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WO2024260432A1 - Composant de cyclisation d'arn linéaire et son utilisation - Google Patents

Composant de cyclisation d'arn linéaire et son utilisation Download PDF

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Publication number
WO2024260432A1
WO2024260432A1 PCT/CN2024/100495 CN2024100495W WO2024260432A1 WO 2024260432 A1 WO2024260432 A1 WO 2024260432A1 CN 2024100495 W CN2024100495 W CN 2024100495W WO 2024260432 A1 WO2024260432 A1 WO 2024260432A1
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rna
cyclization
linear
motif
circular
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Chinese (zh)
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田宝磊
董原辰
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Beijing Supracirc Biotechnology Co Ltd
Institute of Chemistry CAS
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Beijing Supracirc Biotechnology Co Ltd
Institute of Chemistry CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present application belongs to the field of biomedicine technology, and specifically relates to a method for preparing single-stranded circular RNA.
  • Circular RNA is a type of single-stranded RNA characterized by a covalently closed topological structure, so there is no free end. Compared with linear RNA, circular RNA is not easily digested by exonucleases and exhibits higher stability, so it has many practical application prospects both in vivo and in vitro. For example, it can be used as a small RNA (microRNA, miRNA) sponge, protein binding skeleton, RNA expression vector and protein regulatory factor. Therefore, how to artificially synthesize circular RNA has attracted widespread attention.
  • microRNA small RNA
  • the commonly used methods for in vitro synthesis of circular RNA currently include chemical ligation, intron exon arrangement (PIE) and enzyme ligation.
  • the chemical ligation method mainly uses chemical reagents such as cyanogen bromide (BrCN) and morpholino derivatives such as 2-(N-morpholino)-ethanesulfonic acid (MES) to connect the hydroxyl and phosphate groups at both ends of the linear RNA chain, thereby realizing the cyclization of the linear RNA chain.
  • chemical reagents such as cyanogen bromide (BrCN) and morpholino derivatives such as 2-(N-morpholino)-ethanesulfonic acid (MES)
  • MES 2-(N-morpholino)-ethanesulfonic acid
  • such methods are generally only applicable to the cyclization of very small linear RNA chains ( ⁇ 100nt), and due to the presence of 2′ hydroxyl groups in the RNA chain, it is very easy to generate 2′-5′
  • the PIE rule is realized by utilizing two ester exchange reactions that determine the splicing site in the intron self-splicing reaction.
  • a key limitation of the existing PIE method is that the circular RNA product based on the PIE method will retain some exons of the natural group I intron gene, and a large number of splicing intermediate byproducts will be produced in the reaction system.
  • this method since this method has very high requirements for the secondary structure of introns, it greatly increases the difficulty of sequence design.
  • the enzyme ligation method mainly uses a series of DNA and RNA ligases (T4 DNA ligase, T4 RNA ligase 1, T4 RNA ligase 2) derived from TA phage. These enzymes require linear RNA substrates containing There is a 5' monophosphate for cyclization.
  • T4 DNA ligase T4 RNA ligase 1, T4 RNA ligase 2
  • T4 DNA ligase T4 DNA ligase 1, T4 RNA ligase 2
  • the present invention provides a method for realizing cyclization based on an RNA molecule connected with a specific sequence using itself as a template.
  • the RNA sequence to be cyclized relies on the complementary base pairing between the cyclization motifs, so that the 3' end and the 5' end of the RNA to be cyclized are extremely close in space, so that by adding RNA ligase, the head and tail ends of the RNA are covalently connected to achieve the cyclization of the target RNA sequence.
  • the present invention provides a linear RNA for circularization. In another aspect.
  • the present invention provides a circularization component for circularizing linear RNA. In another aspect, the present invention provides a method for preparing circular RNA.
  • the present invention provides an expression vector, which expresses a precursor linear RNA for circularization in vitro.
  • the present invention provides the circularization component for circularizing linear RNA, the linear RNA for circularization or the expression vector, for use in treating, preventing or diagnosing a disease, or for use in preparing a drug for treating or preventing a disease or a diagnostic or detection agent.
  • the present invention provides a universal and simple RNA cyclization method and components thereof, which can efficiently cyclize RNA chains of different lengths and sequences.
  • the method uses itself as a template, does not require the introduction of additional splint chains, and does not generate by-products.
  • the sequence design of the method is simple, and there is no need to consider the secondary structure of the sequence to be cyclized.
  • the stability of the circular RNA prepared by this method is greatly improved compared to the linear precursor, and does not affect the original function of the RNA sequence, and can reduce the generation of polymer impurities.
  • the present invention provides a linear RNA for cyclization, wherein the cyclized linear RNA comprises the following structure from the 5' end to the 3' end:
  • the circulation promoter sequence has the following structure from the 5' end to the 3' end:
  • the GGGA- is completely complementary or partially complementary to the -X 1 X 2 X 3 X 4 -, so that the circularized promoter forms a stem-loop structure.
  • the lock unit is completely complementary to the key unit, so that the 5′ end of the cyclization promoter sequence and the 3′ end of the key unit sequence form a cyclase action nick.
  • GGGA- and -X 1 X 2 X 3 X 4 - are fully complementary or have at least 3 complementary pairs, preferably with only a mismatch between G and X 3 .
  • X 1 is selected from the base U, A, C or G, preferably the base U; X 2 , X 3 , X 4 are each independently selected from the base G or C.
  • the non-complementary region length of the cyclic promoter is 3-15nt. In some embodiments, the non-complementary region sequence is not complementary to GGGA-, -X1X2X3X4- and the target RNA. In some embodiments, the ratio of bases C and A in the non-complementary region is 66%-100%. In some embodiments, the non-complementary region sequence in the cyclic promoter is -CCAAC- from the 5' end to the 3' end. In some embodiments, the cyclic promoter is GGGACCAACUCUC (SEQ ID NO: 1) from the 5' end to the 3' end. In some embodiments, the cyclic promoter can be replaced with GGGACCAACUCCC (SEQ ID NO: 2).
  • the 5' end of the linear RNA used for circularization is triphosphorylated or monophosphorylated.
  • the lock motif and the key motif complement each other to form a pairing structure of at least 3 bp (basepairs). In some embodiments, the lock motif and the key motif complement each other to form a pairing structure of 3-15 bp or 3-9 bp. Specifically, 3, 4, 5, 6, 7, 8, Paired structures of 9, 10, 11, 12, 13, 14 or 15 bp.
  • the GC ratio in the paired structure formed by the key motif and the key motif is between 20% and 70%.
  • the structure of the lock motif from 5' end to 3' end is UAG. In some embodiments, the sequence of the key motif from 5' end to 3' end is CUA. In some embodiments, the sequence of the lock motif from 5' end to 3' end is UAGUAG, and the sequence of the key motif from 5' end to 3' end is CUACUA.
  • GGGACCAACUCCC SEQ ID NO: 2 can be substituted in the circularization promoter.
  • GGGACCAACUCCC SEQ ID NO: 2 can be substituted in the circularization promoter.
  • the circularization promoter sequence, the lock motif sequence, and the key motif sequence in the linear RNA for circularization form the following structural fragments after circularization:
  • a structure is formed that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% homologous to the above structural fragments.
  • the target RNA sequence (i.e., the RNA sequence to be circularized)
  • the length is more than 50nt, preferably 100nt to 10000nt, or greater than 200nt, 300nt, 400nt, 500nt, 600nt, 700nt, 800nt, 900nt, 1000nt, 1500nt, 2000nt, 3000nt, 4000nt, 5000nt, 6000nt, 7000nt, 8000nt, 9000nt, 10000nt.
  • the target RNA sequence regulates the target nucleic acid in the subject, or directly encodes and expresses the target polypeptide or protein. In some embodiments, the target RNA sequence regulates the function of pre-mRNA, mRNA, miRNA, LncRNA, circRNA and tRNA in the subject, or expresses a polypeptide or protein encoded by a single-stranded circular RNA.
  • the linear RNA used for cyclization and any element therein comprise unmodified, partially modified or completely modified nucleosides.
  • the modification includes, but is not limited to, 5'-position pyrimidine modification, 8'-position purine modification, modification at the cytosine exocyclic amine, and substitution of 5-bromo-uracil; and 2'-position sugar modification, including but not limited to sugar-modified ribonucleotides, nucleotide analogs are also intended to include nucleotides having bases such as inosine, quercetin, xanthine; sugars such as 2'-methyl ribose; non-natural phosphodiester linkages such as methylphosphonates, phosphorothioates, and peptide linkages.
  • Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine, and 6-methyladenosine.
  • the present invention provides a circularization component for circularizing linear RNA, which comprises: a circularization promoter, a lock motif and a key motif.
  • the circularized promoter, the lock motif and the key motif form a linear structure with the target RNA as follows:
  • the circularized promoter has the following structure from the 5' end to the 3' end:
  • the GGGA- is completely complementary or partially complementary to the -X 1 X 2 X 3 X 4 -, so that the circularized promoter forms a stem-loop structure.
  • the lock motif is selected from a sequence that is completely complementary to the key motif from the 3' end to the 5' end. List.
  • the circularizing promoter, lock motif, and key motif have the same definitions as described above for circularized linear RNA.
  • the circularization promoter, the lock motif and the key motif in the circularization assembly of the circularized linear RNA form a circular intermediate RNA precursor chain with the following structure after hybridization: A cyclase action incision is formed between the circularization promoter and the key motif.
  • the present invention provides a method for preparing circular RNA, comprising the following steps:
  • Step 1 Connecting the circularized promoter, lock motif, target RNA sequence and key motif of the present invention in sequence to obtain a DNA template chain connected with a circular component;
  • Step 2 performing in vitro transcription on the DNA template chain to obtain the linear RNA chain for cyclization of the present invention
  • Step 3 convert the 5′-terminal triphosphate of the RNA sequence obtained in step 2 into a monophosphate to obtain a circular RNA precursor chain;
  • Step 4 placing the circular RNA precursor chain in a buffer solution and annealing it to form an RNA precursor chain in a circular intermediate state;
  • Step 5 The RNA precursor chain in the circular intermediate state described in step 4 is treated with RNA ligase to connect the interfaces of the 5′ end and the 3′ end to obtain a covalently closed circular RNA.
  • PCR method can be selected for step 1.
  • solid phase synthesis method can be used to replace the steps 1 and 2 to obtain the linear RNA chain for circularization.
  • step 3 the linear RNA chain is treated with Apyrase enzyme to convert the 5′-terminal triphosphate of the RNA sequence obtained in step 2 into a monophosphate to obtain a circular RNA precursor chain.
  • step 5 is to obtain the RNA precursor chain in a circular intermediate state by annealing after step 4.
  • the RNA ligase is selected from T4 RNA ligase 1 (T4 RNA ligase 1) or T4 RNA ligase 2 (T4 RNA Ligase 2).
  • the amount of T4 RNA ligase used in step 5 is 5-50 U/ ⁇ M circular intermediate RNA precursor chain.
  • the amount of T4 RNA ligase used can be selected from 5, 10, 20, 30, 40, 50 U/ ⁇ M circular intermediate RNA precursor chain.
  • the method of preparing circular RNA further comprises a purification step 6:
  • Enzyme digestion step Use RNase R to treat the connected circular RNA system to completely remove the unreacted linear long single strands;
  • Product extraction step use one or more of the RNA purification kit method, ethanol precipitation ultrafiltration method or polyacrylamide gel electrophoresis method to purify and extract circular RNA.
  • the RNA Clean & Concentrator Kit (RCC) RNA purification kit is used to extract the enzymatically purified circular RNA.
  • the circular RNA is precipitated by ethanol, and then the circular RNA is enriched by ultrafiltration and desalting.
  • the polyacrylamide gel electrophoresis the circular RNA is extracted after gel cutting and purification using polyacrylamide gel electrophoresis.
  • the present invention provides an expression vector for expressing a precursor linear RNA for circularization in vitro.
  • the expression vector is selected from a linear DNA fragment, a plasmid vector, a viral vector, a bacterial artificial chromosome, a yeast artificial chromosome.
  • the linear DNA fragment is preferably a PCR product or a linear plasmid fragment.
  • the present invention provides the aforementioned linear RNA for circularization, circularization component for circularization of linear RNA or expression vector, and use thereof in preparing circularized RNA.
  • the present invention provides the aforementioned linear RNA for circularization, the circularization component or expression vector for circularizing linear RNA, and its use in treating, preventing or diagnosing diseases or in preparing drugs for treating or preventing diseases or diagnostic or detection reagents.
  • nucleic acid combinations and compositions disclosed herein are listed below. These definitions apply to the terms used throughout this specification and claims, except where otherwise limited in specific cases, and these terms are used either individually or as part of a larger group.
  • the terms “optional” and “optionally” include both the cases of being selected or not being selected.
  • “optionally modified” includes both the cases of being modified and not being modified.
  • RNA circular polyribonucleotide
  • circular RNA refers to polyribonucleotides that form a circular structure through covalent bonds.
  • cyclization efficiency refers to a measurement of the resulting circular polyribonucleotide compared to its linear starting material.
  • nucleotide refers to a ribonucleotide, a deoxyribonucleotide, a modified form thereof, or an analog thereof.
  • Nucleotides include substances including purines (e.g., adenine, hypoxanthine, guanine, and derivatives and analogs thereof) and pyrimidines (e.g., cytosine, uracil, thymine, and derivatives and analogs thereof).
  • Nucleotide analogs include There are modified nucleotides in the chemical structure of bases, sugars and/or phosphates, including but not limited to, 5'-position pyrimidine modification, 8'-position purine modification, modification at the cytosine exocyclic amine and substitution of 5-bromo-uracil; and 2'-position sugar modification, including but not limited to sugar-modified ribonucleotides. Nucleotide analogs are also intended to include nucleotides with bases such as inosine, quercetin, xanthine; sugars such as 2'-methyl ribose; non-natural phosphodiester linkages such as methylphosphonates, thiophosphates and peptide linkages.
  • Nucleotide analogs include 5-methoxyuridine, 1-methylpseudouridine and 6-methyladenosine.
  • modified ribonucleosides include 5-methylcytidine, 5-methoxyuridine, 1-methylpseudouridine, N6-methyladenosine and/or pseudouridine. In some embodiments, such modified nucleosides provide additional stability and resistance to immune activation.
  • the term "complementary” refers to the ability to pair between two sequences containing naturally or non-naturally occurring bases or their analogs by base stacking and specific hydrogen bonding. For example, if a base at one position of a nucleic acid is able to form a hydrogen bond with a base at a corresponding position of a target, the bases are considered to be complementary to each other at that position. Nucleic acids may contain universal bases or inert abasic spacers that do not provide positive or negative contributions to hydrogen bonding. Base pairing may include canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., G:U Wobble base pairing and Hoogsteen base pairing).
  • adenosine-type bases are complementary to thymidine-type bases (T) or uracil-type bases (U)
  • cytosine-type bases are complementary to guanosine-type bases (G)
  • universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U or T and are considered to be complementary thereto.
  • Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U or T. See Watkins and SantaLucia, Nucl. Acids Research, 2005; 33(19): 6258-6267.
  • the circular RNA provided herein has a higher functional stability. Compared with mRNA with 5 moU modification, optimized UTR, cap and/or polyA tail, the circular RNA provided in this article has higher functional stability.
  • FIG1 shows a schematic diagram of RNA chain self-circularization.
  • FIG2 shows the polyacrylamide gel electrophoresis characterization of the 215 nt circular RNA prepared based on the present self-circularization strategy and the corresponding circularization efficiency.
  • Figure 3 shows the RNase R and RNase H enzyme digestion verification of the 215nt circular RNA prepared based on this self-circularization strategy.
  • FIG. 4 shows the interface sequencing of the 215 nt circular RNA prepared based on the present self-circularization strategy.
  • Figure 5 shows a schematic diagram of the RNA precursor chain in the circular intermediate state described in step 4 of Example 1, where components 1, 2 and 3 correspond to the circularized promoter, component 4 corresponds to the lock motif, and component 5 corresponds to the key motif.
  • FIG6 shows the polymerization degree verification of circular RNA prepared based on the present self-circularization strategy.
  • FIG. 7 shows the polyacrylamide gel electrophoresis characterization of circular RNAs of different sizes prepared based on the present self-circularization strategy.
  • FIG8 shows the circularization efficiency of circular RNAs of different sizes prepared based on the present self-circularization strategy.
  • Figure 9 shows the polyacrylamide gel electrophoresis characterization of circular RNA prepared at different enzyme connection times in the 4M2 group without the "key motif-lock motif” sequence and the 4P group containing the "key motif-lock motif” sequence.
  • Figure 10 shows the circularization efficiency of circular RNA prepared at different enzyme connection times in the 4M2 group without a "key motif-lock motif” sequence and the 4P group containing a "key motif-lock motif” sequence.
  • the present invention provides a universal and simple RNA looping method, which can efficiently RNA chains of different lengths and sequences are cyclized.
  • This method uses itself as a template, does not require the introduction of additional splint chains, and does not generate by-products.
  • this method is simple to design sequences, and does not require consideration of the secondary structure of the sequence to be cyclized.
  • the stability of the circular RNA prepared by this method is greatly improved compared to the linear precursor, and does not affect the original function of the RNA sequence.
  • the experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified.
  • the experimental methods in the following examples that do not specify specific conditions are usually carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.
  • the materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified.
  • Step 1 construction of dsDNA template : 2.5 ⁇ L forward primer (10 ⁇ M), 2.5 ⁇ L reverse primer (10 ⁇ M), 1 ⁇ L DNA template (1 ng), 19 ⁇ L ddH 2 O and 19 ⁇ L 2 ⁇ Phusion plus mixer were added to 50 ⁇ L PCR reaction system for PCR amplification and adding circularization components, which included the circularization promoter, lock motif and key motif described in Table 1.
  • the obtained double-stranded DNA was separated and purified by agarose gel electrophoresis, and verified by TA cloning and Sanger sequencing. Finally, the final dsDNA template was obtained by PCR reaction again using the sequenced verified plasmid (TOPO-TA/Blunt Vector) as a template.
  • Step 2 5' triphosphorylated linear ssRNA was transcribed in vitro using dsDNA as a template .
  • the reaction system (20 ⁇ L) contained 500 ng of linear dsDNA template, 4 ⁇ L of T7 transcription 5 ⁇ buffer, 8 ⁇ L of rNTP (25 mM ATP, CTP, UTP and 3 mM GTP), 2 ⁇ L of enzyme mixture (T7) and nuclease-free water.
  • the reaction system was incubated at 37°C for 4 hours. 1 ⁇ L of RNase-Free DNase was added to the reaction system, and the reaction was incubated at 37°C for 30 minutes to remove the template dsDNA.
  • the reaction system was purified using the RNA Clean & Concentrator Kit (RCC) to obtain 5' triphosphorylated linear precursor ssRNA.
  • RRCC RNA Clean & Concentrator Kit
  • Step 3 Convert 5' triphosphorylated linear ssRNA to monophosphorylated form :
  • the reaction system (10 ⁇ L) contains 2 ⁇ M 5' triphosphorylated linear ssRNA, 0.1 U Apyrase, 20 U RiboLock RNase inhibitor and DEPC water.
  • 1 ⁇ Apyrase reaction buffer (20 mM 4-morpholineethanesulfonic acid (MES), 50 mM NaCl, 5 mM CaCl 2 , 1 mM dl-dithiothreitol (DTT) and 0.05% 20, (pH 7.5)).
  • the reaction system was incubated at 37°C for 2 hours and inactivated at 65°C for 20 minutes to obtain the monophosphorylated linear ssRNA.
  • Step 4 annealing of linear ssRNA :
  • the reaction system (40 ⁇ L) contained 0.5 ⁇ M monophosphorylated linear ssRNA, and the solution used was 1 ⁇ TE ⁇ Mg 2+ buffer (10 mM Tris-HCl, 0.1 mM EDTA, 5 mM Mg 2+ , pH 7.6).
  • the reaction system was incubated at 75°C for 5 minutes and cooled to 25°C at a rate of 1°C/min to obtain the RNA precursor chain in the circular intermediate state .
  • T4 Rnl2 enzymatically ligates the RNA precursor chain in the circular intermediate state to obtain circular ssRNA :
  • the reaction system (80 ⁇ L) contains 0.25 ⁇ M RNA precursor chain in the circular intermediate state, 10 U T4 Rnl2, 40 U RiboLock RNase inhibitor and DEPC water, and the solution used in the reaction is 1 ⁇ T4 Rnl2 reaction buffer (50 mM Tris-HCl, 2 mM MgCl 2 , 1 mM dl-dithiothreitol (DTT) and 400 ⁇ M adenosine triphosphate (ATP), pH 7.5).
  • the reaction system is incubated at 37°C for 2 hours and inactivated with proteinase K to obtain the crude circular ssRNA product.
  • Step 6 purification of circular ssRNA :
  • the reaction system (20 ⁇ L) consists of 500 ng of crude circular ssRNA, 2U RNase R and DEPC water, and the reaction solution is 1 ⁇ RNase R reaction buffer (20 mM Tris-HCl, 100 mM KCl and 0.1 mM MgCl 2 , pH 8.0).
  • the reaction system is incubated at 37°C for 30 minutes and at 70°C for 5 minutes.
  • the reaction system is purified using the RNA Clean & Concentrator Kit (RCC) to obtain circular ssRNA.
  • RRCC RNA Clean & Concentrator Kit
  • Step 7 denaturing PAGE analysis and yield assessment : Samples were electrophoresed with denaturing polyacrylamide gels containing 8M urea (denaturing PAGE). Depending on the length of the precursor RNA, the gel range was 4% to 10%, and the ratio of acrylamide to bisacrylamide was 19:1 to 39:1. The gel was post-stained with Sybr Gold TM and then imaged using a UV gel imager from BIO-RAD (Hercules, USA). Quantitative data were obtained using Image Lab software.
  • I circular RNA
  • I (tot) the band intensity of all bands in the lane (composed of circular RNA, linear precursor RNA and polymerized byproducts).
  • the measurement results are shown in Figure 2.
  • RNase R and RNase H were used to identify the product, and the measurement results are shown in Figure 3.
  • ⁇ represents circular RNA
  • - represents linear RNA precursor
  • -- represents linear RNA dimer.
  • step 1 the target RNA of the same length is used to replace the target RNA in step 1 of Example 1
  • TAGTAG SEQ ID NO: 5
  • CTACTA SEQ ID NO: 6
  • step 1 of Example 1 The same method as in step 1 of Example 1 is used to prepare the dsDNA full sequences of 173nt, 240nt, 375nt, 525nt, 675nt, 895nt and 1125nt in length as described in Table 2 (the sequences marked with _ in Table 2 correspond to target RNAs of 148nt, 215nt, 350nt, 500nt, 650nt, 870nt, and 1100nt, respectively), and the corresponding circular RNA is prepared using the methods of steps 2 to 6 of Example 1.
  • the yield of circularized RNA is determined using the method of step 7 of Example 1, and the test results are shown in Figures 7-8. The results show that the circularization method of the present invention is suitable for the circularization of linear RNAs of different lengths.
  • Example 3 Effect of enzyme connection time on cyclization efficiency
  • step 5 the yield of circularized RNA at different time points within 0-60 min of incubation was calculated according to the method of step 7 of the embodiment to evaluate the effect of the incubation time of the enzyme-linked reaction in step 5 on the cyclization efficiency. The test results are shown in Figures 9-10.

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Abstract

L'invention concerne un composant de cyclisation d'ARN linéaire et un procédé de cyclisation de chaîne d'ARN. Des primitives de formation d'anneau sont liées à deux extrémités d'une séquence d'ARN à soumettre à une formation d'anneau, et en fonction de l'interaction d'appariement complémentaire de bases entre les primitives de formation d'anneau, les extrémités 3' et 5' de la séquence d'ARN sont extrêmement proches dans l'espace, et sont liées au moyen d'une ARN ligase pour achever la cyclisation d'un ARN cible. L'ARN cyclisé préparé selon le procédé présente une stabilité supérieure à celle d'un ARN linéaire.
PCT/CN2024/100495 2023-06-21 2024-06-20 Composant de cyclisation d'arn linéaire et son utilisation Pending WO2024260432A1 (fr)

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CN104328185A (zh) * 2014-10-31 2015-02-04 江汉大学 一种监控总rna中线状rna消除的方法
CN111321143A (zh) * 2020-02-16 2020-06-23 中国海洋大学 一种制备环状rna的方法
CN112204141A (zh) * 2018-02-22 2021-01-08 美国陶氏益农公司 短/小发夹rna分子
WO2022232291A1 (fr) * 2021-04-27 2022-11-03 Factor Bioscience Inc. Arn circulaire

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104328185A (zh) * 2014-10-31 2015-02-04 江汉大学 一种监控总rna中线状rna消除的方法
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