentration of 20 nM to 11 concentration points.

Hep3B cells were cultured at 37°C with 5% _CO2 in MEM (minimum essential medium) containing 10% fetal bovine serum. Twenty-four hours before transfection, Hep3B cells were inoculated into 96-well plates at a density of 10,000 cells per well. Each well contained 100 µl of medium.

Cells were transfected with siRNA at final concentrations of 20 nM, 6.67 nM, 2.22 nM, 0.741 nM, 0.247 nM, 0.0823 nM, 0.0274 nM, 0.00914 nM, 0.00305 nM, 0.00102 nM, and 0.000339 nM using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product instructions. Total cellular RNA was extracted from cells 24 h after treatment using a high-throughput cellular RNA extraction kit, and RNA reverse transcription and quantitative real-time PCR detection were performed. Human ApoC3 mRNA levels were measured and corrected based on the level of the internal reference gene ACTIN.

Results are expressed relative to the remaining percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC ₅₀ inhibition results are presented in Table 58.

Table 58. Multidose inhibitory activity of siRNA against human ApoC3 in Hep3B cells

No. 20 nM 6.67 nM 2.22 nM 0.741 nM 0.247 nM 0.0823 nM TRD006884 2% 2.0% 3.5% 4.3% 12.6% 20.5% TRD006885 3% 5.1% 9.7% 13.5% 26.9% 41.5% TRD006886 5% 8.6% 14.0% 20.6% 53.2% 88.1% TRD006887 4% 10.9% 9.6% 25.5% 45.5% 82.2% TRD006888 3% 6.1% 10.6% 19.8% 12.4% 30.1% TRD006925 7% 2.1% 7.2% 12.0% 12.9% 33.1% TRD006966 4% 3.6% 12.1% 28.1% 40.0% 115.0% TRD006927 3% 5.5% 5.1% 7.0% 13.9% 33.9% TRD006928 3% 4.9% 3.6% 5.2% 11.0% 19.8% TRD006936 4% 5.8% 6.8% 24.7% 22.8% 58.2% TRD006937 10% 12.8% 17.6% 24.4% 19.1% 53.9% TRD006964 15% 21.2% 28.6% 20.2% 16.4% 54.4% TRD006965 36% 23.7% 30.3% 36.2% 37.7% 61.7% TRD006971 6% 3.5% 2.9% 2.5% 5.1% 16.1% TRD006973 5% 3.0% 5.0% 9.6% 29.8% 56.1% TRD006975 5% 2.1% 3.9% 7.4% 16.7% 53.5%

No. 0.0274 nM 0.00914 nM 0.00305 nM 0.00102 nM 0.000339 nM IC ₅₀ value for Hep3B cells (nM) TRD006884 39.8% 92.8% 97.5% 102.3% 106.3% 0.024 TRD006885 83.2% 115.9% 141.0% 118.3% 124.5% 0.0692 TRD006886 110.0% 190.3% 187.7% 162.3% 145.3% 0.2089 TRD006887 123.6% 160.3% 148.0% 179.4% 134.5% 0.195 TRD006888 53.1% 69.0% 94.0% 100.5% 73.5% 0.0288 TRD006925 53.7% 84.5% 94.6% 94.1% 118.4% 0.0363 TRD006966 187.9% 159.1% 130.2% 101.0% 118.8% 0.2089 TRD006927 65.7% 107.2% 121.5% 140.9% 106.7% 0.0457 TRD006928 42.4% 72.6% 99.9% 120.3% 102.4% 0.0214 TRD006936 88.2% 136.7% 106.2% 96.9% 111.7% 0.1 TRD006937 111.7% 116.2% 114.8% 109.3% 107.3% 0.0871 TRD006964 97.0% 101.5% 111.3% 108.0% 89.6% 0.0933 TRD006965 117.9% 156.2% 133.6% 147.2% 121.7% 0.1096 TRD006971 35.0% 57.8% 102.0% 104.3% 95.9% 0.0148 TRD006973 78.7% 114.1% 118.0% 124.7% 114.6% 0.0955 TRD006975 101.3% 108.0% 110.5% 131.6% 111.8% 0.0912

Example 29. Inhibition of human ApoC3 in primary human hepatocytes (PHH) by siRNA - inhibitory activity at 11 concentration points

Primary human hepatocyte (PHH) viability screening was performed using test compounds in primary human hepatocytes (PHH) using 11 concentration gradients. Each siRNA sample for transfection was serially diluted 3-fold from an initial final concentration of 20 nM to 11 concentration points.

Primary human hepatocytes (PHH) were cryopreserved in liquid nitrogen. Twenty-four hours prior to transfection, primary human hepatocytes (PHH) were thawed and then inoculated into a 96-well plate at a density of 40,000 cells per well. Each well contained 100 µl of medium.

Cells were transfected with siRNA at final gradient concentrations of 20 nM, 6.67 nM, 2.22 nM, 0.741 nM, 0.247 nM, 0.0823 nM, 0.0274 nM, 0.00914 nM, 0.00305 nM, 0.00102 nM, and 0.000339 nM using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product instructions. 24 h after treatment, total cellular RNA was extracted from the cells using a high-throughput cellular RNA extraction kit, and RNA reverse transcription and quantitative real-time PCR detection were performed. Human ApoC3 mRNA levels were measured and corrected based on the level of the internal reference gene ACTIN.

The results are expressed relative to the remaining percentage of human ApoC3 mRNA expression in cells treated with control siRNA. IC ₅₀ inhibition results are presented in Table 59. All of them can effectively inhibit human ApoC3 mRNA expression.

Table 59. Multidose inhibitory activity of siRNA against human ApoC3 in primary human hepatocytes (PHH)

Double chain number 20 nM 6.67 nM 2.22 nM 0.741 nM 0.247 nM 0.0823 nM TRD006884 7% 5.5% 9.2% 28.2% 22.4% 46.8% TRD006885 6% 10.2% 11.5% 11.1% 23.3% 30.3% TRD006888 7% 5.8% 10.1% 13.8% 20.2% 40.8% TRD006925 5% 6.1% 9.2% 13.3% 13.5% 26.2% TRD006928 10% 7.1% 8.0% 7.5% 12.5% 25.5% TRD006937 5% 7.9% 9.5% 17.3% 26.2% 54.2% TRD006886 6.1% 9.2% 10.3% 14.1% 22.0% 39.9% TRD006971 3.9% 5.6% 6.2% 6.9% 9.4% 14.2% TRD006964 5% 6.1% 17.0% 17.6% 28.2% 63.7%

Double chain number 0.0274 nM 0.00914 nM 0.00305 nM 0.00102 nM 0.000339 nM IC ₅₀ value for primary human hepatocyte (nM) TRD006884 80.2% 97.8% 105.7% 112.9% 85.3% 0.0756 TRD006885 64.5% 85.2% 115.5% 98.5% 112.2% 0.0427 TRD006888 66.9% 99.8% 95.3% 107.6% 106.1% 0.0574 RD006925 86.9% 84.2% 107.3% 89.9% 80.3% 0.0336 TRD006928 44.8% 86.5% 107.2% 107.7% 111.6% 0.0267 TRD006937 84.7% 114.7% 110.1% 118.6% 107.4% 0.0953 TRD006886 78.2% 100.0% 115.3% 111.1% 115.2% 0.0631 TRD006971 19.6% 42.8% 69.5% 95.3% 105.3% 0.0071 TRD006964 81.3% 113.8% 112.6% 90.9% 97.1% 0.1202

Example 30. Inhibition of monkey ApoC3 in primary monkey hepatocytes by siRNA - inhibitory activity at 11 concentration points

A primary screen for monkey hepatocyte viability was performed using test compounds in primary monkey hepatocytes using 11 concentration gradients. Each siRNA sample for transfection was serially diluted 3-fold from an initial final concentration of 20 nM to 11 concentration points.

Cells were transfected with siRNA at final gradient concentrations of 20 nM, 6.67 nM, 2.22 nM, 0.741 nM, 0.247 nM, 0.0823 nM, 0.0274 nM, 0.00914 nM, 0.00305 nM, 0.00102 nM, and 0.000339 nM using Lipofectamine RNAi MAX (ThermoFisher, 13778150) according to the product instructions. Treatment solutions with the above concentrations were prepared in advance and added to a 96-well plate. Primary monkey hepatocytes were cryopreserved in liquid nitrogen. Primary monkey hepatocytes were thawed and then inoculated into a 96-well plate (with miRNA samples) at a density of 30,000 cells per well. Each well contained 100 µl of medium.

Twenty-four hours after reverse transfection, the culture medium was changed, and culturing was continued for another 24 hours. Total cellular RNA was then extracted from the cells using a high-throughput cellular RNA extraction kit, and reverse transcription of the RNA and quantitative real-time PCR detection were performed. Monkey ApoC3 mRNA levels were measured and adjusted based on the level of the internal reference gene GAPDH.

The results are expressed relative to the remaining percentage of monkey ApoC3 mRNA expression in cells treated with control siRNA. IC ₅₀ inhibition results are presented in Table 61. All of them were able to effectively inhibit monkey ApoC3 mRNA expression in primary monkey hepatocytes.

Example 31 In vivo testing of siRNA agents in ApoC3 transgenic mice

To evaluate the in vivo effect of some ApoC3 siRNA agents, ApoC3 transgenic mice (The Jackson Laboratory, 006907-B6; CBA-Tg (ApoC3) 3707Bres/J) were purchased and used. Experiments were performed with ApoC3 transgenic mice, and serum levels of human ApoC3 protein, triglycerides, and total cholesterol were measured as recommended by the kit manufacturers (Roche Cobas C311: CHOL2 &TRIGL; MSD Human ApoC3 Antibody Kit (B21ZV-3)).

For normalization, ApoC3 protein, triglyceride, and total cholesterol levels for each animal at a given time point were divided by the pre-treatment expression level in that animal to determine the "normalized to pre-dose" expression ratio.

ApoC3 protein, triglyceride, and total cholesterol levels can be measured at various times before and after administration of ApoC3 siRNA agents. Unless otherwise noted herein, blood samples were collected from the submandibular region into centrifuge tubes containing sodium heparin. After the blood samples were thoroughly mixed with sodium heparin, the tubes were centrifuged at 3000×g for 5 minutes to separate serum and stored at 4°C.

The ApoC3 transgenic mouse model described above was used. On day 0, each mouse received a single subcutaneous injection of 200 μl of the corresponding siRNA agent dissolved in PBS (1×) or control (PBS (1×)) (i.e., the vehicle group), which included the administration groups shown in Table 62 below.

Injections of ApoC3 siRNA agents were performed between the skin and muscle (i.e., subcutaneous injections). Six mice were tested in each group (n=6). Serum was collected from mice on day -2 (pre-dose blood sampling with overnight fasting) and on days 7, 14, 21, 28, 35, and 42. Mice fasted overnight before each collection. ApoC3 protein, triglyceride, and total cholesterol levels in serum were determined using a device according to the agent manufacturer's recommendations.

ApoC3 protein, triglyceride, and total cholesterol levels were normalized for each animal. For normalization, ApoC3 protein, triglyceride, and total cholesterol levels for each animal at a given time point were divided by the animal's pre-treatment expression level (in this case, day -2) to determine the "normalized to pre-treatment" expression ratio.

The experimental data are shown below in Tables 63-65 and Figs. 7-9. Each of the ApoC3 siRNA agents in each of the administration groups (i.e., groups 2-6) demonstrated a significant reduction in ApoC3 protein levels, triglycerides, and total cholesterol compared to the control (group 1).

Example 32. Evaluation of various modifications in positions 9 and 10 of the AS chain

In this experiment, the in vivo inhibition efficiency of the siRNA conjugates of the present invention with 2'-fluoro modifications at different sites against the mRNA expression level of the target gene was investigated.

Male C57BL/6 mice aged 6 to 8 weeks were randomized into groups of 6, 3 mice per time point, and each group of mice received test conjugates (TRD007047 and TRD006870), control conjugate (TRD002218), and PBS.

All animals received a single dose by subcutaneous injection based on their body weight. The siRNA conjugates were administered at a dose of 1 mg/kg (based on siRNA) in a volume of 5 ml/kg. Mice were sacrificed 7 days after administration, and their livers were collected and later preserved with RNA (Sigma Aldrich). Liver tissue was then homogenized using a tissue homogenizer, and total RNA was extracted from the liver tissue using a tissue RNA extraction kit (FireGen Biomedicals, FG0412), following the procedure described in the instructions. Total RNA was reverse transcribed to cDNA, and the level of TTR mRNA expression in liver tissue was measured using quantitative real-time fluorescence PCR. In the fluorescence quantitative PCR method, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as an internal reference gene, and the mRNA expression levels of TTR and GAPDH were measured using Taqman primers-probes for TTR and GAPDH, respectively.

The TTR mRNA expression level was calculated according to the equation below:

TTR mRNA expression = [(TTR mRNA expression in the test group/GAPDH mRNA expression in the test group)/(TTR mRNA expression in the control group/GAPDH mRNA expression in the control group)] × 100%

The compounds are shown in Table 66, the grouping of the tested compounds in mice is shown in Table 67, and the sequences of the detection primers are shown in Table 68.

At 28 days after administration, the in vivo inhibition efficiency of the siRNA conjugates of the present invention with F modifications at different sites against the mRNA expression level of the target gene was shown in Table 69. The siRNA compounds with F modifications at different sites inhibited a greater expression of TTR mRNA than the positive control compound TRD002218 at day 28 after administration. The 9F and 10F modifications showed high inhibition efficiency, and the inhibitory effects did not differ significantly, indicating that the 9F and 10F modifications can mediate higher efficiency of siRNA inhibition.

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SEQUENCE LIST

<110> TOJIE BIOTECH (SHANGHAI) CO., LTD.

<120> MODIFIED siRNA WITH REDUCED OFF-TARGET ACTIVITY

<130> 721092CPCT

<140> PCT/CN2021/110509

<141> 2021-08-04

<150> CN202010772542.6

<151> 2020-08-04

<150> CN202110244977.8

<151> 2021-03-05

<150> CN202110361502.7

<151> 2021-04-02

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<210> 6

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 6

augugagga uucuugucaa from 21

<210> 7

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 7

augugagga uucuugucaa from 21

<210> 8

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 8

augugagga uucuugucaa from 21

<210> 9

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 9

augugagga uucuugucaa from 21

<210> 10

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 10

augugagga uucuugucaa from 21

<210> 11

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 11

augugagga uucuugucaa from 21

<210> 12

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 12

augugagga uucuugucaa from 21

<210> 13

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 13

gaacuacucc cuuucuuca 19

<210> 14

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 14

ugaagaaagg gaguaguucu u 21

<210> 15

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 15

ccauuuguuc agugguucg 19

<210> 16

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 16

ugaaccacug aacaaauggc a 21

<210> 17

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 17

caccucugca cgucgcaug 19

<210> 18

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 18

uaugcgacgu gcagagguga a 21

<210> 19

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 19

caguguucuu gcucuauaaa 19

<210> 20

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 20

uuauagagca agaacacugu u 21

<210> 21

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 21

ugaagaaagg gaguaguucu u 21

<210> 22

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 22

ugaagaaagg gaguaguucu u 21

<210> 23

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> n represents Id, and the bases in other positions represent

are chemically modified bases.

<400> 23

ugaaganagg gaguaguucu u 21

<210> 24

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 24

ugaagaaagg gaguaguucu u 21

<210> 25

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 25

ugaagaaagg gaguaguucu u 21

<210> 26

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 26

ugaagaaagg gaguaguucu u 21

<210> 27

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 27

ugaagaaagg gaguaguucu u 21

<210> 28

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 28

ugaaccacug aacaaauggc a 21

<210> 29

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 29

ugaaccacug aacaaauggc a 21

<210> 30

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> n represents Id, and the bases in other positions represent

are chemically modified bases.

<400> 30

ugaaccncug aacaaauggc a 21

<210> 31

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 31

ugaaccacug aacaaauggc a 21

<210> 32

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 32

ugaaccacug aacaaauggc a 21

<210> 33

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 33

ugaaccacug aacaaauggc a 21

<210> 34

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 34

ugaaccacug aacaaauggc a 21

<210> 35

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 35

uaugcgacgu gcagagguga a 21

<210> 36

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 36

uaugcgacgu gcagagguga a 21

<210> 37

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> n represents Id, and the bases in other positions represent

are chemically modified bases.

<400> 37

uaugcgncgu gcagagguga a 21

<210> 38

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 38

uaugcgacgu gcagagguga a 21

<210> 39

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 39

uaugcgacgu gcagagguga a 21

<210> 40

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 40

uaugcgacgu gcagagguga a 21

<210> 41

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 41

uaugcgacgu gcagagguga a 21

<210> 42

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 42

uuauagagca agaacacugu u 21

<210> 43

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 43

uuauagagca agaacacugu u 21

<210> 44

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> n represents Id, and the bases in other positions represent

are chemically modified bases.

<400> 44

uuauagngca agaacacugu u 21

<210> 45

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 45

uuauagagca agaacacugu u 21

<210> 46

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 46

uuauagagca agaacacugu u 21

<210> 47

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 47

uuauagagca agaacacugu u 21

<210> 48

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 48

uuauagagca agaacacugu u 21

<210> 49

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 49

caguguucuu gcucuauaaa 19

<210> 50

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 50

uuauagagca agaacacugu u 21

<210> 51

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 51

caguguucuu gcucuauaaa 19

<210> 52

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 52

uuauagagca agaacacugu u 21

<210> 53

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 53

caguguucuu gcucuauaaa 19

<210> 54

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 54

uuauagagca agaacacugu u 21

<210> 55

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 55

caguguucuu gcucuauaaa 19

<210> 56

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 56

uuauagagca agaacacugu u 21

<210> 57

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 57

caguguucuu gcucuauaaa 19

<210> 58

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 58

uuauagagca agaacacugu u 21

<210> 59

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 59

caguguucuu gcucuauaaa 19

<210> 60

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 60

uuauagagca agaacacugu u 21

<210> 61

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 61

caguguucuu gcucuauaaa 19

<210> 62

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 62

uuauagagca agaacacugu u 21

<210> 63

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 63

gugugcacuu cgcuucacc 19

<210> 64

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 64

agugaagcga agugcacacg g 21

<210> 65

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 65

gugugcacuu cgcuucacc 19

<210> 66

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 66

agugaagcga agugcacacg g 21

<210> 67

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 67

gugugcacuu cgcuucacc 19

<210> 68

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 68

agugaagcga agugcacacg g 21

<210> 69

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 69

cuuuugucuu uggguauau 19

<210> 70

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 70

auauacccaa agacaaaaga a 21

<210> 71

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 71

cuuuugucuu uggguauau 19

<210> 72

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 72

auauacccaa agacaaaaga a 21

<210> 73

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 73

uuaccaauuu ucuuuuguu 19

<210> 74

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 74

aacaaaagaa aauugguaac a 21

<210> 75

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 75

uuaccaauuu ucuuuuguu 19

<210> 76

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 76

aacaaaagaa aauugguaac a 21

<210> 77

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 77

cgugugcacu ucgcuucac 19

<210> 78

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 78

augaagcgaa gugcacacgg u 21

<210> 79

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 79

cgugugcacu ucgcuucac 19

<210> 80

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 80

augaagcgaa gugcacacgg u 21

<210> 81

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 81

ugucuuuggg uauacauuu 19

<210> 82

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 82

aaauguauac ccaaagacaa a 21

<210> 83

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 83

ugucuuuggg uauacauuu 19

<210> 84

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 84

aaauguauac ccaaagacaa a 21

<210> 85

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 85

cuuuugucuu uggguauac 19

<210> 86

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 86

auauacccaa agacaaaaga a 21

<210> 87

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 87

cuuuugucuu uggguauac 19

<210> 88

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 88

auauacccaa agacaaaaga a 21

<210> 89

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 89

caucuucuug uugguucuu 19

<210> 90

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 90

aagaaccaac aagaagauga g 21

<210> 91

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 91

caucuucuug uugguucuu 19

<210> 92

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 92

aagaaccaac aagaagauga g 21

<210> 93

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 93

ugucugcggc guuuuauca 19

<210> 94

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 94

ugauaaaacg ccgcagacac a 21

<210> 95

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 95

ugucugcggc guuuuauca 19

<210> 96

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 96

ugauaaaacg ccgcagacac a 21

<210> 97

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 97

ugcacuucgc uucaccucu 19

<210> 98

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 98

agaggugaag cgaagugcac a 21

<210> 99

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 99

ugcacuucgc uucaccucu 19

<210> 100

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 100

agaggugaag cgaagugcac a 21

<210> 101

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 101

gcacuucgcu ucaccucug 19

<210> 102

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 102

uagaggugaa gcgaagugca from 21

<210> 103

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 103

gcacuucgcu ucaccucug 19

<210> 104

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 104

uagaggugaa gcgaagugca from 21

<210> 105

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 105

ggcgcugaau ccugcggac 19

<210> 106

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 106

auccgcagga uucagcgccg a 21

<210> 107

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 107

ggcgcugaau ccugcggac 19

<210> 108

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 108

auccgcagga uucagcgccg a 21

<210> 109

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 109

gugugcacuu cgcuucaca 19

<210> 110

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 110

ugugaagcga agugcacacu u 21

<210> 111

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 111

gugugcacuu cgcuucaca 19

<210> 112

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 112

ugugaagcga agugcacacu u 21

<210> 113

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 113

gugugcacuu cgcuucaca 19

<210> 114

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 114

ugugaagcga agugcacacu u 21

<210> 115

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 115

gccucugccc gagcuucaa 19

<210> 116

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 116

uugaagcucg ggcagaggcc a 21

<210> 117

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 117

uugaagnucg ggcagaggcc a 21

<210> 118

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 118

gcuucaugca ggguuacau 19

<210> 119

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 119

auguaacccu gcaugaagcu g 21

<210> 120

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 120

auguaanccu gcaugaagcu g 21

<210> 121

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 121

ugagcagcgu gcaggaguu 19

<210> 122

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 122

aacuccugca cgcugcucag u 21

<210> 123

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 123

aacuccngca cgcugcucag u 21

<210> 124

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 124

caguucccug aaagacuau 19

<210> 125

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 125

auagucuuuc agggaacuga a 21

<210> 126

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 126

auagucnuuc agggaacuga a 21

<210> 127

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 127

aagucccaccu gccuaucca 19

<210> 128

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 128

uggauaggca gguggacuug g 21

<210> 129

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 129

uggauangca gguggacuug g 21

<210> 130

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 130

ucucagugcu cuccuaccu 19

<210> 131

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 131

agguaggaga gcacugagaa u 21

<210> 132

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 132

agguagnaga gcacugagaa u 21

<210> 133

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 133

ggcaugcugg ccucccaau 19

<210> 134

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 134

auugggaggc cagcaugccu g 21

<210> 135

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 135

auugggnggc cagcaugccu g 21

<210> 136

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 136

gcaugcuggc cucccaaua 19

<210> 137

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 137

uauugggagg ccagcaugcc u 21

<210> 138

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 138

uauuggnagg ccagcaugcc u 21

<210> 139

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 139

cuggccuccc aauaaagcu 19

<210> 140

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 140

agcuuuauug ggaggccagc a 21

<210> 141

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 141

agcuuunuug ggaggccagc a 21

<210> 142

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 142

ggccucccaa uaaagcuga 19

<210> 143

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 143

ucagcuuuau ugggaggcca g 21

<210> 144

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 144

ucagcunuau ugggaggcca g 21

<210> 145

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 145

uaaagcugga caagaagcu 19

<210> 146

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 146

agcuucuugu ccagcuuuau u 21

<210> 147

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 147

agcuucnugu ccagcuuuau u 21

<210> 148

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 148

uauucucagu gcucuccua 19

<210> 149

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 149

uaggagagca cugagaauac u 21

<210> 150

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 150

uaggagngca cugagaauac u 21

<210> 151

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 151

ccguuaagga caaguucuu 19

<210> 152

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 152

aagaacuugu ccuuaacggu g 21

<210> 153

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 153

aagaacnugu ccuuaacggu g 21

<210> 154

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 154

cugcgagcuc cugggucu 19

<210> 155

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 155

agacccaagg agcucgcagg a 21

<210> 156

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 156

agacccnagg agcucgcagg a 21

<210> 157

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 157

acaguauucu cagugcucu 19

<210> 158

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 158

agagcacuga gaauacuguc from 21

<210> 159

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 159

agagcanuga gaauacuguc from 21

<210> 160

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 160

uucucagugc ucuccuacu 19

<210> 161

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 161

aguaggag cacugagaau a 21

<210> 162

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 162

aguaggngag cacugagaau a 21

<210> 163

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 163

aagggacagu auucucagu 19

<210> 164

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 164

acugagaaua cugucccuuu u 21

<210> 165

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 165

acugagnaua cugucccuuu u 21

<210> 166

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 166

aauaaagcug gacaagaaa 19

<210> 167

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 167

uuucuugucc agcuuuauug g 21

<210> 168

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 168

uuucuunucc agcuuuauug g 21

<210> 169

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 169

gacaaguucu cugaguucu 19

<210> 170

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 170

agaacucaga gaacuugucc u 21

<210> 171

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 171

agaacunaga gaacuugucc u 21

<210> 172

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 172

cgaggaugcc ucccuucuu 19

<210> 173

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 173

aagaagggag gcauccucgg from 21

<210> 174

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 174

aagaagngag gcauccucgg from 21

<210> 175

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 175

acuacuggag caccguuaa 19

<210> 176

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 176

uuaacggugc uccaguaguc u 21

<210> 177

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 177

uuaacgnugc uccaguaguc u 21

<210> 178

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 178

aaaagcugg acaagaagu 19

<210> 179

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 179

acuucuuguc cagcuuuauu g 21

<210> 180

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 180

acuucunguc cagcuuuauu g 21

<210> 181

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 181

agggacagua uucucagua 19

<210> 182

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 182

uacugagaau acugucccuu u 21

<210> 183

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 183

uacuganaau acugucccuu u 21

<210> 184

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 184

gccucccaau aaagcugga 19

<210> 185

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 185

uccagcuuua uugggaggcc a 21

<210> 186

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 186

uccagcnuua uugggaggcc a 21

<210> 187

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 187

ugcuggccuc ccaauaaaa 19

<210> 188

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 188

uuuuauuggg aggccagcau g 21

<210> 189

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 189

uuuuaunggg aggccagcau g 21

<210> 190

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 190

auucucagug cucuccuau 19

<210> 191

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 191

auaggagagc acugagaaua from 21

<210> 192

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 192

auagganagc acugagaaua from 21

<210> 193

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 193

uucaguuccc ugaaagacu 19

<210> 194

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 194

agucuuucag ggaacugaag from 21

<210> 195

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 195

agucuuncag ggaacugaag from 21

<210> 196

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 196

caugcuggcc ucccaauaa 19

<210> 197

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 197

uuauugggag gccagcaugc from 21

<210> 198

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 198

uuauugngag gccagcaugc from 21

<210> 199

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 199

uauucucagu gcucuccuu 19

<210> 200

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 200

uaggagagca cugagaauac u 21

<210> 201

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 201

uaggagngca cugagaauac u 21

<210> 202

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 202

uauucucagu gcucuccuc 19

<210> 203

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 203

uaggagagca cugagaauac u 21

<210> 204

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 204

uaggagngca cugagaauac u 21

<210> 205

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 205

uauucucagu gcucuccug 19

<210> 206

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 206

uaggagagca cugagaauac u 21

<210> 207

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 207

uaggagngca cugagaauac u 21

<210> 208

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 208

ccguuaagga caaguucua 19

<210> 209

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 209

aagaacuugu ccuuaacggu g 21

<210> 210

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 210

aagaacnugu ccuuaacggu g 21

<210> 211

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 211

ccguuaagga caaguucuc 19

<210> 212

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 212

aagaacuugu ccuuaacggu g 21

<210> 213

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 213

aagaacnugu ccuuaacggu g 21

<210> 214

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 214

ccguuaagga caaguucug 19

<210> 215

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 215

aagaacuugu ccuuaacggu g 21

<210> 216

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 216

aagaacnugu ccuuaacggu g 21

<210> 217

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 217

aauaaagcug gacaagaau 19

<210> 218

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 218

uuucuugucc agcuuuauug g 21

<210> 219

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 219

uuucuunucc agcuuuauug g 21

<210> 220

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 220

aauaaagcug gacaagaac 19

<210> 221

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 221

uuucuugucc agcuuuauug g 21

<210> 222

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 222

uuucuunucc agcuuuauug g 21

<210> 223

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 223

aauaaagcug gacaagaag 19

<210> 224

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 224

uuucuugucc agcuuuauug g 21

<210> 225

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 225

uuucuunucc agcuuuauug g 21

<210> 226

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 226

gcaccguuaa ggacaagua 19

<210> 227

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 227

aacuuguccu uaacggugcu from 21

<210> 228

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 228

aacuugnccu uaacggugcu from 21

<210> 229

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 229

gcaccguuaa ggacaaguc 19

<210> 230

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 230

aacuuguccu uaacggugcu from 21

<210> 231

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 231

aacuugnccu uaacggugcu from 21

<210> 232

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 232

gcaccguuaa ggacaagug 19

<210> 233

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 233

aacuuguccu uaacggugcu from 21

<210> 234

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 234

aacuugnccu uaacggugcu from 21

<210> 235

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 235

gacaaguucu cugaguuca 19

<210> 236

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 236

agaacucaga gaacuugucc u 21

<210> 237

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 237

agaacunaga gaacuugucc u 21

<210> 238

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 238

gacaaguucu cugaguucc 19

<210> 239

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 239

agaacucaga gaacuugucc u 21

<210> 240

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 240

agaacunaga gaacuugucc u 21

<210> 241

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 241

gacaaguucu cugaguucg 19

<210> 242

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 242

agaacucaga gaacuugucc u 21

<210> 243

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 243

agaacunaga gaacuugucc u 21

<210> 244

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 244

auucucagug cucuccuaa 19

<210> 245

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 245

auaggagagc acugagaaua from 21

<210> 246

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 246

auagganagc acugagaaua from 21

<210> 247

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 247

auucucagug cucuccuac 19

<210> 248

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 248

auaggagagc acugagaaua from 21

<210> 249

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 249

auagganagc acugagaaua from 21

<210> 250

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 250

auucucagug cucuccuag 19

<210> 251

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 251

auaggagagc acugagaaua from 21

<210> 252

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 252

auagganagc acugagaaua from 21

<210> 253

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 253

gcaccguuaa ggacaaguu 19

<210> 254

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 254

aacuuguccu uaacggugcu from 21

<210> 255

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n represents W’

<400> 255

aacuugnccu uaacggugcu from 21

<210> 256

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 256

ccguuaagga caaguucuu 19

<210> 257

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 257

aagaacuugu ccuuaacggu g 21

<210> 258

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 258

aagaacnugu ccuuaacggu g 21

<210> 259

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 259

auucucagug cucuccuau 19

<210> 260

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 260

auaggagagc acugagaaua from 21

<210> 261

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 261

auagganagc acugagaaua from 21

<210> 262

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 262

aauaaagcug gacaagaaa 19

<210> 263

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 263

uuucuugucc agcuuuauug g 21

<210> 264

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 264

uuucuunucc agcuuuauug g 21

<210> 265

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 265

gacaaguucu cugaguucu 19

<210> 266

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 266

agaacucaga gaacuugucc u 21

<210> 267

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 267

agaacunaga gaacuugucc u 21

<210> 268

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 268

uauucucagu gcucuccua 19

<210> 269

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 269

uaggagagca cugagaauac u 21

<210> 270

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 270

uaggagngca cugagaauac u 21

<210> 271

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 271

auucucagug cucuccuau 19

<210> 272

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 272

auaggagagc acugagaaua from 21

<210> 273

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 273

auagganagc acugagaaua from 21

<210> 274

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 274

uauucucagu gcucuccug 19

<210> 275

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 275

uaggagagca cugagaauac u 21

<210> 276

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 276

uaggagngca cugagaauac u 21

<210> 277

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 277

gacaaguucu cugaguucc 19

<210> 278

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 278

agaacucaga gaacuugucc u 21

<210> 279

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 279

agaacunaga gaacuugucc u 21

<210> 280

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 280

gcaccguuaa ggacaaguc 19

<210> 281

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 281

aacuuguccu uaacggugcu from 21

<210> 282

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (7)..(7)

<223> n is W’

<400> 282

aacuugnccu uaacggugcu from 21

<210> 283

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 283

gccucugccc gagcuucaa 19

<210> 284

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 284

uugaagcucg ggcagaggcc a 21

<210> 285

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 285

cgaggaugcc ucccuucuu 19

<210> 286

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 286

aagaagggag gcauccucgg from 21

<210> 287

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 287

gcuucaugca ggguuacau 19

<210> 288

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 288

auguaacccu gcaugaagcu g 21

<210> 289

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 289

ugagcagcgu gcaggaguu 19

<210> 290

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 290

aacuccugca cgcugcucag u 21

<210> 291

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 291

uucaguuccc ugaaagacu 19

<210> 292

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 292

agucuuucag ggaacugaag from 21

<210> 293

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 293

caguucccug aaagacuau 19

<210> 294

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 294

auagucuuuc agggaacuga a 21

<210> 295

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 295

acuacuggag caccguuaa 19

<210> 296

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 296

uuaacggugc uccaguaguc u 21

<210> 297

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 297

gcaccguuaa ggacaaguu 19

<210> 298

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 298

aacuuguccu uaacggugcu from 21

<210> 299

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 299

ccguuaagga caaguucuu 19

<210> 300

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 300

aagaacuugu ccuuaacggu g 21

<210> 301

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 301

gacaaguucu cugaguucu 19

<210> 302

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 302

agaacucaga gaacuugucc u 21

<210> 303

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 303

aagucccaccu gccuaucca 19

<210> 304

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 304

uggauaggca gguggacuug g 21

<210> 305

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 305

cugcgagcuc cugggucu 19

<210> 306

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 306

agacccaagg agcucgcagg a 21

<210> 307

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 307

aagggacagu auucucagu 19

<210> 308

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 308

acugagaaua cugucccuuu u 21

<210> 309

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 309

agggacagua uucucagua 19

<210> 310

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 310

uacugagaau acugucccuu u 21

<210> 311

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 311

acaguauucu cagugcucu 19

<210> 312

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 312

agagcacuga gaauacuguc from 21

<210> 313

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 313

uauucucagu gcucuccua 19

<210> 314

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 314

uaggagagca cugagaauac u 21

<210> 315

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 315

auucucagug cucuccuau 19

<210> 316

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 316

auaggagagc acugagaaua from 21

<210> 317

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 317

uucucagugc ucuccuacu 19

<210> 318

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 318

aguaggag cacugagaau a 21

<210> 319

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 319

ucucagugcu cuccuaccu 19

<210> 320

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 320

agguaggaga gcacugagaa u 21

<210> 321

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 321

ggcaugcugg ccucccaau 19

<210> 322

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 322

auugggaggc cagcaugccu g 21

<210> 323

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 323

gcaugcuggc cucccaaua 19

<210> 324

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 324

uauugggagg ccagcaugcc u 21

<210> 325

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 325

caugcuggcc ucccaauaa 19

<210> 326

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 326

uuauugggag gccagcaugc from 21

<210> 327

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 327

ugcuggccuc ccaauaaaa 19

<210> 328

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 328

uuuuauuggg aggccagcau g 21

<210> 329

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 329

cuggccuccc aauaaagcu 19

<210> 330

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 330

agcuuuauug ggaggccagc a 21

<210> 331

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 331

ggccucccaa uaaagcuga 19

<210> 332

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 332

ucagcuuuau ugggaggcca g 21

<210> 333

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 333

gccucccaau aaagcugga 19

<210> 334

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 334

uccagcuuua uugggaggcc a 21

<210> 335

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 335

aauaaagcug gacaagaaa 19

<210> 336

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 336

uuucuugucc agcuuuauug g 21

<210> 337

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 337

aaaagcugg acaagaagu 19

<210> 338

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 338

acuucuuguc cagcuuuauu g 21

<210> 339

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 339

uaaagcugga caagaagcu 19

<210> 340

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 340

agcuucuugu ccagcuuuau u 21

<210> 341

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 341

uauucucagu gcucuccua 19

<210> 342

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 342

uaggagagca cugagaauac u 21

<210> 343

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 343

ccguuaagga caaguucuu 19

<210> 344

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 344

aagaacuugu ccuuaacggu g 21

<210> 345

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 345

cugcgagcuc cugggucu 19

<210> 346

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 346

agacccaagg agcucgcagg a 21

<210> 347

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 347

acaguauucu cagugcucu 19

<210> 348

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 348

agagcacuga gaauacuguc from 21

<210> 349

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 349

uucucagugc ucuccuacu 19

<210> 350

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 350

aguaggag cacugagaau a 21

<210> 351

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 351

aagggacagu auucucagu 19

<210> 352

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 352

acugagaaua cugucccuuu u 21

<210> 353

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 353

aauaaagcug gacaagaaa 19

<210> 354

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 354

uuucuugucc agcuuuauug g 21

<210> 355

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 355

gacaaguucu cugaguucu 19

<210> 356

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 356

agaacucaga gaacuugucc u 21

<210> 357

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 357

cgaggaugcc ucccuucuu 19

<210> 358

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 358

aagaagggag gcauccucgg from 21

<210> 359

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 359

acuacuggag caccguuaa 19

<210> 360

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 360

uuaacggugc uccaguaguc u 21

<210> 361

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 361

aaaagcugg acaagaagu 19

<210> 362

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 362

acuucuuguc cagcuuuauu g 21

<210> 363

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 363

agggacagua uucucagua 19

<210> 364

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 364

uacugagaau acugucccuu u 21

<210> 365

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

vvvvvvvvvvvvbases.

<400> 365

gccucccaau aaagcugga 19

<210> 366

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 366

uccagcuuua uugggaggcc a 21

<210> 367

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 367

ugcuggccuc ccaauaaaa 19

<210> 368

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 368

uuuuauuggg aggccagcau g 21

<210> 369

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 369

auucucagug cucuccuau 19

<210> 370

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 370

auaggagagc acugagaaua from 21

<210> 371

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 371

uucaguuccc ugaaagacu 19

<210> 372

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 372

agucuuucag ggaacugaag from 21

<210> 373

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 373

caugcuggcc ucccaauaa 19

<210> 374

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 374

uuauugggag gccagcaugc from 21

<210> 375

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 375

auucucagug cucuccuau 19

<210> 376

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 376

auaggagagc acugagaaua from 21

<210> 377

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 377

uauucucagu gcucuccuu 19

<210> 378

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 378

uaggagagca cugagaauac u 21

<210> 379

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 379

uauucucagu gcucuccuc 19

<210> 380

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 380

uaggagagca cugagaauac u 21

<210> 381

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 381

uauucucagu gcucuccug 19

<210> 382

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 382

uaggagagca cugagaauac u 21

<210> 383

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 383

ccguuaagga caaguucua 19

<210> 384

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 384

aagaacuugu ccuuaacggu g 21

<210> 385

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 385

ccguuaagga caaguucuc 19

<210> 386

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 386

aagaacuugu ccuuaacggu g 21

<210> 387

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 387

ccguuaagga caaguucug 19

<210> 388

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 388

aagaacuugu ccuuaacggu g 21

<210> 389

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 389

aauaaagcug gacaagaau 19

<210> 390

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

vvgrounds.

<400> 390

uuucuugucc agcuuuauug g 21

<210> 391

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 391

aauaaagcug gacaagaac 19

<210> 392

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 392

uuucuugucc agcuuuauug g 21

<210> 393

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 393

aauaaagcug gacaagaag 19

<210> 394

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 394

uuucuugucc agcuuuauug g 21

<210> 395

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 395

gacaaguucu cugaguuca 19

<210> 396

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 396

agaacucaga gaacuugucc u 21

<210> 397

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 397

gacaaguucu cugaguucc 19

<210> 398

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 398

agaacucaga gaacuugucc u 21

<210> 399

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 399

gacaaguucu cugaguucg 19

<210> 400

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 400

agaacucaga gaacuugucc u 21

<210> 401

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 401

auucucagug cucuccuaa 19

<210> 402

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 402

auaggagagc acugagaaua from 21

<210> 403

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 403

auucucagug cucuccuac 19

<210> 404

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 404

auaggagagc acugagaaua from 21

<210> 405

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 405

auucucagug cucuccuag 19

<210> 406

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 406

auaggagagc acugagaaua from 21

<210> 407

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 407

gcaccguuaa ggacaagua 19

<210> 408

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 408

aacuuguccu uaacggugcu from 21

<210> 409

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 409

gcaccguuaa ggacaaguc 19

<210> 410

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 410

aacuuguccu uaacggugcu from 21

<210> 411

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 411

gcaccguuaa ggacaagug 19

<210> 412

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 412

aacuuguccu uaacggugcu from 21

<210> 413

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 413

gcaccguuaa ggacaaguu 19

<210> 414

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 414

aacuuguccu uaacggugcu from 21

<210> 415

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 415

ccguuaagga caaguucuu 19

<210> 416

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 416

aagaacuugu ccuuaacggu g 21

<210> 417

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 417

auucucagug cucuccuau 19

<210> 418

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 418

auaggagagc acugagaaua from 21

<210> 419

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 419

aauaaagcug gacaagaaa 19

<210> 420

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 420

uuucuugucc agcuuuauug g 21

<210> 421

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 421

gacaaguucu cugaguucu 19

<210> 422

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 422

agaacucaga gaacuugucc u 21

<210> 423

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 423

uauucucagu gcucuccua 19

<210> 424

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 424

uaggagagca cugagaauac u 21

<210> 425

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 425

uauucucagu gcucuccug 19

<210> 426

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 426

uaggagagca cugagaauac u 21

<210> 427

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 427

gacaaguucu cugaguucc 19

<210> 428

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 428

agaacucaga gaacuugucc u 21

<210> 429

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 429

gcaccguuaa ggacaaguc 19

<210> 430

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 430

aacuuguccu uaacggugcu from 21

<210> 431

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 431

gcaccguuaa ggacaagug 19

<210> 432

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 432

aacuuguccu uaacggugcu from 21

<210> 433

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 433

tgcctccctt ctcagcttca 20

<210> 434

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 434

gggaactgaa gccatcggtc 20

<210> 435

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 435

atgaagcacg ccaccaagac cgcca 25

<210> 436

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 436

acgtggacat ccgcaaagac 20

<210> 437

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 437

tcttcattgt gctgggtgcc 20

<210> 438

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 438

aacacagtgc tgtctggcgg cacca 25

<210> 439

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 439

gcctgcctgc tctgttcatc 20

<210> 440

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 440

aagccaagaa gggaggtgtc from 21

<210> 441

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 441

ttgttgctgc cgtgctgtca ctcctgg 27

<210> 442

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 442

tcaagatcgt cagcaacgcc 20

<210> 443

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 443

acagtcttct gggtggcagt 20

<210> 444

<211> 25

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 444

accaactgct tagcacccct ggcca 25

<210> 445

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 445

caguguucuu gcucuauaaa 19

<210> 446

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 446

uuauagagca agaacacugu u 21

<210> 447

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 447

caguguucuu gcucuauaaa 19

<210> 448

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 448

uuauagagca agaacacugu u 21

<210> 449

<211> 19

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(19)

<223> The bases in these positions are chemically modified

grounds.

<400> 449

caguguucuu gcucuauaaa 19

<210> 450

<211> 21

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<223> The bases in these positions are chemically modified

grounds.

<400> 450

uuauagagca agaacacugu u 21

<210> 451

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 451

gggaagaccg cggagtct 18

<210> 452

<211> 30

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 452

cagttctact ctgtacactc cttctacaaa 30

<210> 453

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 453

ctgcacgggc tcaccacaga tga 23

<210> 454

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 454

cggcaaattc aacggcacag 20

<210> 455

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 455

ccacgacata ctcagcaccg 20

<210> 456

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic oligonucleotide

<400> 456

accatcttcc aggagcgaga ccccact 27

<---

Claims (71)

1. A small interfering RNA (siRNA) for inhibiting the expression of a target gene or its mRNA, containing a sense strand and an antisense strand, where each strand contains from 15 to 35 nucleotides; the antisense strand contains a chemical modification of formula (I) or a tautomeric modification thereof in at least one of the nucleotide positions 5, 6 or 7 of its 5' region: (I), where Y is selected from the group consisting of O, NH and S; each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each of R ₄ , R ₄ ' and R ₅ is independently H or C ₁ -C ₆ alkyl; J ₂ is H or C ₁ -C ₆ alkyl; n is 0, 1 or 2; m is 0, 1 or 2; s is 0 or 1; R ₃ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and p is 1, 2 or 3; Q ₁ is , and Q ₂ is R ₂ ; or Q ₁ is R ₂ , and Q ₂ is ; Where: R ₁ is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and q is 1, 2 or 3; J ₁ is H or C ₁ -C ₆ alkyl; R ₂ is selected from the group consisting of H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-alkylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, halogen, methoxy, ethoxy, N ₃ , C ₂ -C ₆ alkenyl and C ₂ -C ₆ alkynyl, and r is 1, 2 or 3; B represents the base; where the chemical modification of formula (I) is not ; and when X is NH-CO, R ₁ is not H; in addition to the nucleotide modified by the chemical modification of formula (I) or a tautomeric modification thereof, at least one nucleotide modified in another manner is also contained in at least one of the sense strand and/or the antisense strand, wherein each of the nucleotides modified in another manner is independently selected from the group consisting of a 2'-methoxy-modified nucleotide, a 2'-fluoro-modified nucleotide and a 2'-deoxy-modified nucleotide. 2. miRNA according to paragraph 1, where: each X is independently selected from the group consisting of CR ₄ (R ₄ '), S, NR ₅ and NH-CO, wherein each of R ₄ , R ₄ ' and R ₅ is independently H, methyl, ethyl, n-propyl or isopropyl; each J ₁ and each J ₂ is independently H or methyl; R ₃ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _p R ₆ , where R ₆ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and p is 1 or 2; R ₁ is selected from the group consisting of H, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl and (CH ₂ ) _q R ₇ , where R ₇ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and q is 1 or 2; R ₂ is selected from the group consisting of H, OH, F, Cl, NH ₂ , methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, vinyl, allyl, ethynyl, propargyl, S-CH ₃ , NCH ₃ (CH ₃ ), OCH ₂ CH ₂ OCH ₃ , -O-methylamino, -O-ethylamino and (CH ₂ ) _r R ₈ , where R ₈ is selected from the group consisting of OH, F, Cl, methoxy, ethoxy, N ₃ , vinyl, allyl, ethynyl and propargyl, and r is 1 or 2. 3. The miRNA of claim 1 or 2, wherein the chemical modification is selected from any of the following structures: , , , , , , , , , , . 4. The miRNA of any one of claims 1 to 3, wherein the antisense strand comprises a chemical modification of formula (I) or a tautomeric modification thereof at nucleotide position 7 of its 5' region. 5. The miRNA according to any one of paragraphs 1-4, where: in the direction from the 5'-end to the 3'-end, each of the nucleotides at positions 2, 6 and 14 of the antisense strand independently represents a 2'-fluoro-modified nucleotide; or in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 14 and 16 of the antisense strand is independently a 2'-fluoro-modified nucleotide; or in the 5' to 3' direction, each of the nucleotides at positions 2, 6, 12 and 14 of the antisense strand is independently a 2'-fluoro-modified nucleotide; or in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 12, 14, 16, and 18 of the antisense strand is independently a 2'-fluoro-modified nucleotide; or in the 5' to 3' direction, each of the nucleotides at positions 2, 4, 6, 9, 12, 14, 16, and 18 of the antisense strand is independently a 2'-fluoro-modified nucleotide; or in the direction from the 5'-end to the 3'-end, each of the nucleotides at positions 2, 4, 6, 10, 12, 14, 16 and 18 of the antisense strand independently represents a 2'-fluoro-modified nucleotide. 6. The miRNA according to any one of paragraphs 1-5, where: The sense strand has a nucleotide sequence of the formula shown below: 5'-N _a N _{a N a} N _a XN _a N _b N _b N _b N _a N _a N _a N _a N _a N _a N _a N _a N _a N _a -3'; where each X is independently N _a or N _b ; each N _a and each N _b is independently a modified nucleotide or an unmodified nucleotide, and the modifications on N _a and N _b are different; and/or, The antisense strand has a nucleotide sequence of the formula shown below: 5'-N _a 'N _b 'N _a 'X'N _a 'N _b 'W'N _a 'X'Y'N _a 'X'N _a 'N _b 'N _a 'X'N _a 'X'N _a 'N _a 'N _a '-3'; wherein each X' is independently N _a ' or N _b ' and Y' is N _a ' or N _b '; each N _a ' and each N _b ' is independently a modified nucleotide or an unmodified nucleotide, wherein the modifications on N _a ' and N _b ' are different; W' is a nucleotide containing a chemical modification of formula (I) or a tautomeric modification thereof; N _a is a 2'-methoxy-modified nucleotide and N _b is a 2'-fluoro-modified nucleotide; and/or N _a ' is a 2'-methoxy-modified nucleotide, and N _b ' is a 2'-fluoro-modified nucleotide. 7. The miRNA of claim 6, wherein the antisense strand has a nucleotide sequence of the formula given below: 5'-N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'W'N _a 'X'Y'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _b 'N _a 'N _a 'N _a '-3'; where each X' is independently N _a ' or N _b ', and Y' is N _a ' or N _b '; N _a ' is a 2'-methoxy-modified nucleotide; N _b ' is a 2'-fluoro-modified nucleotide. 8. The miRNA according to any one of paragraphs 1-7, wherein the sense strand has a nucleotide sequence of the formula given below: 5'-N _a N _a N _a N _{a N a} N _a N _b N _b N _{b N a N a N a N a} N _{a N a} N _a N _a N _a N _a -3' or 5'-N _{a N} a N _a N _a N _{b N a} N _b N _b N _{b N a} N _a N _a N _a N _a N _a N _a N _a N _a N _a -3'; where N _a is a 2'-methoxy-modified nucleotide and N _b is a 2'-fluoro-modified nucleotide. 9. The miRNA of any one of claims 6 to 8, wherein W' is a nucleotide containing a chemical modification selected from the group consisting of the following structures or a tautomeric modification thereof; , or ; where B is the base corresponding to position 7 of the 5' region of the antisense strand. 10. The miRNA of any one of claims 1 to 9, wherein at least one phosphoester group in the sense strand and/or antisense strand is a phosphoester group with a modification group. 11. A siRNA conjugate for inhibiting the expression of a target gene or its mRNA in the liver, comprising: miRNA according to any one of claims 1-10 and a conjugated group associated with miRNA; wherein the conjugated group comprises a pharmaceutically acceptable targeting ligand and a linker, and The miRNA, linker and targeting ligand are sequentially linked covalently or non-covalently; the targeting ligand binds to the asialoglycoprotein receptor; and the targeting ligand comprises a galactose cluster or a galactose derivative cluster, wherein the galactose derivative is selected from the group consisting of N-acetyl-galactosamine, N-trifluoroacetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine and N-isobutyryl-galactosamine. 12. A pharmaceutical composition for inhibiting the expression of a target gene or its mRNA, containing a therapeutically effective amount of siRNA according to any one of claims 1-10 and a pharmaceutically acceptable excipient and/or adjuvant. 13. A method for inhibiting the expression of a target gene or its mRNA, comprising: administering to a subject in need thereof an effective amount or an effective dose of siRNA according to any one of claims 1-10 and/or a pharmaceutical composition according to claim 12.