WO2017132945A1 - Design and application of mirancer molecule - Google Patents

Abstract

Provided is a single-stranded RNA sequence for binding to a target microRNA. The single-stranded RNA sequence includes a 5'-end arm and a 3'-end arm. The 5'-end arm can bind, through complementary base pairing, with the target microRNA, and the 3'-end arm can bind, through complementary base pairing, with the 5'-end arm.

Description

Design and application of miRancer molecules Technical field

The invention relates to RNA molecules and uses thereof. In particular, the present invention relates to oligomeric RNA molecules capable of specifically binding and enhancing the expression and activity of microRNAs and uses thereof.

Background technique

A mature microRNA (miRNA) is a small RNA of about 22 nucleotides (nt) in length (e.g., about 18-25 nucleotides) that has a variety of important regulatory roles in the cell. miRNAs are widely found in eukaryotes, and they function in conjunction with mRNA molecules that are complementary to their sequences (eg, leading to gene silencing), an important strategy for regulating gene expression. The results show that miRNAs participate in the regulation of life activities such as development, apoptosis, proliferation and differentiation, and can play a role in the diagnosis and treatment of diseases, especially cancer.

Summary of the invention

In some embodiments, provided herein is a single-stranded RNA sequence that binds to a target microRNA, preferably the single-stranded RNA sequence enhances the activity of a target microRNA (referred to herein as a miRancer molecule) having 5 An 'end arm> and a 3' end arm, the 5' end arm binding to a target microRNA by base complementary pairing, the 3' end arm binding to the 5' end arm by base complementary pairing,

The single-stranded RNA sequence has the following formula 1 from the 5' end to the 3' end:

(n _5-11 )(n _0-3 )(n _6-7 )(n ₁ )(n _11-21 )(n _0-2 ),

Wherein n is a contiguous nucleotide or an analog thereof, and the number thereafter is the number of nucleotides or the like, and each n may be independently selected from the following nucleotides: A, U, G, and C. Or a nucleotide analog thereof, wherein n _{1 is} complementary to the nucleotide at the first position of the 5' end of the target microRNA, and the corresponding n _6-7 corresponds to the second to seventh of the target microRNA from the 5' end to the 3' end Bit or position 2-8, such that (n _5-11 )(n _0-3 )(n _6-7 ) is the 5' end arm and (n _11-21 )(n _0-2 ) is the 3' end arm Where n _0-3 is the inserted C or G to form a GC pair between the 5' end arm and the 3' end arm.

In some embodiments, the single-stranded RNA molecules provided herein are capable of binding to a target microRNA and enhancing the activity of the microRNA. The inventors referred to the RNA molecule capable of enhancing microRNA activity as a microRNA enhancer, referred to as miRancer. The enhanced microRNA activity can include, for example, one or more of stabilizing the microRNA, extending the half-life of the microRNA, altering expression of a target gene regulated by the microRNA, and the like. Methods for measuring the expression of target genes regulated by microRNAs and microRNAs are known in the art.

In some embodiments, the 5' end arm and the target microRNA are from the 5' end to the 3' end in addition to the inserted ( _n0-3 ) as a gap in the RNA molecule described herein. At least 50%, 60%, 70%, 80%, 90%, or 100% identity of the complementary strand of nucleotides 2-18, preferably wherein (n6-7) and the target microRNA are from the 5' end The complementary strand of nucleotides 2-7 or 2-8 of the 3' end has at least 80%, 90%, or 100% identity, preferably (n _11-21 ) and (n _5-11 ) ( The number of unpaired nucleotides in n _0-3 )(n _6-7 ) is less than 6, 5, 4, 3, 2, or 1 pair, preferably all unpaired nucleotides are around (n ₁ ). In some embodiments, (n _5-11 )(n _0-3 )(n _6-7 ) and the target microRNA are at least 50%, 60 from nucleotides 2-18 of the 5' end to the 3' end. %, 70%, 80%, 90%, 95% or 100% complementary. In some embodiments, (n6-7) and the target microRNA are at least 80%, 90%, 95%, 99% from positions 5-7 or 2-8 of the 5' end to the 3' end. Or 100% complementary. In some embodiments, (n _11-21 ) and (n _5-11 )(n _0-3 )(n _6-7 ) are at least 50%, 60%, 70%, 80%, 90%, 95% or 100% complementary.

In some embodiments, provided herein is a vector, such as a plasmid, comprising the single-stranded RNA sequence.

In some embodiments, provided herein is a vector, such as a plasmid, comprising a polynucleotide sequence of the single-stranded RNA sequence.

In some embodiments, provided herein is a synthetic or recombinant RNA molecule of the single-stranded RNA sequence.

In some embodiments, provided herein is a cell comprising the vector.

In some embodiments, provided herein, the single-stranded RNA sequence, the vector, or the synthetic RNA molecule is used to specifically increase expression of an endogenous and/or exogenous target microRNA or other small RNA and/ Or the method of use and/or use of the activity.

In some embodiments, provided herein, the single-stranded RNA sequence, the vector, or the synthetic RNA molecule is used to specifically modulate (eg, increase) expression of a gene targeted by the target microRNA in vitro and/or in vivo. Use and / or use. In some embodiments, provided herein, the single-stranded RNA sequence can be specifically regulated (eg, increased) by integration into a region of the genome that regulates microRNA-targeted gene expression, including, for example, the 5'UTR region of the gene. Expression of a gene targeted by the target microRNA.

In some embodiments, provided herein are methods and/or uses of the single-stranded RNA sequence, the vector, or the synthetic RNA molecule for sensing the target microRNA or other small RNA.

In some embodiments, provided herein, the single-stranded RNA sequence, the vector, or the synthetic RNA molecule is used to sense the target microRNA or other small RNA to modulate the target microRNA or other small Use of expression of RNA-targeted genes.

In some embodiments, provided herein is a method comprising a method for specifically increasing the expression and/or activity of an endogenous and/or exogenous target microRNA or other small RNA for use in vitro and/or Or a method of specifically modulating expression of a gene targeted by said target microRNA in vivo, for use as a sensor for sensing said target microRNA or other small RNA, and for sensing said target microRNA or other small RNA Thereby modulating any one or more of the expression methods of the target microRNA or other small RNA-targeted gene, the method comprising providing the single-stranded RNA sequence, the vector, or the synthesis provided herein The step of contacting the RNA molecule with a sample (eg, a sample of cells, blood, tissue, etc.) containing the target microRNA or other small RNA. In some embodiments, the single-stranded RNA sequence, the vector, or the synthetic RNA molecule is introduced into a cell of interest. In some embodiments, the target microRNA or other small RNA is sensed by interacting with a single-stranded RNA sequence, the vector, or the synthetic RNA molecule, with a target microRNA released after cell lysis, and can be used The presence of the microRNA in the sample is detected.

In some embodiments, provided herein is a method of producing a single-stranded RNA sequence that binds to a target microRNA, the method comprising selecting a target microRNA (eg, selecting a corresponding microRNA according to a gene of interest desired to be modulated), and then generating the single sheet provided herein A stranded RNA sequence, the vector, or the synthetic RNA molecule.

In some embodiments, provided herein is a composition comprising the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule, preferably the composition can be used for the purposes described herein and method. In some embodiments, provided herein is a kit comprising the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule, and instructions for use, preferably the kit can be used herein The purpose and method of description. In some embodiments, the kit includes various reagents such as buffers and the like suitable for storing the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule. In some embodiments, the kit includes various reagents, such as enzymes, transformation or transfection reagents, suitable for performing the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule to react with a target microRNA, . In some embodiments, the kit comprises, by the single-stranded RNA sequence, the vector, and/or the synthetic RNA molecule, modulating expression of a gene of interest by reacting with a target microRNA (eg, enhancing expression of a gene of interest) ) Reagents.

In some embodiments, provided herein are isolated or recombinant polynucleotides that are capable of being transcribed into the single-stranded RNA sequence, wherein the polynucleotide comprises the single-stranded RNA expression vector, wherein the expression vector comprises the encoding DNA sequence of single-stranded RNA.

In some embodiments, provided herein is a method of binding to a target microRNA and enhancing the activity of the microRNA, the method comprising introducing the single-stranded RNA sequence, the vector, or the synthetic RNA molecule into a target cell step. The enhanced microRNA activity can include, for example, one or more of stabilizing the microRNA, extending the half-life of the microRNA, altering expression of a target gene regulated by the microRNA, and the like.

DRAWINGS

Figure 1: Different forms of the miR-7 binding sequence.

Figure 2: Different forms of the miR-9 binding sequence.

Figure 3: Schematic representation of the miR-7 reporter and miR-9 reporter.

Figure 4: Luciferase activity of miR-7 reporter or miR-9 reporter after overexpression of miRancers. *, p < 0.05, **, p < 0.01 by Student's t-test.

Figure 5: Schematic representation of the miR-7 binding sequence for different stem lengths.

Figure 6: Luciferase activity of miR-7 reporter after overexpression of miRancers with different stem lengths **, p<0.01 by Student's t-test.

Figure 7: Luciferase activity of miR-7 reporter or miR-9 reporter after overexpression of miRancer. **, p<0.01 by Student's t-test.

Figure 8: Expression levels of miR-7 or miR-9 after overexpression of miRancer. ***, p < 0.001 by Student's t test.

Figure 9: Schematic representation of a luciferase reporter vector containing a miRancer or sponge sequence.

Figure 10: Effect of overexpression of miR-7 or miR-9 on different luciferase activities. *, p < 0.05, **, p < 0.01, ***, p > 0.001 by Student's t test.

detailed description

The invention now will be described more fully hereinafter with reference to the appended claims In fact, the invention may be embodied in many different forms and should not be construed as being limited to the embodiments shown herein.

1.microRNA enhancer

In some embodiments, the single-stranded RNA molecule provided herein is capable of binding to a target microRNA and enhancing the activity of the microRNA, such an RNA molecule that enhances microRNA activity is referred to herein as a microRNA enhancer, referred to as a microRNA enhancer, miRancer. In some embodiments, enhancing microRNA activity can include, for example, stabilizing the microRNA, extending the half-life of the microRNA, altering (eg, increasing or decreasing) one or more of aspects of target gene expression regulated by the microRNA. Methods for measuring the half-life of microRNAs and expression of target genes are known in the art, see, for example, the methods exemplified in the Examples herein.

In some embodiments, the miRancer is a single-stranded RNA sequence capable of binding to a target microRNA, the single-stranded RNA sequence having a 5'-end and a 3'-arm, the 5'-arm binding to the target microRNA via base-pair pairing The 3' end arm binds to the 5' end arm by a base complementary pairing.

Herein, unless otherwise explicitly stated, the order of nucleotide sequences refers to the order from the 5' end to the 3' end.

In some embodiments, a single-stranded RNA sequence, such as a miRancer molecule, comprises or consists of a molecule of Formula 1 below from the 5' end to the 3' end:

(n _5-11 )(n _0-3 )(n _6-7 )(n ₁ )(n _11-21 )(n _0-2 ),

Wherein n is a contiguous nucleotide or analog thereof, and the number thereafter is the number of nucleotides or analogs thereof, wherein n _{1 is} the nucleotide of the first position at the 5' end of the target microRNA (usually U) Complementary, the corresponding n _6-7 corresponds to position 2-7 or position 2-8 of the target microRNA from the 5' end to the 3' end (referred to as "seed sequence"), thereby (n _5-11 ) (n _{0 -3} ) (n _6-7 ) is a 5' end arm, (n _11-21 ) (n _0-2 ) is a 3' end arm, where n _0-3 is the inserted C or G to the 5' A GC pair is formed between the end arm and the 3' end arm, and (n _0-2 ) may be a 3' end overhang, which may be, for example, a UU.

In some embodiments, (n ₁ ) is A. In some embodiments, wherein the 5' end arm and the target microRNA are from the 5' end to the 3' end of the 2-18th nucleus, except that the inserted ( _n0-3 ) is not calculated as a gap At least 50%, 60%, 70%, 80%, 90%, or 100% of the nucleotides are complementary, for example, the 5' end arm of the miRancer molecule and the 2-18 of the microRNA molecule from 5' to 3' The bit sequence (or, for example, the 2-17th, 2-16th, 2-15th, 2-14th sequence) is 100%, about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92 %, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% complementary. In some embodiments, preferably wherein (n _6-7 ) is 100%, about 99%, 98%, 97% from the 2-7th or 2-8 sequence of the 5' to 3' of the target microRNA molecule. , 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80 %, 75%, 70%, 65%, 60%, 55%, or 50% complementary. In some embodiments, 100%, about 99%, 98%, 97%, 96%, 95 between (n _5-11 )(n _0-3 )(n _6-7 ) and (n _11-21 ) %, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% complementary, forming a stem structure. In some embodiments, it is preferred that the number of unpaired nucleotides in (n _11-21 ) and (n _5-11 )(n _0-3 )(n _6-7 ) is less than 6, 5, 4, 3, 2, or 1 pair, preferably all unpaired nucleotides form a ring structure around (n ₁ ). In some embodiments, the stem structure is 10-18 pairs of paired nucleotides in length, including, for example, 10 pairs, 11 pairs, 12 pairs, 13 pairs, 14 pairs, 15 pairs, 16 pairs, 17 pairs, 18 pairs. Paired nucleotides. In some embodiments, the loop structure comprises 1-13 nucleotides, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleosides acid. In some embodiments, (n _0-2 ) can be a 3' end overhang, which can be, for example, a UU. In some embodiments, ₁ , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleosides immediately adjacent to (n ₁ ) (including (n ₁ )) The acid forms a loop; for example, each of the 5' end and the 3' end of (n ₁ ) has 1, 2, 3, 4, 5 or 6 nucleotides forming a 5' end and a 3' end arm, said 5' The ends and the 3' end arms are not complementary to form a loop. In some embodiments, the (n ₁₎ of the 5 'and 3' ends each having 5 or 6 nucleotides to form a 5 'end and 3' end of the arm, the 5 ' The ends are not complementary to the 3' end arms, thereby forming a loop, and at least 50%, 60%, 70%, 80%, 90%, or 100% complement each other without forming a loop, forming a stem. In some embodiments, the (n ₁₎ of the 5 'and 3' ends each having 5 or 6 nucleotides to form a 5 'end and 3' end of the arm, the 5 ' The ends and the 3' end arms are not complementary to form a loop, and the remaining nucleotides that do not form a loop are 100% complementary to form a stem.

In some embodiments, each n of the nucleotide sequences described herein are independently of one another selected from the group consisting of: A, U, T, G, and C, or nucleotide analogs thereof, eg, if The sequences are RNA sequences, each n being independently of one another selected from the group consisting of: A, U, G and C, or nucleotide analogs thereof.

In some embodiments, (n ₁ ) is A. In some embodiments, (n _6-7 ) is fully complementary to positions 2-7 or 2-8 of the microRNA, and (n _0-3 ) is inserted cytidine (C) or guanosine (G). The corresponding position in the corresponding (n _11-21 ) sequence is a complementary guanosine (G) or cytidine (C) analog thereof to the 5' end arm and the 3' end arm The GC pairing is formed, and the number of the pairings may be 0, 1, 2, and 3, and the inserted C is not counted as a gap when calculating the complementarity between the microRNA and the miRancer. In some embodiments, (n _5-11 ) is 5, 6, 7, 8, 9, 10, or 11 contiguous nucleotides. In some embodiments, (n _6-7 ) is 6 or 7 contiguous nucleotides. In some embodiments, (n _11-21 ) is 16, 17, or 18 contiguous nucleotides, wherein the number of unpaired nucleotides is less than 6, 5, 4, 3, 2, or 1 pair, preferably all Unpaired nucleotides form a loop structure around (n ₁ ); in some embodiments, the stem structure is 10-18 pairs of paired nucleotides in length, including, for example, 10 pairs, 11 pairs, 12 pairs, 13 pairs, 14 pairs, 15 pairs, 16 pairs, 17 pairs, 18 pairs of paired nucleotides; the loop structure comprises 1-13 nucleotides including, for example, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13 nucleotides. In some embodiments, (n _0-2 ) can be a 3' end overhang, which can be, for example, a UU. In some embodiments, the 3 'end overhang can, for example, facilitate binding of Ago2. In some embodiments, the miRancer molecule can further comprise a sequence that promotes binding of Ago2 at the 3' end, such as an HDV ribozyme, which is capable of promoting the removal of additional sequences of the 3' segment of the RNA molecule, resulting in a short 3' end overhang, thereby The binding of RNA molecules to Ago2 is facilitated (see, for example, Renfu Shang et al, NATURE COMMUNICATIONS, 6: 8430, which is incorporated herein by reference in its entirety).

In some embodiments, a single-stranded RNA sequence, such as a miRancer molecule, comprises or consists of a molecule of Formula 2 below from the 5' end to the 3' end:

(n _7-9 )(n ₂ )(n ₇ )(n ₁ )(n _16-18 ),

Wherein n is a contiguous nucleotide or analog thereof, and the number thereafter is the number of nucleotides or analogs thereof, wherein n _{1 is} the nucleotide of the first position at the 5' end of the target microRNA (usually U) Complementary, corresponding n ₇ corresponds to position 2-8 of the target microRNA from the 5' end to the 3' end, wherein n ₂ is an inserted C or G to form a GC between the 5' end arm and the 3' end arm pair. In some embodiments, (n ₇ ) is at least 50%, 60%, 70%, 80%, 90%, or 100% complementary to the 2nd to 8th positions of the target microRNA from the 5' end to the 3' end. In some embodiments, (n ₇₎ with a target microRNA from 5 'to 3' end of the fully complementary to 2-8. In some embodiments, ₁ , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleosides immediately adjacent to (n ₁ ) (including (n ₁ )) The acid forms a loop; for example, each of the 5' end and the 3' end of (n ₁ ) has 1, 2, 3, 4, 5 or 6 nucleotides forming a 5' end and a 3' end arm, said 5' The ends and the 3' end arms are not complementary to form a loop. In some embodiments, the (n ₁₎ of the 5 'and 3' ends each having 5 or 6 nucleotides to form a 5 'end and 3' end of the arm, the 5 ' The ends are not complementary to the 3' end arms, thereby forming a loop, and at least 50%, 60%, 70%, 80%, 90%, or 100% complement each other without forming a loop, forming a stem. In some embodiments, the (n ₁₎ of the 5 'and 3' ends each having 5 or 6 nucleotides to form a 5 'end and 3' end of the arm, the 5 ' The ends and the 3' end arms are not complementary to form a loop, and the remaining nucleotides that do not form a loop are 100% complementary to form a stem.

The miRancer provided herein can be an oligonucleotide. The term "oligonucleotide" refers to a molecule formed by covalently linking two or more nucleotides. The term oligonucleotide typically includes oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, and chimeric combinations of these. When referring to a nucleotide or monomer sequence, it may be a base sequence such as, for example, the sequence of A, T (or U), G or C or an analog thereof.

As used herein, "nucleotide" as used herein refers to a moiety comprising a sugar moiety, a base moiety, and a glycoside of a covalently linked group (such as a phosphate or phosphorothioate internucleotide linking group) and includes a naturally occurring nucleotide (such as DNA or RNA) and a modified sugar and/or base moiety Non-naturally occurring nucleotides, which are also referred to herein as "nucleotide analogs." Non-naturally occurring nucleotides include nucleotides having a modified sugar moiety (e.g., a bicyclic nucleotide or a 2&apos; modified nucleotide, such as a 2&apos; substituted nucleotide). A "nucleotide analog" is a variant produced by a naturally occurring nucleotide (such as a DNA or RNA nucleotide) using modifications in the sugar and/or base moiety. Analogs may have no functional or functional impact on the oligonucleotide. For example, by increasing the binding affinity for the target and/or increased resistance to intracellular nucleases and/or increased ease of transport into the cell. In some embodiments, the miRancer molecule comprises 1, 2, 3 or more nucleotide analogs. In some embodiments, the oligomer comprises 3-8 nucleotide analogs, such as 6 or 7 nucleotide analogs. In some embodiments, the nucleotide analog comprises a locked nucleic acid (LNA). For example, one, two, three or more nucleotide analogs of the nucleotide analogs can be LNA.

A modified RNA molecule as defined herein may contain a nucleotide analog/modification, such as a backbone modification, a sugar modification, or a base modification. The backbone modification may be the chemistry of the phosphate ester of the nucleotide backbone contained in the nucleic acid molecule. The phosphate group of the modified modified backbone can be modified by replacing one or more oxygen atoms with different substituents, examples of which include, for example, phosphorothioates. The sugar modification can be a chemical modification of the sugar of the nucleotide of the nucleic acid molecule, for example, the 2' hydroxyl (OH) can be modified or replaced by a number of different "oxy" or "deoxy" substituents. The base modification can be a chemical modification of the base portion of the nucleotide of the nucleic acid molecule. The nucleotide analog or modification may be selected from nucleotide analogs suitable for transcription and/or translation. Nucleotide analogs/modifications may include 2-amino-6-chloropurine nucleoside-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2'-fluorothymidine- 5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 7-deazacyosine-5'-triphosphate, and Pseudouridine-5'-triphosphate and the like. Modified nucleosides may include pyridin-4-one ribonucleoside, 5-aza-uridine, dihydro pseudouridine, 2-thio-dihydrouridine, 2,6-diaminopurine, N2, N2 -Dimethylguanosine, N1-methyl-pseudouridine, 5,6-dihydrouridine, 4-thio-uridine, 5-hydroxy-uridine, deoxy-thymidine, inosine, α- Thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino -6-chloro-indole, pseudo-cytidine, 6-chloro-indole, and the like.

The miRancer molecules provided herein may be included or consist of a total length of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 consecutive nucleotide sequences composition. In some embodiments, the oligonucleotide comprises or consists of a total of about 30-45, such as about 33-40, such as about 33-37, such as about 33-35 contiguous nucleotide sequences in length. . In some embodiments, the oligonucleotides of the invention consist of no more than 40 nucleotides, such as no more than 38 nucleotides, such as no more than 34 nucleotides, such as 33, 35 or 37 Nucleotide.

2. Composition

Provided herein are methods and compositions comprising a miRancer that enhances microRNA activity when expressed in a cell. Such methods and compositions comprise a miRancer expression vector. "miRancer expression vector" refers to a vector comprising a miRancer molecule having a polynucleotide sequence encoding miRancer. The miRancer expression vector is designed to produce a miRancer molecule from the vector.

"MicroRNA" or "miRNA" refers to an oligoribonucleic acid, generally ranging from about 18 to about 25 nucleotides in length, which regulates the expression of a polynucleotide comprising a target sequence. MicroRNAs are non-protein-encoding RNAs that have been identified in animals and plants. The microRNA is initially transcribed into long polyadenylated RNA which is then processed to form a shorter sequence with the ability to form a stable hairpin. Most microRNA genes synthesize pri-miRNAs under the action of RNA polymerase II. In the nucleus, the pri-miRNA is cleaved by the Drosha enzyme to form a stem-loop structure of about 70 nt, that is, a pre-miRNA. Subsequently, the pre-miRNA is cleaved by the Dicer enzyme to produce a microRNA single-stranded structure that forms a mature microRNA.

3.miRancer expression vector

The miRancer expression vector is provided herein. The miRancer expression vector expression vector comprises a polynucleotide that can be transcribed into a miRancer sequence that enhances microRNA activity.

In some embodiments, the miRancer expression vector provides for formation with a similar hair The miRancer molecule that sandwiches the RNA structure. In some embodiments, the miRancer molecule can stabilize a microRNA molecule to modulate the expression activity of the microRNA targeting gene. The miRNA can be derived from any animal or plant.

In some embodiments, the 5' end arm sequence of miRancer and the contiguous nucleotide sequence of the miRNA sequence starting from position 2 can be 100%, at least 99%, 98%, 97%, 96%, 95%, 90% , 85%, 80% or less complementary sequences. In an embodiment, the 5' end-arm sequence of miRancer comprises a sequence that has 1, 2, 3, 4, 5 or more mismatches with a contiguous nucleotide sequence starting from position 2 of the miRNA sequence, and is still sufficient The complement forms a double-stranded structure with the miRNA sequence, generates a miRNA binding sequence and enhances the activity of the microRNA.

The vector miRancer molecule is expressed by miRancer and has a sequence that is sufficiently complementary to the microRNA. A "sufficiently complementary sequence" to a microRNA target sequence means that its complementarity is sufficient to allow the miRancer molecule to bind to the microRNA and increase the activity of the microRNA. In a specific embodiment, the miRancer having sufficient complementarity to the target sequence is 100% complementary to the microRNA target sequence or may be less than 100% complementary to the target sequence (ie, at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or less complementary sequences). In other embodiments, the miRancer and the target sequence can have 1, 2, 3, 4, 5 or up to 6 mismatches as long as the miRancer is sufficiently complementary to the target sequence to increase the activity of the target sequence, such as half-life.

In some embodiments, the miRNA sequence can have a "U" at the 5' end. Optionally, a pair of base pair changes can be added within the 5' end of the miRNA such that the sequence differs from the target sequence by one nucleotide.

"Target sequence" refers to the sequence of the microRNA targeted by the miRancer. The target sequence may be an endogenous sequence or may be an introduced heterologous sequence.

The miRancer produced by the miRancer expression vector is capable of increasing the activity of the microRNA. Methods for determining the activity of a microRNA include measuring changes in the expression of a gene/protein that it targets. In some embodiments, a single miRNA can silence a plurality of proteins/genes or whole proteins and/or gene families in a protein and/or gene family.

"Increase" means normal levels of microRNA relative to wild-type organisms (or Increase in microRNA-targeted gene/protein levels). By "increasing miRancer activity" it can be an increase in expression activity to any statistically significant amount, for example an increase of at least 10%, 15%, 20%, 25%, 30%, 35%, 40 relative to wild-type expression activity. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

The miRancer molecules described herein can be delivered to one or more of an animal such as a mammal, including a human or a plant cell.

4. Polynucleotide encoding miRancer expression vector and preparation method thereof

Provided herein are isolated or recombinant polynucleotides encoding various components of a miRancer expression vector, a miRancer expression vector, together with a different product of a miRancer expression vector processed into a miRancer.

Polynucleotides can be polymers of RNA or DNA, which can be single or double stranded, optionally comprising synthetic, non-natural or modified nucleotide bases. A polynucleotide in the form of a DNA polymer can be composed of one or more fragments of cDNA, genomic DNA, synthetic DNA, or a mixture thereof. Polynucleotides can include ribonucleotides as well as combinations of ribonucleotides and deoxyribonucleotides. The deoxynucleotides and ribonucleotides include naturally occurring molecules and synthetic analogs. Polynucleotides of the invention also encompass all forms of sequences including, but not limited to, single-stranded forms, double-stranded forms, hairpin structures, stem-loop structures, and the like.

The compositions provided herein can comprise isolated or substantially purified polynucleotides. An "isolated" or "purified" polynucleotide is substantially or essentially free of those free components that normally accompany or interact with a polynucleotide in a naturally occurring environment. Thus, an isolated or purified polynucleotide is substantially free of other cellular material, or a culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In some embodiments, the "isolated" polynucleotide is naturally ubiquitously present on the genomic DNA of the organism from which the polynucleotide is derived (ie, located 5' of the polynucleotide and The sequence of the 3' end).

Recombinant polynucleotides comprising a miRancer expression vector and different components thereof are also provided. A recombinant vector comprises a combination of artificial or heterologous nucleic acid sequences, such as non-naturally coexisting regulatory and coding sequences. In other embodiments, the recombinant vector may comprise regulatory sequences derived from different sources And coding sequences, or regulatory sequences and coding sequences derived from the same source but arranged in a manner different from the naturally occurring manner. The carrier can be used alone or in combination with a carrier. If a vector is used, the choice of vector will depend on the method used to transform the host cell. For example, a plasmid vector can be used. In order to successfully transform, screen and propagate a host cell comprising any of the isolated nucleic acid fragments of the invention, a genetic element that can be included on the vector. Therefore, in order to obtain a cell line showing a desired expression level and pattern, screening can be performed by Southern blot analysis, Northern blot analysis, immunoblot analysis of protein expression, phenotypic analysis, and the like.

In a specific embodiment, one or more miRancer expression vectors described herein can be expressed in different cell types in an expression cassette format. The cassette can include 5' and 3' regulatory sequences operably linked to the polynucleotides provided herein. In the 5'-3' direction of transcription, the expression cassette can include a transcriptional and translational initiation region (ie, a promoter), a recombinant polynucleotide provided herein, and a transcriptional and translational termination region (ie, a termination region).

A number of promoters are available for the miRancer expression vectors provided herein. The timing, location and/or level of miRancer expression can be modulated by the use of different promoters in the miRancer expression vector. If desired, the miRancer expression vector may contain a promoter regulatory region (eg, a regulatory region that confers inducible, constitutive, environmental or developmental regulation, or cell or tissue specific/selective expression), a transcriptional initiation site, ribose A body binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.

5. Import method

The methods provided herein comprise introducing a miRancer expression vector into a cell to increase the activity of the target microRNA. The methods provided herein are limited to a particular method as long as the polynucleotide is introduced into the interior of at least one cell of the host. Methods for introducing polynucleotides into host cells are known in the art and include, but are not limited to, virus-mediated methods. Introduction includes the integration of a nucleic acid into a eukaryotic or prokaryotic cell in which the nucleic acid can be integrated into the genome of the cell and includes providing the nucleic acid or protein to the cell. Transformation protocols and protocols for introducing polynucleotide sequences into plants can vary depending on the type of cell being transformed.

6. How to use

A method of increasing microRNA activity by introducing a miRancer expression vector into a cell is provided. The methods provided herein comprise introducing a miRancer expression vector into a cell, wherein the miRancer molecule produced by the miRancer expression vector increases microRNA and/or alters (eg, increases or decreases) the activity of the microRNA-targeted gene. In some embodiments, a miRancer molecule can be integrated into a regulatory region of a gene targeted by a target microRNA in the genome (including, for example, 5'UTR and/or 3'UTR and/or regions upstream, downstream, intron, etc.) Regulate the expression of genes. In some embodiments, the miRancer molecule provided herein is capable of increasing gene expression of a microRNA-targeted gene, which is very surprising, as the role of the microRNA is generally believed to reduce gene expression. In some embodiments, the miRancer molecules provided herein are capable of altering the effect of a target microRNA (eg, the target microRNA reduces expression of some of the genes, and then, by introducing a miRancer molecule or vector provided herein into the regulatory region of the gene, such that microRNA increases gene expression). In some embodiments, the expression of a gene of interest desired to alter its expression can be modulated by the corresponding microRNA encoding the corresponding microRNA of the gene of interest desired to be regulated.

7. Active variants

Also provided herein are active variants of polynucleotides for use in the compositions and methods. "Variant" refers to a substantially similar sequence. In the case of a polynucleotide, a variant includes deletion and/or insertion of one or more nucleotides at one or more internal sites of the polynucleotide, and/or one or more positions in the polynucleotide. A dot has one or more nucleotide substitutions. Variant polynucleotides can include polynucleotides of synthetic origin, such as those produced, for example, by site-directed mutagenesis. Generally, the miRancer expression vector, single-stranded RNA molecule, miRancer molecule disclosed herein has at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 with the prototype polynucleotide. Sequence identity of %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. Sequence alignment methods for alignment are well known in the art. Procedures for aligning sequences and determining sequence identity include CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. You can use the default parameters to make an alignment using these programs.

8. Target microRNA molecule

In some embodiments, the target microRNA to which miRancer is capable of acting is not specifically limited. For example, a target microRNA molecule that is desired to be modulated can be selected from an existing database. In some embodiments, the corresponding microRNA molecule can be selected based on the gene that is desired to be modulated. In addition, genes targeted by microRNAs can be searched for by bioinformatics methods (eg, using miRanda, TargetScan, RNAhybrid, etc.).

The miRancer molecules herein can increase the activity of the microRNA by binding to a target microRNA molecule. In some embodiments, when hybridized to a target microRNA molecule, the oligonucleotide can tolerate 1, 2, 3 or 4 (or more) mismatches and still fully bind to the target, Shows the desired effect (eg, increases the activity of the microRNA). For example, a mismatch can be compensated for by an increased length oligonucleotide sequence and/or an increased number of nucleotide analogs (such as locked nucleic acids (LNA)) present within the nucleotide sequence. In some embodiments, the contiguous nucleotide sequence comprises no more than 3 mismatches (eg, no more than 1 or no more than 2 mismatches) when hybridized to a target microRNA molecule. In some embodiments, the contiguous nucleotide sequence comprises no more than one mismatch when hybridized to a target microRNA molecule.

The miRancer molecule of the invention preferably is at least 80% complementary to the microRNA molecule in the complementary region, 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%, at least 99%, such as at least 100% complementary.

In some embodiments, the miRancer may comprise additional 5' or 3' nucleotides or modifications, such as, independently, 1, 2, 3, 4 or 5 additional nucleotides 5' and/or 3' It is not complementary to the target sequence. In this aspect, in some embodiments, an oligonucleotide of the invention may comprise a contiguous nucleotide sequence joined to another nucleotide at the 5&apos; and or 3&apos; side. In some embodiments, the additional 5&apos; or 3&apos; nucleotide is a naturally occurring nucleotide, such as DNA or RNA. In some embodiments, the additional 5&apos; or 3&apos; nucleotide can be a nucleotide analog. The complementary region may include positions 2-12, 2-13, 2-14, 2-15, 2-16, 2-17 of the microRNA molecule, 2-18 digits.

9. Pharmaceutical composition

The miRancer molecules, vectors, and cells of the present invention can be used in pharmaceutical preparations and compositions, and can also be prepared into kits for convenient use. Suitably, the composition or kit comprises a pharmaceutically acceptable solvent such as water or saline, a diluent, a carrier, a salt or an adjuvant.

The invention also encompasses pharmaceutical compositions and formulations comprising the oligonucleotides of the invention. The pharmaceutical compositions of the invention may be administered in a variety of ways, depending on whether local or systemic treatment is desired, and depending on the area to be treated.

10. Application

The oligonucleotides of the invention can be used as research reagents, for example, for diagnosis, treatment, and prevention. In the study, the oligonucleotides can be used to specifically bind to a target microRNA, which can increase its activity, thereby facilitating functional analysis of the target or its assessment as a target for therapeutic intervention.

In diagnostic agents, the oligonucleotides can be used to detect and quantify target microRNA levels in tissues in a cell by Northern blotting, in situ hybridization, or similar techniques.

For a therapeutic agent, an animal or human suspected of having a disease or condition that can be treated by modulation of the expression of a gene targeted by the target microRNA can be treated by administering an oligonucleotide of the invention. Further provided is a method of treating a mammal (eg, a human) suspected of having or prone to have a disease or condition associated with expression of a gene targeted by a target microRNA, by administering a therapeutically or prophylactically effective amount of one or A variety of oligonucleotides or compositions of the invention. Oligonucleotides or pharmaceutical compositions of the invention are typically administered in an effective amount.

The invention also provides a method for treating a disease, such as a tumor, comprising administering to a patient in need thereof an oligonucleotide molecule described herein or a pharmaceutical composition comprising the molecule.

11. Experimental results

Reagents and materials

The cell line used in the following experiments was the MCF-7 human breast cancer cell line, purchased from ATCC, the medium was RPMI-1640, purchased from Gibco, and the fetal bovine serum FBS was purchased from Gibco. The transfection reagent was lipofectamine 2000, purchased from invitrogen. Plasmid pSilencer 4.1CMV was purchased from Ambion, and plasmid psiCheck2 and dual luciferase assay kits were purchased from promega. Primer synthesis was completed by Shanghai Biotech, and mimics synthesis was completed by Shanghai Jima.

The recombinant plasmid cloning method is a conventional method, and the sequencing is completed by Shanghai Biotech, and the details are explained below.

The dual luciferase reporter assay was performed according to the kit (Promega E1910) instructions.

experiment procedure

1. Design microRNA binding sequences with advanced structure.

First, the binding sequence of the microRNA is designed to have a single-stranded RNA with a similar hairpin structure. The 5' end arm of the hairpin structure binds to the microRNA through base complementary pairing, while the 3' end arm binds to the 5' end arm through base complementary pairing. . In order to prevent the processing of Dicer1 and the occurrence of siRNA-like effects, the preferred double strands are within 18 nucleotides in length.

Then, a 7 nt or 13 nt ring structure is introduced at the corner of the hairpin structure to determine the potential functions that different structures may bring.

Thereafter, two pairs of GCs are inserted in the middle of the hairpin loop structure to stabilize the RNA secondary structure.

Finally, a linear microRNA binding sequence was also designed as a negative control.

Thus, eight different forms of exemplary microRNA binding sequences were obtained (exemplified by miR-7 and miR-9). Figure 1 shows the different forms of the miR-7 binding sequence; Figure 2 shows the different forms of the miR-9 binding sequence.

2. Preparation of an expression vector.

The pSilencer 4.1CMV plasmid was used as an expression vector for small hairpin RNA. Plasmid passage After double digestion with BamHI and HindIII, they were separated by 1% agarose gel, then recovered by Axyprep DNA extraction kit (AP-GX-50) and stored at -20 °C.

3. Prepare the insert sequence.

The following DNA sequences were synthesized:

miR-7 binding sequence A positive:

GATCCAAAATCACTAGTCTTCCAGGAAGACTAGTGATTTTA

miR-7 binding sequence A reverse:

AGCTTAAAATCACTAGTCTTCCTGGAAGACTAGTGATTTTG

miR-7 binding sequence B forward:

GATCCAAAATCACTAGTCTTCCACCTAGACTAGTGATTTA

miR-7 binding sequence B reverse:

AGCTTAAAATCACTAGTCTAGGTGGAAGACTAGTGATTTTG

miR-7 binding sequence C positive:

GATCCAAAATCACTAGTCTTCCACCTTCTCTAGTGATTTTA

miR-7 binding sequence C reverse:

AGCTTAAAATCACTAGAGAAGGTGGAAGACTAGTGATTTTG

miR-7 binding sequence D positive:

GATCCAAAATCACTAGTCTTCCAA

miR-7 binding sequence D reverse:

AGCTTTGGAAGACTAGTGATTTTG

miR-7 binding sequence E positive:

GATCCAATCACTACCGTCTTCCAGGAAGACGGTAGTGATTA

miR-7 binding sequence E reverse:

AGCTTAATCACTACCGTCTTCCTGGAAGACGGTAGTGATTG

miR-7 binding sequence F forward:

GATCCAATCACTACCGTCTTCCACCTAGACGGTAGTGATTA

miR-7 binding sequence F reverse:

AGCTTAATCACTACCGTCTAGGTGGAAGACGGTAGTGATTG

miR-7 binding sequence G positive:

GATCCAATCACTACCGTCTTCCACCTTCTCGGTAGTGATTA

miR-7 binding sequence G reverse:

AGCTTAATCACTACCGAGAAGGTGGAAGACGGTAGTGATTG

miR-7 binding sequence H positive:

GATCCAATCACTACCGTCTTCCAA

miR-7 binding sequence H reverse:

AGCTTTGGAAGACGGTAGTGATTG

miR-9 binding sequence A positive:

GATCCCAGCTAGATAACCAAAGACTTTGGTTATCTAGCTGA

miR-9 binding sequence A reverse:

AGCTTCAGCTAGATAACCAAAGTCTTTGGTTATCTAGCTGG

miR-9 binding sequence B forward:

GATCCCAGCTAGATAACCAAAGAGAATGGTTATCTAGCTGA

miR-9 binding sequence B reverse:

AGCTTCAGCTAGATAACCATTCTCTTTGGTTATCTAGCTGG

miR-9 binding sequence C positive:

GATCCCAGCTAGATAACCAAAGAGAAACCTTATCTAGCTGA

miR-9 binding sequence C reverse:

AGCTTCAGCTAGATAAGGTTTCTCTTTGGTTATCTAGCTGG

miR-9 binding sequence D positive:

GATCCCAGCTAGATAACCAAAGAA

miR-9 binding sequence D reverse:

AGCTTTCTTTGGTTATCTAGCTGG

miR-9 binding sequence E positive:

GATCCGCTAGATACCACCAAAGACTTTGGTGGTATCTAGCA

miR-9 binding sequence E reverse:

AGCTTGCTAGATACCACCAAAGTCTTTGGTGGTATCTAGCG

miR-9 binding sequence F forward:

GATCCGCTAGATACCACCAAAGAGAATGGTGGTATCTAGCA

miR-9 binding sequence F reverse:

AGCTTGCTAGATACCACCATTCTCTTTGGTGGTATCTAGCG

miR-9 binding sequence G positive:

GATCCGCTAGATACCACCAAAGAGAAACCTGGTATCTAGCA

miR-9 binding sequence G reverse:

AGCTTGCTAGATACCAGGTTTCTCTTTGGTGGTATCTAGCG

miR-9 binding sequence H positive:

GATCCGCTAGATACCACCAAAGAA

miR-9 binding sequence H reverse:

AGCTTTCTTTGGTGGTATCTAGCG

These DNA sequences were paired in pairs in a 1×TE buffer as follows:

95 ° C 2 min

Touch down at 0.1°Cevery 8s

4 ° C 30 min

The annealed DNA sequence was ligated between BamHI and HindIII of the pSilencer 4.1 CMV plasmid, and the kit used was a TAKARA ligation kit (D6022). The recombinant plasmid was sequenced to confirm that the sequence was correct.

4. Prepare a microRNA reporter vector.

A binding site for the microRNA was inserted between XhoI and NotI of the psi-CHECK2 plasmid to obtain a microRNA reporter vector. Figure 3 shows a schematic of the miR-7 reporter and miR-9 reporter.

5. Luciferase reporter experiment.

The miR-7 binding sequence and the miR-7 reporter were co-transfected into MCF-7 cells, and then a dual luciferase reporter assay (Promega E1910) was performed as described. Figure 4 shows luciferase activity of miR-7 reporter or miR-9 reporter after overexpression of miRancers. *, p<0.05, **, p<0.01 by Student's t-test.

Type F is selected as a preferred microRNA binding sequence that enhances microRNA activity. All sequences that enhance microRNA activity are called microRNA enhancers, referred to as miRancers.

6. Optimize the miRancer structure

Based on the F-type, we continued to explore the appropriate miRancer structure. By shortening or prolonging the structure of the miRancer Stem region, we obtained a hairpin structure containing 15 bp (miR-7 binding sequence F1) or 13 bp (miR-7 binding sequence F2). Show that both can still work. Figure 5 shows a schematic representation of the miR-7 binding sequence for different stem lengths. Figure 6 shows luciferase activity of miR-7 reporter after overexpression of miRancers of different stem lengths, p < 0.01 by Student's t-test.

7. Chemical synthesis of miRancers.

Furthermore, miRancer (Shanghai Jima) was synthesized by chemical method according to F type. The miRancer designed for the bantam miRNA in Drosophila was used as a control. The inventors have found that chemically synthesized miRancers can also specifically affect the activity and level of microRNAs. Figure 7 shows luciferase activity of miR-7 reporter or miR-9 reporter after overexpression of miRancer. **, p < 0.01 by Student's t-test. Figure 8 shows the expression levels of miR-7 or miR-9 after overexpression of miRancer. ***, p<0.001 by Student's t test.

8. miRancer motif enhances gene expression.

MiRancer-7, miR-7 sponge, miRancer-9, miR-9 sponge were inserted upstream of luciferase, and then co-transfected with miR-7 mimics, miR-9 mimics (Shanghai Jima), respectively, and luciferase containing miRancer was found. It can be stabilized by the corresponding miRNA, while the luciferase containing the sponge is inhibited by the corresponding miRNA. This suggests that miRancer can be used to enhance gene expression. Figure 9 shows a schematic representation of a luciferase reporter vector containing a miRancer or sponge sequence. Figure 10 shows the effect of overexpression of miR-7 or miR-9 on different luciferase activities. *, p < 0.05, **, p < 0.01, ***, p > 0.001 by Student's t test.

Claims (10)

A single-stranded RNA sequence that binds to a target microRNA having a 5' end arm and a 3' end arm, the 5' end arm binding to a target microRNA by base complementary pairing, the 3' end arm Binding to the 5' end arm by base complementary pairing, The single-stranded RNA sequence has the following formula 1 from the 5' end to the 3' end: (n _5-11 )(n _0-3 )(n _6-7 )(n ₁ )(n _11-21 )(n _0-2 ), Wherein n is a contiguous nucleotide or an analog thereof, and the number thereafter is the number of nucleotides or analogs thereof, wherein n _{1 is} complementary to the nucleotide at the first position of the 5' end of the target microRNA, and the corresponding n _6-7 corresponds to the 2-7th or 2-8th position of the target microRNA from the 5' end to the 3' end, such that (n _5-11 )(n _0-3 )(n _6-7 ) is 5' end The arm, (n _11-21 )(n _0-2 ) is the 3' end arm, where n _0-3 is the inserted C or G to form a GC pair between the 5' end arm and the 3' end arm. The single-stranded RNA sequence of claim 1, wherein the 5' end arm and the target microRNA are from the 5' end to the 3' end, except that the inserted ( _n0-3 ) is not calculated as a gap. At least 50%, 60%, 70%, 80%, 90%, or 100% identity of the complementary strand of nucleotides 2-18, preferably wherein (n6-7) and the target microRNA are from the 5' end The complementary strand of nucleotides 2-7 or 2-8 of the 3' end has at least 80%, 90%, or 100% identity, preferably (n _11-21 ) and (n _5-11 ) ( The number of unpaired nucleotides in n _0-3 )(n _6-7 ) is less than 6, 5, 4, 3, 2, or 1 pair, preferably all unpaired nucleotides are around (n ₁ ). A vector, such as a plasmid, comprising the single-stranded RNA sequence of claim 1 or 2. A vector comprising a polynucleotide sequence encoding the single-stranded RNA sequence of claim 1 or 2, such as a plasmid. A synthetic RNA molecule comprising the single-stranded RNA sequence of claim 1 or 2. A cell comprising the vector of claim 3 or 4. Use of the single-stranded RNA sequence of claim 1 or 2, the vector of claim 3 or 4, or the synthetic RNA molecule of claim 5, for use in specifically increasing endogenous and/or exogenous targets Expression and/or activity of a microRNA or other small RNA for the specific regulation of expression of a gene targeted by the target microRNA in vitro and/or in vivo, for use as a sensor for sensing the target microRNA or other small RNA, and Used to sense the target MicroRNAs or other small RNAs thereby modulate the expression of genes targeted by the target microRNA or other small RNA. A method comprising a method for specifically increasing the expression and/or activity of an endogenous and/or exogenous target microRNA or other small RNA for specific regulation of said target in vitro and/or in vivo A method of expressing a gene targeted by a microRNA, for use as a sensor for sensing a target microRNA or other small RNA, and for modulating the target microRNA by sensing the target microRNA or other small RNA Or a method for expressing a gene targeted by a small RNA, the method comprising: the single-stranded RNA sequence of claim 1 or 2, the vector of claim 3 or 4, or the synthetic RNA molecule of claim 5, and a target microRNA or The step of contacting other samples of small RNA. A method of producing a single-stranded RNA sequence that binds to a target microRNA, the method comprising producing the single-stranded RNA sequence of the binding target microRNA of claim 1 or 2. A composition or kit comprising the single-stranded RNA sequence of claim 1 or 2, the vector of claim 3 or 4, and/or the synthetic RNA molecule of claim 5, preferably the composition or kit Use in the use of claim 7 or the method of claim 8.

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