WO2023165009A1 - 一种用于制备环状rna的载体及其应用 - Google Patents

一种用于制备环状rna的载体及其应用 Download PDF

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WO2023165009A1
WO2023165009A1 PCT/CN2022/090198 CN2022090198W WO2023165009A1 WO 2023165009 A1 WO2023165009 A1 WO 2023165009A1 CN 2022090198 W CN2022090198 W CN 2022090198W WO 2023165009 A1 WO2023165009 A1 WO 2023165009A1
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rna
circular rna
preparing
poly
carrier
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胡勇
吕彬
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深圳瑞吉生物科技有限公司
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N2800/00—Nucleic acids vectors
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • C—CHEMISTRY; METALLURGY
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the invention relates to the technical field of molecular biology, in particular to providing a carrier for preparing circular RNA and its application.
  • Circular RNA Circular RNA
  • linear RNA linear RNA
  • the circRNA molecule has a closed circular structure, is not affected by RNA exonuclease, is not easy to degrade, and has more stable expression.
  • Wesselhoeft, R.A. et al. reported a highly efficient RNA circularization tool based on type I self-cleaving intron design.
  • the circularization vector used contained the IRES element (CVB3 IRES) of Coxsackie virus, Gaussia luciferase (GLuc) And the type I intron of Anabaena pre-tRNA, the type I intron of Anabaena pre-tRNA is split and designed on both sides of the sequence to be circularized.
  • the circRNA precursor molecule can be automatically cyclized through the double ester exchange reaction, but a large number of by-products will also be produced in the process of generating circRNA, such as uncirculated cyclic RNA precursor molecules and spliced intron fragments, etc.
  • the mainstream method of enriching and purifying circular RNA is to use RNase R for treatment, but RNase R is unstable in nature, and the difference between different batches of enzymes is large, which may easily lead to unstable purification and enrichment of circular RNA; and RNase R can not only digest linear RNA, but also digest part of circular RNA, resulting in the loss of part of circular RNA during the purification and enrichment of circular RNA.
  • WO2019236673A1 records a method for eukaryotic Circular RNA translated in cells, and the carrier used to prepare this circular RNA is described, which can realize the preparation of circular RNA, but it cannot solve the above-mentioned purification and enrichment of circular RNA while shortening the processing time of RNase R, Improve the enrichment efficiency of circular RNA and reduce the problem of cytotoxicity.
  • One object of the present invention is to provide a vector for preparing circular RNA.
  • Another object of the present invention is to provide a circular RNA precursor molecule.
  • Another object of the present invention is to provide a preparation method of the circular RNA precursor molecule.
  • Another object of the present invention is to provide the application of the carrier in the preparation of circular RNA.
  • Another object of the present invention is to provide a method for preparing circular RNA.
  • the present invention completes the preparation of the circular RNA precursor molecule that introduces the poly-A tail by designing a DNA sequence that can be transcribed into the poly-A tail of the circular RNA precursor molecule in the carrier, and can solve the problem in the process of preparing the circular RNA. Circular RNA enrichment efficiency, circular RNA product purity and/or cytotoxicity after transfection of cells, etc.
  • the present invention provides a vector for preparing circular RNA, which comprises a DNA sequence capable of being transcribed into a circular RNA precursor molecule, and the 3' end of the circular RNA precursor molecule has There are poly A tails.
  • the length of the poly A tail is 5-150 A.
  • the length of the poly A tail is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 21, 22, 23 , 24, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73 , 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 ,99,100,101,102,
  • the vector for preparing circular RNA of the present invention comprises elements arranged in the following order:
  • IRES internal ribosome entry site
  • a DNA sequence capable of being transcribed into said poly-A tail is a DNA sequence capable of being transcribed into said poly-A tail.
  • the carrier for preparing circular RNA of the present invention may further comprise one or more of the following elements:
  • 3' homology arm inserted in the Anabaena pre-tRNA 5' type I intron fragment (can be in the middle of the type intron fragment sequence, or near the 3' position).
  • the specific sequences of the non-coding region, the Anabaena pre-tRNA 5' type I intron fragment, the 5' homology arm, the 5' spacer sequence, the 3' spacer sequence, and the 3' homology arm can be respectively adopted from established methods in the field. known sequence of fragments. For example, fragments or non-coding regions described in WO2019236673A1 can be used.
  • described Anabaena pre-tRNA 3' type I intron fragment is as set forth in SEQ ID NO.1 or SEQ ID NO.2 The 3' type I intron fragment shown.
  • described Anabaena pre-tRNA 3' type I intron fragment is as set forth in SEQ ID NO.1 or SEQ ID NO.2 The 3' type I intron fragment shown.
  • the IRES in the vector for preparing circular RNA of the present invention, is shown in SEQ ID NO.1 or SEQ ID NO.2.
  • described Anabaena pre-tRNA 5' type I intron fragment is as set forth in SEQ ID NO.1 or SEQ ID NO.2 The 5' type I intron fragment is shown.
  • the 5' homology arm is as shown in SEQ ID NO.1 or SEQ ID NO.2 5' homology arm .
  • the 5' spacer sequence such as the 5' spacer sequence shown in SEQ ID NO.1 or SEQ ID NO.2 .
  • the 3' homology arm is as shown in SEQ ID NO.1 or SEQ ID NO.2 3' homology arm .
  • the 3' spacer sequence such as the 3' spacer sequence shown in SEQ ID NO.1 or SEQ ID NO.2 .
  • the carrier for preparing circular RNA of the present invention has the 1st to 1017th nucleotide sequence and/or the 1576th to 1781st nucleotide sequence of the sequence shown in SEQ ID NO.1 Nucleotide sequence.
  • the circular RNA in the vector for preparing circular RNA of the present invention, can encode hFIX, SP-B, VEGF-A, human methylmalonyl-CoA mutase , CFTR, cancer autoantigens, gene editing enzymes, HIV antibody, CD19 antibody, CD22 antibody, CD3 antibody, CLDN6 antibody, Luc2, GLuc, Fluc, eGFP, hEPO, Cas9 endonuclease, 2019-nCoV S protein or tumor suppressor factor.
  • the protein coding region in the vector for preparing circular RNA is a nucleotide fragment encoding these proteins.
  • the circular RNA encodes Luc2 or GLuc.
  • the protein coding region or non-coding region in the vector for preparing circular RNA of the present invention, is the Luc2 gene; In the carrier for preparing the circular RNA, the protein coding region or the non-coding region is the GLuc gene; two reporter genes verify the feasibility of the technical solution of the present invention.
  • the vector for preparing circular RNA of the present invention has the sequence shown in SEQ ID NO.1 or SEQ ID NO.2, and the protein coding region or non-coding region uses a reporter gene sequence , verified the feasibility of the technical solution of the present invention.
  • the present invention also provides a circular RNA precursor molecule, which is obtained by in vitro transcription through the carrier for preparing circular RNA described in the present invention.
  • the present invention also provides the application of the carrier in the preparation of circular RNA.
  • the present invention also provides a preparation method of a circular RNA precursor molecule, which comprises the steps of:
  • RNA in vitro synthesis system including the DNA template described in a, and perform RNA in vitro synthesis to obtain RNA precursor molecules that can be circularized.
  • the poly A tail is used in the preparation of linear mRNA synthesized in vitro to protect the mRNA from exonuclease attacks, aiming at improving the stability of the mRNA and maintaining the activity of the mRNA as a translation template.
  • the prior art does not consider the introduction of poly A tail, the reason is that circular RNA has a closed circular structure, is not easy to be degraded by exonuclease, is more stable than linear RNA, and does not need to introduce poly A Tail, and the sequence introduced into the circRNA precursor molecule may affect the structure of the circRNA precursor molecule and lead to abnormal circularization.
  • the present invention overcomes this prejudice, and creatively introduces the DNA sequence that can be transcribed into the poly-A tail of the circular RNA precursor molecule into the carrier used to prepare the circular RNA, thereby preparing a circular RNA with a poly-A tail RNA precursor molecule.
  • the creative use of this poly-A tail does not affect its cyclization efficiency in the prediction of the secondary structure of the circRNA precursor molecule.
  • it is proved that the structure of the circular RNA precursor molecule will not be seriously affected, and its ring-forming efficiency will not be affected, and the cytotoxicity of the purified circular RNA is compared with that of the linear RNA, which is passed through nucleosides. Acid-modified linear RNAs and circular RNAs without poly A tails purified in the same way were reduced in levels.
  • the present invention first introduces a poly A tail at the 3' end of the circular RNA precursor molecule carrier that has not undergone a circularization reaction by means of vector construction or PCR amplification, and then in the transcription reaction After the end, add GTP with a final concentration of 2mM to the system, and circularize the RNA at 55°C. During this process, the poly A tail at the 3' end of the RNA will be cut off during the diester exchange process. Then, RNase R was used to digest the spliced products at 37°C for 20 min. Finally, MEGAclear Transcription Clean-Up Kit (purchased from Invitrogen) was used to purify the final circular RNA product.
  • the present invention also provides a method for preparing circular RNA, which includes the steps of:
  • the circular RNA precursor molecule is circularized to obtain a circular RNA.
  • the preparation method of circular RNA of the present invention comprises the following steps:
  • RNA in vitro synthesis system including the DNA template and performing RNA in vitro synthesis to obtain the RNA encoding Luc2 and GLuc.
  • the RNA in vitro synthesis system includes the following components in 100 ⁇ L:
  • the procedure for in vitro synthesis of RNA is 37° C. for 4 hours.
  • step 3) after obtaining the circular RNA precursor molecules encoding Luc2 and GLuc also includes: circularization reaction of transcripts, Rnase R digestion reaction, RNA purification reaction, adjusting the RNA concentration of encoding Luc2 and GLuc and packing steps.
  • the "precursor molecule" described in the present invention is a linear RNA molecule produced by in vitro transcription (for example, from the vector provided by the present invention).
  • This precursor RNA molecule contains the complete circRNA sequence, the splicing sequences (intron segments and homology arms) required for RNA circularization, and a polyA tail. These spliced sequences (intronic segments and homology arms) and poly-A tails are removed from the precursor RNA during circularization, resulting in a circRNA and two introns/homology arms and a linear poly-A tail. RNA fragments.
  • the invention provides a carrier for preparing circular RNA, by designing a DNA sequence that can be transcribed into a poly A tail of a circular RNA precursor molecule, and introducing a poly A tail. Therefore, during the circularization process of the circular RNA precursor molecule, the by-products are more easily digested by RNase R, shortening the processing time of RNase R, and reducing the amount of RNase R used. Moreover, it is guaranteed that the introduced poly-A tail will not seriously affect the structure of the circular RNA precursor molecule, so that the circularization efficiency of the RNA is reduced.
  • the cytotoxicity of the circular RNA purified by this method is compared with that of the linear one. Nucleotide-modified linear RNAs and circular RNAs purified in the same way without poly A tails had reduced levels.
  • Fig. 1 is a schematic diagram of the structure of the circRNA precursor encoding Luc2.
  • Fig. 2 is a schematic diagram of the secondary structure of the circular RNA precursor molecule encoding Luc2 without poly-A tail and with poly-A tail predicted by RNAFold based on the minimum free energy.
  • Figure 3A and Figure 3B are the agarose gel electrophoresis detection images of circular RNA precursor molecules without poly-A tail and poly-A tail that encode GLuc and Luc2 respectively after being digested by Rnase R for 20 min .
  • Figure 4 is an agarose gel electrophoresis detection diagram of the time gradient of RNase R digestion after circularization of circular RNA precursor molecules encoding Luc2 without poly A tails and with poly A tails.
  • Fig. 5 is a cell viability detection graph of transfected 293T cells after circularization and purification of circular RNA precursor molecules encoding Luc2 without poly-A tails and with poly-A tails.
  • Fig. 6 is a graph showing luciferase activity detection of transfected 293T cells after circularization and purification of circular RNA precursor molecules encoding Luc2 without poly A tails and with poly A tails.
  • Fig. 7 is an agarose gel electrophoresis detection diagram after circularization of circular RNA precursor molecules encoding Luc2 with poly-A tails of different lengths after being digested by Rnase R for 20 min.
  • the invention provides a method for purifying circular RNA based on type I intron self-splicing.
  • a carrier is used to prepare a circular RNA precursor molecule encoding GLuc, and the carrier includes a sequence shown in SEQ ID NO.1 (a nucleotide fragment composed of a sequence shown in SEQ ID NO.1 Also referred to as Seq1) in the present invention.
  • a circularized vector is used to prepare a circular RNA precursor molecule encoding Luc2, said vector comprising a sequence shown in SEQ ID NO.2 (a core composed of a sequence shown in SEQ ID NO.2
  • the nucleotide fragment is also referred to as Seq2 in the present invention).
  • the circRNA in the circularization vector used includes the IRES element (CVB3 IRES) of Coxsackie virus, luciferase (Luc2), poly A tail at the 3' end and I of Anabaena pre-tRNA.
  • the intron is split and designed on both sides of the sequence to be circularized. The specific structure is shown in Figure 1.
  • the initial concentration of the primer F and primer R1 is preferably 10 ⁇ mol/L; the concentration of the DNA template is preferably 1 ng/ ⁇ L.
  • the sequence of the primer F is as follows:
  • the sequence of the primer R1 is as follows:
  • the PrimeSTAR Max Premix (2 ⁇ ) includes the following components: PrimeSTAR Max DNA Polymerase, dNTPs and Mg 2+ .
  • the PCR amplification program is preferably as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 56°C for 15 s, extension at 72°C for 40 s, 30 cycles; final extension at 72°C for 5 min.
  • the amplification product is preferably detected by agarose gel electrophoresis to determine whether the reaction is successful; the parameters detected by the agarose gel electrophoresis are preferably as follows: 1.5% agarose , 120V, 40min. In the present invention, the reaction is considered successful when the target band appears in the agarose gel electrophoresis.
  • the electrophoresis product is subjected to DNA recovery and extraction.
  • the DNA extraction kit is Gel Extraction Kit (purchased from the Omega brand); the present invention preferably adopts NanoDrop to detect the concentration of the purified template after the purification, and the ratio of 260/280, 260/230 , when 260/280 is between 1.6 and 1.8, the template is considered qualified.
  • an RNA in vitro synthesis system including the DNA template is constructed to perform RNA in vitro synthesis to obtain the precursor circRNA.
  • the RNA in vitro synthesis system includes the following components in 100 ⁇ L:
  • the Enzyme Mix includes T7 RNA polymerase, RNase inhibitor and inorganic pyrophosphatase.
  • the procedure for in vitro synthesis of RNA is preferably 37° C. for 4 hours.
  • the RNA in vitro synthesis is preferably carried out in a constant temperature reactor; the RNA in vitro synthesis system is preferably placed in a 1.5ml RNase-free Tube tube, and multiple tubes are simultaneously reacted at one time; in the RNA in vitro synthesis system Reagents were added in the order described above.
  • the present invention preferably further includes the steps of removing the DNA template, circularizing the RNA, digesting the circRNA with Rnase R, and purifying the circRNA.
  • the removal of the DNA template is preferably achieved by DNase I digestion; the digestion preferably includes mixing DNase I with the solution after the RNA synthesis reaction in vitro; the DNase I and the solution after the RNA synthesis reaction in vitro
  • the volume ratio is preferably 1:20; in the present invention, after the mixing, preferably, the solution is collected by centrifugation to the bottom of the RNase-free Tube tube.
  • the rotational speed of the centrifugation is preferably 800-1200 rpm, more preferably 1000 rpm; the centrifugation time is preferably 8-12 s, more preferably 10 s.
  • the temperature of the digestion is preferably 37° C.; the time of the digestion is preferably 1 h.
  • residual DNA fragment detection is performed.
  • the circularization of the RNA is preferably achieved by adding GTP to a final concentration of 2 mM, and then heating the reaction at 55° C. for 8 minutes. A portion of the RNA is diluted in 95% formamide loading and denatured at 70° C. for 3 After minutes, cool to room temperature. RNA was then detected using a 1.5% agarose gel.
  • RNA column purification is performed on the product after RNase R treatment.
  • the RNA column purification kit is MEGAclear Transcription Clean-Up Kit, and a part of the RNA is diluted in 95% formamide loading and denatured at 70°C for 3 minutes Then, cool to room temperature. RNA was then detected using a 1.5% agarose gel.
  • the quality detection of the circRNA includes the concentration of circRNA and the ratio of 260/280 and 260/230 of RNA.
  • the range of 260/280 is 1.8-2.1, and the range of 260/230 is greater than 2.0, the circRNA is considered qualified.
  • cell viability and luciferase expression detection are performed on the synthesized circRNA, and the specific operations are as follows:
  • Cell preparation Prepare cells for detection 3 days in advance.
  • the 293T cells purchased from the cell bank of the Chinese Academy of Sciences were passaged in cell culture flasks to ensure that the cells were in the logarithmic growth phase when used.
  • Cell dilution Take the cell suspension, dilute it to 2 ⁇ 10 5 cells/ml with DMEM medium containing 10% FBS, and mix by pipetting.
  • RNA inoculation 0.5ml of cell suspension was added to a 24-well plate. For each RNA sample, 3 wells of parallel cells should be prepared, and 1 well for blank control. The 24-well plate was placed in a (37 ⁇ 1)°C, (5 ⁇ 0.5)% CO 2 incubator for overnight cultivation.
  • RNA sample (circRNA synthesized by Seq1 and Seq2 transcription), negative control Linear Luc RNA or The Linear Luc RNA modified by methylpseudouracil (N1) was mixed gently with 25 ⁇ L opti-MEM, then added to the opti-MEM medium containing Lipofectamine MessengerMax, and immediately placed on a vortex shaker for 10 times , 1s each time, mix thoroughly, and let stand for 5min.
  • Cell viability detection Add 50 ⁇ L of Cell Counting Kit-8 (CCK-8) solution (purchased from MedChemExpress brand) to each well, and gently shake the culture plate after adding the reagents to help mix. Put the culture plate into the incubator and incubate for 1-4h, and measure the absorbance (OD) at 450nm with a microplate reader.
  • CCK-8 Cell Counting Kit-8
  • Luciferase activity detection In the present invention, preferably, Bright-LiteTM Luciferase Assay system detection kit (purchased from Vazyme brand) is selected for luciferase activity detection. Mix the two components of the solution and the substrate in the kit evenly, take a detection reagent equal to the volume of the culture medium to mix the cells, and detect it with a microplate reader after 2 minutes.
  • Bright-LiteTM Luciferase Assay system detection kit purchased from Vazyme brand
  • Design vectors contain elements in the following order:
  • CVB3 IRES Coxsackievirus internal ribosome entry site
  • a DNA sequence that can be transcribed into the poly-A tail of the GLuc circular RNA precursor molecule is transcribed into the poly-A tail of the GLuc circular RNA precursor molecule.
  • the carrier sequence used to prepare GLuc circular RNA is shown in SEQ ID NO.1.
  • Embodiment 2 is used to prepare the design of the carrier of Luc2 circular RNA
  • Design vectors contain elements in the following order:
  • CVB3 IRES Coxsackievirus internal ribosome entry site
  • a DNA sequence that can be transcribed into a polyA-tailed circRNA precursor molecule is transcribed into a polyA-tailed circRNA precursor molecule.
  • the vector sequence used to prepare Luc2 circular RNA is shown in SEQ ID NO.2.
  • the 1st-206th nucleotide sequence is 3'Intron
  • the 5th-24th nucleotide sequence is the 5'Homology arms inserted into the 3'Intron
  • the 207th-276th nucleotide sequence The sequence is 5'Spacers
  • the 277th-1017th nucleotide sequence is the IRES sequence
  • the 1018th-2670th nucleotide sequence is the Luc gene
  • the 2671-2710th nucleotide sequence is 3'Spacers
  • the 2711-2876th nucleotide sequence is 5'Intron
  • the 2841-2860th nucleotide sequence is the 3'Homology arms inserted into the 5'Intron
  • the 2877-2976th nucleotide sequence is a poly A tail.
  • Embodiment 3 Amplification and purification of DNA template
  • the plasmid DNAs of the synthesized luciferase GLuc and Luc2 were respectively amplified as DNA templates, and the reaction system was as follows:
  • the PCR amplification system is calculated as 50 ⁇ L, and each component is as follows:
  • PrimeSTAR Max Premix (2 ⁇ ) includes the following components: PrimeSTAR Max DNA Polymerase, dNTPs and Mg 2+ .
  • the initial concentration of primer F and primer R is preferably 10 ⁇ mol/L; the concentration of the DNA template is preferably 1 ng/ ⁇ L.
  • primer F The sequence of primer F is shown in SEQ ID NO.3;
  • primer R1 The sequence of primer R1 is shown in SEQ ID NO.4;
  • the amplified DNA fragment encoding GLuc is Seq1 (its nucleotide sequence is shown in SEQ ID NO.1), and the amplified DNA fragment encoding Luc2 is Seq2 (its nucleotide sequence is shown in SEQ ID NO.2 shown);
  • the sequence of another primer R2 is as follows: CTAGATATGCTGTTATCCGTCGATT (SEQ ID NO.5) (commissioned by Sangon Bioengineering Co., Ltd. to synthesize), the amplified DNA fragment encoding GLuc is Seq3 (its nucleotide sequence is as shown in SEQ ID NO.1 1-1781 of SEQ ID NO.2), the amplified DNA fragment encoding Luc2 is Seq4 (its nucleotide sequence is shown in 1-2876 of SEQ ID NO.2).
  • the amplification program of the above PCR is as follows: pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s, annealing at 56°C for 15s, extension at 72°C for 40s, 30 cycles; final extension at 72°C for 5min.
  • the total volume of PCR is 300 ⁇ L.
  • reaction solution was combined into a 1.5ml Tube tube. Take 2 ⁇ L for DNA agarose gel electrophoresis detection to confirm the success of the reaction (1.5% agarose, 120V, 40min).
  • Eligibility criteria a single band appears in the electrophoresis test, and the size is correct.
  • Agarose gel electrophoresis first configure two 1.5% agarose gels (weigh 1.5g agarose and add it to 100ml of TAE solution), then carry out agarose gel electrophoresis to the above remaining PCR products, and then perform agarose gel electrophoresis on the blu-ray instrument Carry out cutting glue recovery under, and take weight. Seq1, Seq2, Seq3 and Seq4 were recovered and purified according to the Gel Extraction Kit DNA extraction kit.
  • Eligibility criteria 260/280 between 1.8 and 2.1, 260/230 between 1.6 and 2.2.
  • RNA in vitro synthesis system is constructed to synthesize RNA in vitro to obtain the precursor circular RNA.
  • the RNA synthesis system in vitro includes the following components in 100 ⁇ L:
  • the RNA synthesis in vitro is preferably carried out in a constant temperature reactor; the procedure for the RNA synthesis in vitro is preferably 37° C. for 4 hours.
  • the predicted objects include:
  • RNA precursor molecule encoding luciferase without a poly A tail (transcribed from the nucleotide sequence consisting of 1-2876 positions of SEQ ID NO.2).
  • RNA precursor molecule encoding luciferase with a poly A tail (transcribed from SEQ ID NO.2).
  • the circularization of the RNA is preferably achieved by adding GTP to a final concentration of 2 mM, then heating the reaction at 55°C for 8 minutes, taking a portion of the RNA and diluting it in 95% formamide loading, denaturing at 70°C for 3 minutes, and then cooling to room temperature, and then use a 1.5% agarose gel to detect RNA.
  • RNA 100 ⁇ g of RNA was diluted with DEPC H 2 O (final volume 86 ⁇ L), then heated at 70° C. for 3 minutes, then cooled on ice for 2 minutes. Add 20 U of Rnase R and 10 ⁇ L of 10 ⁇ Rnase R buffer (purchased from Lucigen brand), and incubate at 37° C. for 20 minutes. Part of the RNA was diluted in 95% formamide loading, denatured at 70°C for 3 minutes, cooled to room temperature, and then detected by 1.5% agarose gel. Three groups of parallel experiments were carried out, and 300 ng of RNA was taken from each group to be detected by agarose gel electrophoresis three times.
  • FIG. 3A The results of agarose gel electrophoresis are shown in Figure 3A and Figure 3B.
  • lanes 1-3 are products after circularization of circular RNA without poly A tails
  • lanes 4-12 are products of lanes 1-3 treated with Rnase R for 20 minutes
  • Lane 13 is the RiboRuler High Range RNA Ladder
  • lanes 14-16 are products after circularization of circular RNA with a poly A tail
  • lanes 17-25 are products of lanes 14-16 after being treated with RNase R for 20 minutes.
  • lane 1 is the RiboRuler High Range RNA Ladder
  • lanes 2-4 are the products of circular RNA without poly A tails
  • lanes 5-13 are the products of lanes 2-4
  • lanes 14-16 are products after circularization of circular RNA with a poly A tail
  • lanes 17-25 are products of lanes 14-16 after being treated with Rnase R for 20 minutes.
  • the introduction of poly A tails does not actually affect the circularization efficiency of RNA, and the circular RNA obtained after RNase R digestion is higher than that of RNA without poly A tails after RNase R digestion.
  • Thermo Fisher MEGAclear Transcription Clean-Up Kit mainly including:
  • RNA concentration of Seq1 recovered by the RNA purification kit was 1180ng/ ⁇ L
  • the RNA concentration of Seq2 was 1025ng/ ⁇ L
  • the RNA concentration of Seq3 was 1285ng/ ⁇ L
  • the RNA concentration of Seq4 was 1204ng/ ⁇ L.
  • Embodiment 7 Circular RNA precursor molecule comparative test
  • the product purity of the circularized vector with poly A tail and the circularized vector without poly A tail provided by the present invention was digested with Rnase R at different time gradients.
  • the final reaction result is shown in Figure 4.
  • the circularized vector with poly-A tail can be treated with 1 U of RNase R after circularization to obtain circular RNA with higher purity than that without poly-A tail within 20 minutes.
  • Cell preparation Prepare cells for detection 3 days in advance.
  • the 293T cells purchased from the cell bank of the Chinese Academy of Sciences were passaged in cell culture flasks to ensure that the cells were in the logarithmic growth phase when used.
  • Cell dilution Take the cell suspension, dilute it to 2 ⁇ 10 5 cells/ml with DMEM medium containing 10% FBS, and mix by pipetting.
  • RNA inoculation 0.5ml of cell suspension was added to a 24-well plate. For each RNA sample, 3 wells of parallel cells should be prepared, and 1 well for blank control. The 24-well plate was placed in a (37 ⁇ 1)°C, (5 ⁇ 0.5)% CO 2 incubator for overnight cultivation.
  • RNA samples (circRNA synthesized by Seq1 and Seq2 transcription: circRNA Luc and circRNA Luc+A ), the negative control Linear Luc RNA or the Linear Luc RNA (N1) modified by methylpseudouracil (N1) were mixed with 25 ⁇ L opti-MEM by gentle pipetting, and then added to the opti-MEM medium containing Lipofectamine MessengerMax , and immediately place it on a vortex shaker for 10 times, 1s each time, mix thoroughly, and let stand for 5min. Three parallel experiments were performed in each group.
  • the cytotoxicity of transfected cells produced by introducing poly A tails to the precursor circular RNA molecules is less than that of linear RNAs and linear RNAs with nucleotide modifications.
  • Example 9 The expression of luciferase in the circular RNA precursor molecule encoding luciferase for 24 hours
  • the cells were replaced with 0.5 mL of fresh medium (90% DMEM+10% FBS) per well.
  • Luciferase detection was performed after 24 hours of culture, and the Bright-LiteTM Luciferase Assay system detection kit was selected for luciferase activity detection. After mixing the solution in the kit and the substrate components evenly in the dark room, take 0.5mL of the detection reagent and cells to mix and lyse. After 2min, take 150 ⁇ L of the lysate and add it to the pre-prepared white plate, quickly The luciferase activity was detected by a microplate reader, and each group was subjected to 2 parallel experiments. The detection results of luciferase activity are shown in Table 3 and Figure 6.
  • Example 10 The impact of poly A tails of different lengths on cyclization efficiency
  • the plasmid DNA of the synthesized luciferase Luc2 was amplified as a DNA template, and the reaction system was as follows:
  • the PCR amplification system is calculated as 50 ⁇ L, and each component is as follows:
  • PrimeSTAR Max Premix (2 ⁇ ) includes the following components: PrimeSTAR Max DNA Polymerase, dNTPs and Mg 2+ .
  • the initial concentration of primer F and primer R is preferably 10 ⁇ mol/L; the concentration of the DNA template is preferably 1 ng/ ⁇ L.
  • primer F The sequence of primer F is shown in SEQ ID NO.3;
  • primer R1 The sequence of primer R1 is shown in SEQ ID NO.4;
  • the sequence of the primer R3 is as follows:
  • the sequence of the primer R4 is as follows:
  • the sequence of the primer R5 is as follows:
  • the sequence of the primer R6 is as follows:
  • the sequence of the primer R7 is as follows:
  • the sequence of the primer R8 is as follows:
  • the sequence of the primer R9 is as follows:
  • the amplification program of the above PCR is as follows: pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s, annealing at 56°C for 15s, extension at 72°C for 40s, 30 cycles; final extension at 72°C for 5min.
  • the total volume of PCR is 300 ⁇ L.
  • reaction solution was combined into a 1.5ml Tube tube. Take 2 ⁇ L for DNA agarose gel electrophoresis detection to confirm the success of the reaction (1.5% agarose, 120V, 40min).
  • Eligibility criteria a single band appears in the electrophoresis test, and the size is correct.
  • Agarose gel electrophoresis first configure two 1.5% agarose gels (weigh 1.5g agarose and add it to 100ml of TAE solution), then carry out agarose gel electrophoresis to the above remaining PCR products, and then perform agarose gel electrophoresis on the blu-ray instrument Carry out cutting glue recovery under, and take weight.
  • the Gel Extraction Kit DNA extraction kit Seq5, Seq6, Seq7, Seq8, Seq9, Seq10 and Seq11 (corresponding to the fragments amplified by the amplification primers R3-R9 respectively) were recovered and purified.
  • Eligibility criteria 260/280 between 1.8 and 2.1, 260/230 between 1.6 and 2.2.
  • RNA in vitro synthesis system is constructed to perform RNA in vitro synthesis to obtain the precursor circular RNA.
  • the RNA synthesis system in vitro includes the following components in 100 ⁇ L:
  • the RNA synthesis in vitro is preferably carried out in a constant temperature reactor; the procedure for the RNA synthesis in vitro is preferably 37° C. for 4 hours.
  • Circularization of the RNA is preferably achieved by adding GTP to a final concentration of 2 mM and then heating the reaction at 55°C for 8 minutes.
  • the RNA concentration of Seq5 recovered by the RNA purification kit is 1428ng/ ⁇ L
  • the RNA concentration of Seq6 is 1556ng/ ⁇ L
  • the RNA concentration of Seq7 is 1542ng/ ⁇ L
  • the RNA concentration of Seq8 is 1391ng/ ⁇ L
  • the RNA concentration of Seq9 is 1516ng/ ⁇ L
  • the RNA concentration of Seq10 was 1331ng/ ⁇ L
  • the RNA concentration of Seq11 was 1173ng/ ⁇ L.
  • RNA Take an equal volume of RNA and dilute it in 95% formamide loading, denature at 70°C for 3 minutes, cool to room temperature, and then use 1.5% agarose gel to detect RNA products.
  • Circularized vectors with poly A tails of different lengths can be treated with 1 U of RNase R after circularization to obtain circular RNA with high purity within 20 minutes.

Abstract

一种用于制备环状RNA的载体，其包含能够转录为环状RNA前体分子的DNA序列，所述环状RNA前体分子的3'端带有多聚A尾。带有多聚A尾的环状RNA前体分子进行体外环化得到环状RNA的过程中，Rnase R的处理时间缩短，降低了环状RNA的损失，在提高环状RNA的富集效率的同时提高产物的纯度；并且所述体外环化后的RNA分子在转染到真核细胞中时细胞毒性降低。提供用于制备环状RNA载体的制备方法，以及所述环状RNA前体分子的制备方法。

Description

一种用于制备环状RNA的载体及其应用 技术领域
本发明涉及分子生物学技术领域，尤其涉及提供一种用于制备环状RNA的载体及其应用。
背景技术
环状RNA(circular RNA,circRNA)与传统的线性RNA(linear RNA)不同，circRNA分子呈封闭环状结构，不受RNA外切酶影响，不易降解，表达更稳定。Wesselhoeft,R.A.等曾报道基于I型自剪切内含子设计的高效RNA环化工具，其使用的环化载体包含了柯萨奇病毒的IRES元件(CVB3 IRES)、Gaussia荧光素酶(GLuc)和Anabaena pre-tRNA的I型内含子，将Anabaena pre-tRNA的I型内含子拆分后设计在待环化序列的两侧。在GTP和镁离子存在的条件下，环状RNA前体分子能通过双酯交换反应实现自动环化，但是在生成环状RNA的过程中也会产生大量的副产物，例如未成环的环状RNA前体分子以及被剪切下来的内含子片段等。目前，主流的富集纯化环状RNA的方法是利用Rnase R进行处理，但Rnase R在性质上不稳定，不同批次酶的差异性大，易导致环状RNA纯化富集效果不稳定；而且Rnase R不仅能消化线性RNA，还能消化部分环状RNA，导致环状RNA纯化富集过程中部分环状RNA的丢失。因此，对于纯化富集环状RNA，需要尽量缩短Rnase R的处理时间以减少环状RNA的损失，在提高环状RNA的富集效率的同时提高产物的纯度，最终在转染细胞时降低其带来的细胞毒性。
M.Puttaraju等在Group I permuted intron-exon(PIE)sequences self-splice to produce circular exon中报道了通过I型内含子的设计实现环状RNA的制备，WO2019236673A1记载了一种用于在真核细胞中翻译的环状RNA，并对制备此种环状RNA使用的载体进行了描述，能够实现环状RNA的制备，但均无法解决上述纯化富集环状RNA的同时缩短Rnase R处理时间、提高环状RNA的富集效率、降低细胞毒性的问题。
发明内容
本发明的一个目的在于提供一种用于制备环状RNA的载体。
本发明的另一目的在于提供一种环状RNA前体分子。
本发明的另一目的在于提供所述环状RNA前体分子的制备方法。
本发明的另一目的在于提供所述载体在制备环状RNA中的应用。
本发明的另一目的在于提供一种环状RNA的制备方法。
本发明通过在载体中设计可以转录为环状RNA前体分子多聚A尾的DNA序列，完成引入多聚A尾的环状RNA前体分子的制备，可在制备环状RNA过程中，解决环状RNA富集效率、环状RNA产物纯度和/或转染细胞后带来的细胞毒性等问题。
具体而言，一方面，本发明提供了一种用于制备环状RNA的载体，其包含能够转录为环状RNA前体分子的DNA序列，所述环状RNA前体分子的3’端带有多聚A尾。
根据本发明的具体实施方案，本发明的用于制备环状RNA的载体中，所述的多聚A尾，长度为5～150个A。具体地，所述多聚A尾长度为5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、20、21、22、23、24、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、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99、100、101、102、103、104、105、106、107、108、109、110、111、112、113、114、115、116、117、118、119、120、121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、145、146、147、148、149或150个A。
根据本发明的具体实施方案，本发明的用于制备环状RNA的载体，包含以下顺序排列的元件：
Anabaena pre-tRNA 3’I型内含子片段；
内部核糖体进入位点(IRES)；
蛋白质编码区或非编码区；
Anabaena pre-tRNA 5’I型内含子片段；
能转录为所述多聚A尾的DNA序列。
根据本发明的具体实施方案，本发明的用于制备环状RNA的载体，还可包含以下元件中的一种或多种：
5’同源臂；插入在所述的Anabaena pre-tRNA 3’I型内含子片段中(可以是在型内含子片段序列的中间，或是靠近5’的位置)；
5’间隔子序列；在所述的Anabaena pre-tRNA 3’I型内含子片段与所述的内部核糖体进入位点之间；
3’间隔子序列；在所述的蛋白质编码区或非编码区与在所述的Anabaena pre-tRNA  5’I型内含子片段之间；
3’同源臂；插入在所述的Anabaena pre-tRNA 5’I型内含子片段中(可以是在型内含子片段序列的中间，或是靠近3’的位置)。
根据本发明的具体实施方案，本发明的用于制备环状RNA的载体中，所述Anabaena pre-tRNA 3’I型内含子片段、内部核糖体进入位点(IRES)、蛋白质编码区或非编码区、Anabaena pre-tRNA 5’I型内含子片段、5’同源臂、5’间隔子序列、3’间隔子序列、3’同源臂的具体序列可各自采用所属领域的已知片段序列。例如可采用WO2019236673A1记载的片段或非编码区。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述Anabaena pre-tRNA 3’I型内含子片段如SEQ ID NO.1或SEQ ID NO.2中所示的3’I型内含子片段。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述Anabaena pre-tRNA 3’I型内含子片段如SEQ ID NO.1或SEQ ID NO.2中所示的3’I型内含子片段。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述IRES如SEQ ID NO.1或SEQ ID NO.2中所示的IRES。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述Anabaena pre-tRNA 5’I型内含子片段如SEQ ID NO.1或SEQ ID NO.2中所示的5’I型内含子片段。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述5’同源臂如SEQ ID NO.1或SEQ ID NO.2中所示的5’同源臂。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述5’间隔子序列如SEQ ID NO.1或SEQ ID NO.2中所示的5’间隔子序列。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述3’同源臂如SEQ ID NO.1或SEQ ID NO.2中所示的3’同源臂。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述3’间隔子序列如SEQ ID NO.1或SEQ ID NO.2中所示的3’间隔子序列。
在本发明的一些具体实施方案中，本发明的用于制备环状RNA的载体，其具有SEQ ID NO.1所示序列的第1～1017位核苷酸序列和/或第1576～1781位核苷酸序列。
在本发明的一些具体实施方案中，本发明的用于制备环状RNA的载体中，所述环 状RNA可编码hFIX、SP-B、VEGF-A、人甲基丙二酰辅酶A变异酶、CFTR、癌症自身抗原、基因编辑酶、HIV抗体、CD19抗体、CD22抗体、CD3抗体、CLDN6抗体、Luc2、GLuc、Fluc、eGFP、hEPO、Cas9核酸内切酶、新型冠状病毒S蛋白或肿瘤抑制因子。换而言之，所述用于制备环状RNA的载体中的蛋白质编码区为编码这些蛋白的核苷酸片段。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体中，所述环状RNA编码Luc2或GLuc。
在本发明的一些具体实施方案中，本发明的用于制备环状RNA的载体中，蛋白质编码区或非编码区为Luc2基因；在本发明的另一些具体实施方案中，本发明的用于制备环状RNA的载体中，蛋白质编码区或非编码区为GLuc基因；两种报告基因验证了本发明技术方案的可行性。
根据本发明的一些具体实施方案，本发明的用于制备环状RNA的载体，其具有SEQ ID NO.1或SEQ ID NO.2所示序列，蛋白质编码区或非编码区使用了报告基因序列，验证了本发明技术方案的可行性。
另一方面，本发明还提供了一种环状RNA前体分子，其是通过本发明所述的用于制备环状RNA的载体进行体外转录得到的。
另一方面，本发明还提供了所述的载体在制备环状RNA中的应用。
另一方面，本发明还提供了种环状RNA前体分子的制备方法，其包括步骤：
a.将本发明所述的用于制备环状RNA的载体进行PCR扩增，获得带有多聚A尾的体外表达RNA的DNA模板；
b.构建包括a所述的DNA模板在内的RNA体外合成体系，进行RNA的体外合成获得可以环化的RNA前体分子。
本发明中，将多聚A尾在体外合成线性mRNA的制备中使用，用于保护mRNA免受核酸外切酶的攻击，旨在提升mRNA的稳定性，并且维持mRNA作为翻译模板的活性。环状RNA的载体设计中，现有技术均未考虑引入多聚A尾，原因在于环状RNA呈闭合环状结构，不易被核酸外切酶降解，比线性RNA更加稳定，无需引入多聚A尾，而且在环状RNA前体分子中引入序列后可能存在影响环状RNA前体分子的结构、导致环化异常的问题。本发明克服了这种偏见，创造性地将可以转录为环状RNA前体分子多聚A尾的DNA序列引入了用于制备环状RNA的载体，从而制备了带有多聚A尾的环状RNA前体分子。这种多聚A尾的创造性使用，在环状RNA前体分子二级结构预 测中说明未影响其环化效率。在实施例的试验中证明，并不会严重影响环状RNA前体分子的结构、不会影响其成环效率，而且纯化得到的环状RNA产生的细胞毒性相比于线性RNA、经过核苷酸修饰的线性RNA以及通过同样方式纯化得到的不带多聚A尾的环状RNA水平降低。
在本发明的一些具体实施方案中，本发明首先通过载体构建或PCR扩增的方式在未发生环化反应的环状RNA前体分子载体的3'末端引入多聚A尾，随后在转录反应结束后向体系中加入终浓度为2mM的GTP，并在55℃条件下环化RNA，在这个过程中RNA 3'末端带有的多聚A尾将在双酯交换过程中被剪切下来。然后，利用Rnase R在37℃处理20min以消化剪接产物。最后使用MEGAclear Transcription Clean-Up Kit(购买自Invitrogen品牌)纯化得到最终环状RNA产物。
另一方面，本发明还提供了一种环状RNA的制备方法，其包括步骤：
以本发明所述的用于制备环状RNA的载体制备环状RNA前体分子；
对环状RNA前体分子进行环化，得到环状RNA。
根据本发明的一些具体实施方案，本发明的环状RNA的制备方法，包括以下步骤：
1)合成转录所述环状RNA前体分子中的编码的荧光素酶(Luc2)以及Gaussia荧光素酶(GLuc)RNA的质粒DNA(合成自生工生物工程有限公司)；
2)以所述合成获得的质粒为模板进行PCR扩增获得带有多聚A尾的体外表达RNA的DNA模板；
3)构建包括所述DNA模板的RNA体外合成体系进行RNA的体外合成获得所述编码Luc2以及GLuc的RNA。
优选地，所述RNA体外合成体系以100μL计，包括以下组分：
Figure PCTCN2022090198-appb-000001
优选地，所述RNA体外合成的程序为37℃，4h。
优选地，步骤3)获得所述编码Luc2以及GLuc的环状RNA前体分子后还包括：转录产物的环化反应，Rnase R消化反应，RNA纯化反应，调节所述编码Luc2以及GLuc的RNA浓度以及分装的步骤。
本发明中所述的“前体分子”，通过体外转录(例如，由本发明提供的载体)产生的线性RNA分子。该前体RNA分子包含完整的circRNA序列，使RNA环化所需的剪接序列(内含子片段和同源臂)以及多聚A尾。这些剪接序列(内含子片段和同源臂)及多聚A尾在环化过程中从前体RNA中除去，产生circRNA和两个内含子/同源臂及带有多聚A尾的线性RNA片段。
本发明的有益效果
本发明提供一种用于制备环状RNA的载体，通过设计可以转录为环状RNA前体分子多聚A尾的DNA序列，在体外合成环状RNA前体分子的3’末端引入了多聚A尾。从而使得环状RNA前体分子在环化过程中，副产物更易于被Rnase R消化、缩短了Rnase R的处理时间、减少了Rnase R的使用量。而且保证引入的多聚A尾并不会严重影响环状RNA前体分子的结构，使得RNA的环化效率降低，通过这种方法纯化得到的环状RNA产生的细胞毒性相比于线性的，经过核苷酸修饰的线性RNA以及通过同样方式纯化的不带有多聚A尾的环状RNA水平降低。
附图说明
图1为编码Luc2的环状RNA前体分子的结构示意图。
图2为通过RNAFold基于最小自由能预测的编码Luc2不带有多聚A尾以及带有多聚A尾的环状RNA前体分子的二级结构示意图。
图3A和图3B为分别编码GLuc和Luc2的不带有多聚A尾以及带有多聚A尾的环状RNA前体分子环化后通过Rnase R消化20min后的琼脂糖凝胶电泳检测图。
图4为编码Luc2不带有多聚A尾以及带有多聚A尾的环状RNA前体分子环化后通过Rnase R消化时间梯度的琼脂糖凝胶电泳检测图。
图5为编码Luc2不带有多聚A尾以及带有多聚A尾的环状RNA前体分子环化纯化后转染293T细胞的细胞活力检测图。
图6为编码Luc2不带有多聚A尾以及带有多聚A尾的环状RNA前体分子环化纯化后转染293T细胞的荧光素酶活性检测图。
图7为编码Luc2带有不同长度多聚A尾的环状RNA前体分子环化后通过Rnase R消化20min后的琼脂糖凝胶电泳检测图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。下文中未详细注明的操作方法，可以按照所属领域的常规操作或是仪器厂商建议的条件进行。
本发明提供了一种基于I型内含子自剪接后环状RNA纯化的方法。在一些具体实施方案中，是使用一种载体制备编码GLuc的环状RNA前体分子，所述载体包括SEQ ID NO.1所示序列(SEQ ID NO.1所示序列组成的核苷酸片段本发明中亦称为Seq1)。在另一些具体实施方案中，是使用一种环化载体制备编码Luc2的环状RNA前体分子，所述载体包括SEQ ID NO.2所示序列(SEQ ID NO.2所示序列组成的核苷酸片段本发明中亦称为Seq2)。
在本发明中，所述使用的环化载体中circRNA包含了柯萨奇病毒的IRES元件(CVB3 IRES)、荧光素酶(Luc2)、3'末端的多聚A尾以及Anabaena pre-tRNA的I型内含子，将其拆分后设计在待环化序列的两侧，具体结构如图1所示。
在本发明中，所述引物F和引物R1(合成自生工生物工程有限公司)的初始浓度优选为10μmol/L；所述DNA模板的浓度优选为1ng/μL。
所述引物F的序列如下：
Figure PCTCN2022090198-appb-000002
所述引物R1的序列如下：
Figure PCTCN2022090198-appb-000003
在本发明中，所述PrimeSTAR Max Premix(2×)包括以下组分：PrimeSTAR Max DNA Polymerase，dNTPs和Mg 2+。在本发明中，所述PCR的扩增程序优选如下：预变性98℃3min；变性98℃10s，退火56℃15s，延伸72℃40s，30个循环；最后延伸72℃，5min。
在本发明中，所述PCR扩增反应结束后，优选地对扩增产物进行琼脂糖凝胶电泳检测以确定反应是否成功；所述琼脂糖凝胶电泳检测的参数优选如下：1.5％琼脂糖，120V，40min。在本发明中，当琼脂糖凝胶电泳出现目的条带认为反应成功。
本发明在所述PCR扩增反应结束后，优选地，将所述电泳产物进行DNA回收提取。在本发明中，所述DNA提取试剂盒为Gel Extraction Kit(购买自Omega品牌)；本发明在所述纯化后优选地采用NanoDrop检测纯化后模板的浓度，以及260/280、260/230 的比值，当260/280在1.6～1.8之间，认为模板合格。
本发明在获得所述DNA模板后，构建包括所述DNA模板的RNA体外合成体系进行RNA的体外合成获得所述前体circRNA。优选地，所述RNA体外合成体系以100μL计，包括以下组分：
Figure PCTCN2022090198-appb-000004
在本发明中，所述Enzyme Mix包括T7RNA聚合酶，RNA酶抑制剂和无机焦磷酸酶。在本发明中，所述RNA体外合成的程序优选为37℃，4h。在本发明中，所述RNA体外合成优选在恒温反应器中进行；所述RNA体外合成体系优选置于1.5ml RNase-free Tube管中，一次同时反应多管；所述RNA体外合成体系中的反应试剂按照上述顺序添加。
本发明在所述RNA体外合成结束后，优选地，还包括去除DNA模板、RNA的环化、Rnase R消化和纯化circRNA的步骤。
在本发明中，所述去除DNA模板优选通过DNase I消化实现；所述消化优选包括将DNase I与RNA体外合成反应后的溶液混合后进行；所述DNase I与RNA体外合成反应后的溶液的体积比优选为1:20；本发明在所述混合后，优选地，进行离心将溶液收集至RNase-free Tube管底部。在本发明中，所述离心的转速优选为800～1200rpm，更优选为1000rpm；所述离心的时间优选为8～12s，更优选为10s。所述消化的温度优选为37℃；所述消化的时间优选为1h。本发明在所述消化结束后，优选地，进行DNA片段残留检测。
在本发明中，所述RNA的环化优选通过将GTP添加至终浓度为2mM，然后将反应在55℃加热8分钟实现，取部分RNA稀释在95％甲酰胺loading中，在70℃变性3分钟后，冷却至室温。然后使用1.5％琼脂糖凝胶检测RNA。
本发明在环化RNA后，需要进行Rnase R的消化以去除环化过程中产生的副产物， 用DEPC H2O稀释20μg RNA(最终体积为86μL)，然后在70℃加热3分钟，然后在冰上冷却2分钟。加入20U Rnase R和10μL的10x Rnase R缓冲液，并在37℃温育20分钟。取部分RNA稀释在95％甲酰胺loading中，在70℃变性3分钟后，冷却至室温。然后使用1.5％琼脂糖凝胶检测RNA。
在本发明中，对进行Rnase R处理后的产物进行柱纯化，优选地，RNA柱纯化试剂盒为MEGAclear Transcription Clean-Up Kit，取部分RNA稀释95％甲酰胺loading中，在70℃变性3分钟后，冷却至室温。然后使用1.5％琼脂糖凝胶检测RNA。
在本发明中，对所述circRNA进行质量检测，其中包括circRNA的浓度、RNA的260/280、260/230的比值。当260/280范围为1.8～2.1，260/230范围为大于2.0时，认为circRNA合格。
在本发明中，对所述合成的circRNA进行细胞活力以及荧光素酶表达检测，具体操作如下：
1)细胞准备：提前3天准备检测用细胞。取购自中科院细胞库的293T细胞，传代于细胞培养瓶内，保证使用时细胞处于对数生长期。
2)细胞消化计数：取生长状态良好的293T细胞，去掉培养基，以2ml PBS清洗细胞后，加入体积百分含量为0.25％的胰酶在37℃消化1min，然后加入含10％FBS的DMEM培养基中和胰酶，吹打细胞并转至15ml离心管，反复吹打混匀，然后取20μL的细胞悬液使用台盼蓝染色，并通过细胞计数仪进行计数。
3)细胞稀释：取细胞悬液，用含10％FBS的DMEM培养基稀释到2×10 5个/ml，吹打混匀。
4)细胞接种：取0.5ml细胞悬液加到24孔板内。每个RNA样品需要准备3孔平行细胞，空白对照1孔。将24孔板放入(37±1)℃、(5±0.5)％CO 2培养箱培养过夜。
5)细胞转染：接种完细胞后约24h，观察24孔板内的细胞状态，汇合度在70％左右。在生物安全柜内，配制所需体积的90％DMEM+10％FBS培养基。转染前30min弃掉孔板的培养基，每孔加入0.5ml新鲜培养基(95％opti-MEM+5％FBS)。
a)配制转染体系：取25μL opti-MEM，加入0.75μL Lipofectamine MessengerMax在室温下混匀后静置10分钟，取0.5μg RNA样品(Seq1、Seq2转录合成的circRNA),阴性对照Linear Luc RNA或通过甲基假尿嘧啶修饰(N1)的Linear Luc RNA分别与25μL opti-MEM轻轻吹打混匀，再加入到含有Lipofectamine MessengerMax的opti-MEM培养基中，立即置于漩涡振荡器上振荡10次，每次1s，充分混匀，静置5min。
b)将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。
c)换液：转染后6h换液，吸掉旧的培养基，每孔换为500μL新鲜培养基(90％DMEM+10％FBS)。
6)细胞活力检测：向每孔中加入50μL Cell Counting Kit-8(CCK-8)溶液(购买自MedChemExpress品牌)，在加完试剂后轻轻晃动培养板以帮助混匀。将培养板放入培养箱中孵育1-4h，用酶标仪测定450nm处的吸光度(OD)。
7)荧光素酶活性检测：在本发明中，优选地，选用Bright-LiteTM Luciferase Assay system检测试剂盒(购买自Vazyme品牌)进行荧光素酶活性检测。将试剂盒中溶液与底物两组分进行混合均匀，取与培养液等体积的检测试剂进行混合细胞，2min后通过酶标仪进行检测。
实施例1 用于制备GLuc环状RNA的载体的设计、制备
设计载体包含以下顺序排列的元件：
5’同源臂；
Anabaena pre-tRNA 3’I型内含子片段；
5’间隔子序列；
柯萨奇病毒内部核糖体进入位点(CVB3 IRES)；
GLuc编码区；
3’间隔子序列；
Anabaena pre-tRNA 5’I型内含子片段；
3’同源臂；
可以转录为GLuc环状RNA前体分子多聚A尾的DNA序列。
用于制备GLuc环状RNA的载体序列如SEQ ID NO.1所示。
Figure PCTCN2022090198-appb-000005
Figure PCTCN2022090198-appb-000006
根据载体序列进行质粒DNA的制备(可委托生工生物工程有限公司合成)。
实施例2 用于制备Luc2环状RNA的载体的设计
设计载体包含以下顺序排列的元件：
5’同源臂；
Anabaena pre-tRNA 3’I型内含子片段；
5’间隔子序列；
柯萨奇病毒内部核糖体进入位点(CVB3 IRES)；
荧光素酶Luc2编码区；
3’间隔子序列；
Anabaena pre-tRNA 5’I型内含子片段；
3’同源臂；
可以转录为环状RNA前体分子多聚A尾的DNA序列。
用于制备Luc2环状RNA的载体序列如SEQ ID NO.2所示。
Figure PCTCN2022090198-appb-000007
Figure PCTCN2022090198-appb-000008
SEQ ID NO.2中，第1-206位核苷酸序列为3’Intron，第5-24位核苷酸序列为插入3’Intron中的5’Homology arms，第207-276位核苷酸序列为5’Spacers，第277-1017位核苷酸序列为IRES序列，第1018-2670位核苷酸序列为Luc基因，第2671-2710位核苷酸序列为3’Spacers，第2711-2876位核苷酸序列为5’Intron，第2841-2860位核苷酸 序列为插入5’Intron中的3’Homology arms，第2877-2976位核苷酸序列为多聚A尾。
根据载体序列进行质粒DNA的制备(可委托生工生物工程有限公司合成)。
实施例3 DNA模板的扩增及纯化
对合成得到的荧光素酶GLuc以及Luc2的质粒DNA分别进行DNA模板的扩增，反应体系如下：
反应体积，50μL(为单个管的反应体积，一次同时反应多管)，所述PCR扩增的体系以50μL计，各组分如下：
Figure PCTCN2022090198-appb-000009
PrimeSTAR Max Premix(2×)包括以下组分：PrimeSTAR Max DNA Polymerase，dNTPs和Mg 2+。引物F和引物R的初始浓度优选为10μmol/L；所述DNA模板的浓度优选为1ng/μL。
引物F的序列如SEQ ID NO.3所示；
引物R1的序列如SEQ ID NO.4所示；
扩增所得的编码GLuc的DNA片段为Seq1(其核苷酸序列如SEQ ID NO.1所示)，扩增所得的编码Luc2的DNA片段为Seq2(其核苷酸序列如SEQ ID NO.2所示)；
另一引物R2的序列如下：CTAGATATGCTGTTATCCGTCGATT(SEQ ID NO.5)(委托生工生物工程有限公司合成)，扩增所得的编码GLuc的DNA片段为Seq3(其核苷酸序列如SEQ ID NO.1的第1-1781位所示)，扩增所得的编码Luc2的DNA片段为Seq4(其核苷酸序列如SEQ ID NO.2的第1-2876位所示)。
上述PCR的扩增程序如下：预变性98℃3min；变性98℃10s，退火56℃15s，延伸72℃40s，30个循环；最后延伸72℃，5min。PCR总体系为300μL。
反应结束后，将反应液合并于1.5ml Tube管中。取2μL进行DNA琼脂糖凝胶电泳检测以确定反应成功(1.5％琼脂糖，120V，40min)。
合格标准：电泳检测出现单一的条带，且大小正确。
测定结果：条带大小单一，大小符合要求。
琼脂糖凝胶电泳：先配置两块1.5％的琼脂糖凝胶(称取1.5g琼脂糖加入100ml的 TAE溶液中)，随后将上述剩余的PCR产物进行琼脂糖凝胶电泳，随后在蓝光仪下进行切胶回收，并称取重量。按照Gel Extraction Kit DNA提取试剂盒对Seq1，Seq2，Seq3以及Seq4进行回收纯化。
合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。
测定结果：Seq1浓度为237ng/μL，260/280＝1.89，260/230＝1.80；Seq2浓度为360ng/μL，260/280＝1.90，260/230＝1.70；Seq3浓度为196ng/μL，260/280＝1.89，260/230＝1.81；Seq4浓度为223ng/μL，260/280＝1.90，260/230＝2.10。
最终用RNase-free水稀释至150ng/μL。
实施例4 环状RNA前体分子的体外合成
在获得所述Seq1，Seq2，Seq3以及Seq4的DNA模板后，构建RNA体外合成体系进行RNA的体外合成获得所述前体环状RNA。所述RNA体外合成体系以100μL计，包括以下组分：
Figure PCTCN2022090198-appb-000010
所述RNA体外合成优选在恒温反应器中进行；所述RNA体外合成的程序优选为37℃，4h。
体外合成带有多聚A尾的RNA的示意图结果如图1所示。
实施例5 环状RNA前体分子二级结构预测
通过RNAFold网站基于最小自由能进行二级结构预测，预测对象包括：
1)编码荧光素酶不带有多聚A尾的环状RNA前体分子(SEQ ID NO.2的第1-2876位组成的核苷酸序列转录而成)。
2)编码荧光素酶带有多聚A尾的环状RNA前体分子(SEQ ID NO.2转录而成)。
结果如图2所示，说明：对于在前体环状RNA序列后添加多聚A尾对前体环状RNA 分子的二级结构并未产生较大的影响。
实施例6 环化RNA的制备及纯化
1)DNase I消化去除DNA模板
向RNA体外合成后的Tube管中各加入5μL DNase I。上下颠倒10次混匀，1000rpm离心10s。重新置于恒温反应器中，37℃，1h。
2)RNA的体外环化
所述RNA的环化优选通过将GTP添加至终浓度为2mM，然后将反应在55℃加热8分钟实现，取部分RNA稀释在95％甲酰胺loading中，在70℃变性3分钟后，冷却至室温，然后使用1.5％琼脂糖凝胶检测RNA。
3)Rnase R消化
用DEPC H 2O稀释100μg RNA(最终体积为86μL)，然后在70℃加热3分钟，然后在冰上冷却2分钟。加入20U Rnase R和10μL的10x Rnase R缓冲液(购买自Lucigen品牌)，并在37℃温育20分钟。取部分RNA稀释在95％甲酰胺loading中，在70℃变性3分钟后，冷却至室温，然后使用1.5％琼脂糖凝胶检测RNA。进行3组平行实验，每组分别取300ng的RNA进行3次琼脂糖凝胶电泳检测，琼脂糖凝胶电泳结果如图3A和图3B所示。图3A中，从左至右，泳道1-3为不带有多聚A尾的环状RNA环化后的产物，泳道4-12为泳道1-3产物利用Rnase R处理20min后的产物，泳道13为RiboRuler High Range RNA Ladder，泳道14-16为带有多聚A尾的环状RNA环化后的产物，泳道17-25为泳道14-16产物利用Rnase R处理20min后的产物。图3B中，从左至右，泳道1为RiboRuler High Range RNA Ladder，泳道2-4为不带有多聚A尾的环状RNA环化后的产物，泳道5-13为泳道2-4产物利用Rnase R处理20min后的产物，泳道14-16为带有多聚A尾的环状RNA环化后的产物，泳道17-25为泳道14-16产物利用Rnase R处理20min后的产物。多聚A尾的引入实际不会影响RNA的环化效率，而且通过Rnase R消化后相比于不带有多聚A尾的RNA经过Rnase R消化后所得到的环状RNA纯度更高。
4)环状RNA的纯化
按照赛默飞MEGAclear Transcription Clean-Up Kit的说明书进行，主要包括：
向样品中加入350μL结合缓冲液并轻轻混合；
向样品中加入250μL的100％乙醇并轻轻混合；
将样品加入纯化柱内，14,000×g离心1分钟；
用2×500μL洗涤液洗涤，14,000×g离心1分钟；
将50μL洗脱液加入至纯化柱内，在设置为70℃的金属浴中孵育5分钟，14,000×g离心1分钟，回收洗脱的RNA。
实际通过RNA纯化试剂盒回收到的Seq1的RNA浓度为1180ng/μL，Seq2的RNA浓度为1025ng/μL，Seq3的RNA浓度为1285ng/μL，Seq4的RNA浓度为1204ng/μL。
实施例7 环状RNA前体分子比较试验
检测本发明提供的带多聚A尾的环化载体以及不带多聚A尾的环化载体在不同时间梯度使用Rnase R消化的产物纯度。
取120μg的带多聚A尾以及不带多聚A尾的环化载体环化后的RNA，在37℃的反应条件下，使用1U的Rnase R分别在0，1，5，10，20和30分钟进行消化处理，最终反应体系如表1所示。取等体积的RNA稀释在95％甲酰胺loading中，在70℃变性3分钟后，冷却至室温，然后使用1.5％琼脂糖凝胶检测RNA产物。
表1带有多聚A尾与无多聚A尾环化载体使用Rnase R消化的反应体系
Figure PCTCN2022090198-appb-000011
最终反应结果如图4所示。带有多聚A尾的环化载体在环化后使用1U的Rnase R处理能在20min内得到相比于不带多聚A尾的纯度更高的环状RNA。
实施例8 编码荧光素酶的环状RNA转染细胞后细胞活力的检测
1)细胞准备：提前3天准备检测用细胞。取购自中科院细胞库的293T细胞，传代于细胞培养瓶内，保证使用时细胞处于对数生长期。
2)细胞消化计数：取生长状态良好的293T细胞，去掉培养基，以2ml PBS清洗细 胞后，加入体积百分含量为0.25％的胰酶在37℃消化1min，然后加入含10％FBS的DMEM培养基中和胰酶，吹打细胞并转至15ml离心管，反复吹打混匀，然后取20μL的细胞悬液使用台盼蓝染色，并通过细胞计数仪进行计数。
3)细胞稀释：取细胞悬液，用含10％FBS的DMEM培养基稀释到2×10 5个/ml，吹打混匀。
4)细胞接种：取0.5ml细胞悬液加到24孔板内。每个RNA样品需要准备3孔平行细胞，空白对照1孔。将24孔板放入(37±1)℃、(5±0.5)％CO 2培养箱培养过夜。
5)细胞转染：接种完细胞后约24h，观察24孔板内的细胞状态，汇合度在70％左右。在生物安全柜内，配制所需体积的90％DMEM+10％FBS培养基。转染前30min弃掉孔板的培养基，每孔加入0.5ml新鲜培养基(95％opti-MEM+5％FBS)。
a)配制转染体系：取25μL opti-MEM，加入0.75μL Lipofectamine MessengerMax在室温下混匀后静置10分钟，取0.5μg RNA样品(Seq1、Seq2转录合成的circRNA：circRNA Luc以及circRNA Luc+A)，阴性对照Linear Luc RNA或通过甲基假尿嘧啶修饰(N1)的Linear Luc RNA(N1)分别与25μL opti-MEM轻轻吹打混匀，再加入到含有Lipofectamine MessengerMax的opti-MEM培养基中，立即置于漩涡振荡器上振荡10次，每次1s，充分混匀，静置5min。每组进行3次平行实验。
b)将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。
c)换液：转染后6h换液，吸掉旧的培养基，每孔换为500μL新鲜培养基(90％DMEM+10％FBS)。
6)细胞活力检测：
向24孔板中每孔加入50μL CCK-8溶液，在加完试剂后轻轻晃动培养板以帮助混匀。将培养板放入培养箱中孵育2h，用酶标仪测定450nm处的吸光度(OD)。吸光度测量结果如表2所示。
表2在450nm处吸光度(OD)的测量结果
RNA Control Linear Luc RNA Linear Luc RNA(N1) circRNA Luc circRNA Luc+A
1 0.28 0.2273 0.2599 0.2592 0.2624
2 0.2815 0.2488 0.2542 0.2517 0.2616
3 0.2871 0.2472 0.2339 0.2359 0.2575
细胞活力结果示意图如图5所示，结果表明，细胞毒性大小为：
Linear Luc RNA＜Linear Luc RNA(N1)＜circRNA Luc+A，circRNA Luc与 circRNA Luc+A之间无显著性差异。
通过对环状RNA前体分子引入多聚A尾在转染细胞后所产生的细胞毒性小于线性RNA以及带核苷酸修饰的线性RNA。
实施例9 编码荧光素酶的环状RNA前体分子24小时的荧光素酶的表达
检测细胞活性后，对细胞进行换液处理，更换为每孔0.5mL的新鲜培养基(90％DMEM+10％FBS)。在培养24小时后进行荧光素酶检测，选用Bright-LiteTM Luciferase Assay system检测试剂盒进行荧光素酶活性检测。在暗室里将试剂盒中溶液与底物两组分进行混合均匀后，取0.5mL的检测试剂与细胞进行混合裂解，在2min后取150μL体积的裂解液加入到预先准备好的白板中，快速通过酶标仪进行检测荧光素酶的活性检测，每组进行2次的平行实验。荧光素酶的活性检测结果如表3以及图6所示。
表3编码荧光素酶的RNA细胞表达水平检测
RNA Control circRNA Luc circRNA Luc+A
1 4009 1061048 1446024
2 7717 1028856 1304428
3 3998 1055645 1465278
4 7706 1011514 1296290
通过荧光素酶的活性检测结果显示，circRNA Luc+A在荧光素酶的表达水平上高于circRNA Luc。
实施例10 不同长度的多聚A尾对环化效率的影响
检测本发明提供的带不同长度多聚A尾的环化载体使用Rnase R消化20min的产物纯度比较。
对合成得到的荧光素酶Luc2的质粒DNA进行DNA模板的扩增，反应体系如下：
反应体积，50μL(为单个管的反应体积，一次同时反应多管)，所述PCR扩增的体系以50μL计，各组分如下：
Figure PCTCN2022090198-appb-000012
PrimeSTAR Max Premix(2×)包括以下组分：PrimeSTAR Max DNA Polymerase，dNTPs和Mg 2+。引物F和引物R的初始浓度优选为10μmol/L；所述DNA模板的浓度 优选为1ng/μL。
引物F的序列如SEQ ID NO.3所示；
引物R1的序列如SEQ ID NO.4所示；
所述引物R3的序列如下：
Figure PCTCN2022090198-appb-000013
所述引物R4的序列如下：
Figure PCTCN2022090198-appb-000014
所述引物R5的序列如下：
Figure PCTCN2022090198-appb-000015
所述引物R6的序列如下：
Figure PCTCN2022090198-appb-000016
所述引物R7的序列如下：
Figure PCTCN2022090198-appb-000017
所述引物R8的序列如下：
Figure PCTCN2022090198-appb-000018
所述引物R9的序列如下：
Figure PCTCN2022090198-appb-000019
上述PCR的扩增程序如下：预变性98℃3min；变性98℃10s，退火56℃15s，延伸72℃40s，30个循环；最后延伸72℃，5min。PCR总体系为300μL。
反应结束后，将反应液合并于1.5ml Tube管中。取2μL进行DNA琼脂糖凝胶电泳检测以确定反应成功(1.5％琼脂糖，120V，40min)。
合格标准：电泳检测出现单一的条带，且大小正确。
测定结果：条带大小单一，大小符合要求。
琼脂糖凝胶电泳：先配置两块1.5％的琼脂糖凝胶(称取1.5g琼脂糖加入100ml的TAE溶液中)，随后将上述剩余的PCR产物进行琼脂糖凝胶电泳，随后在蓝光仪下进行切胶回收，并称取重量。按照Gel Extraction Kit DNA提取试剂盒对Seq5，Seq6， Seq7，Seq8，Seq9，Seq10以及Seq11(分别对应扩增引物R3-R9扩增出的片段)进行回收纯化。
合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。
测定结果：Seq5浓度为255ng/μL，260/280＝1.91，260/230＝1.80；Seq6浓度为196ng/μL，260/280＝1.90，260/230＝2.01；Seq7浓度为312ng/μL，260/280＝1.90，260/230＝1.81；Seq8浓度为291ng/μL，260/280＝1.90，260/230＝2.10；Seq9浓度为165ng/μL，260/280＝1.90，260/230＝2.01；Seq10浓度为165ng/μL，260/280＝1.90，260/230＝2.10；Seq11浓度为152ng/μL，260/280＝1.90，260/230＝2.10。
最终用RNase-free水稀释至150ng/μL。
在获得所述Seq5，Seq6，Seq7，Seq8，Seq9，Seq10以及Seq11的DNA模板后，构建RNA体外合成体系进行RNA的体外合成获得所述前体环状RNA。所述RNA体外合成体系以100μL计，包括以下组分：
Figure PCTCN2022090198-appb-000020
所述RNA体外合成优选在恒温反应器中进行；所述RNA体外合成的程序优选为37℃，4h。
1)DNase I消化去除DNA模板
向RNA体外合成后的Tube管中各加入5μL DNase I。上下颠倒10次混匀，1000rpm离心10s。重新置于恒温反应器中，37℃，1h。
2)RNA的体外环化
所述RNA的环化优选通过将GTP添加至终浓度为2mM，然后将反应在55℃加热8分钟实现环化。
3)环状RNA的纯化
将环化后的产物稀释至100μL体系；
向样品中加入350μL结合缓冲液并轻轻混合；
向样品中加入250μL的100％乙醇并轻轻混合；
将样品加入纯化柱内，14,000×g离心1分钟；
用2×500μL洗涤液洗涤，14,000×g离心1分钟；
将40μL洗脱液加入至纯化柱内，在设置为70℃的金属浴中孵育5分钟，14,000×g离心1分钟，回收洗脱的RNA。
实际通过RNA纯化试剂盒回收到的Seq5的RNA浓度为1428ng/μL，Seq6的RNA浓度为1556ng/μL，Seq7的RNA浓度为1542ng/μL，Seq8的RNA浓度为1391ng/μL，Seq9的RNA浓度为1516ng/μL，Seq10的RNA浓度为1331ng/μL，Seq11的RNA浓度为1173ng/μL。
取20μg的分别带有5，10，15，25，50，75，100以及125个腺嘌呤核糖核苷酸的环化载体环化后的RNA，在37℃的反应条件下，使用1U的Rnase R进行消化处理20分钟，最终反应体系如表4所示。取等体积的RNA稀释在95％甲酰胺loading中，在70℃变性3分钟后，冷却至室温，然后使用1.5％琼脂糖凝胶检测RNA产物。
表4带有不同长度多聚A尾的环化载体使用Rnase R消化的反应体系
Figure PCTCN2022090198-appb-000021
最终反应结果如图7所示。带有不同长度多聚A尾的环化载体在环化后使用1U的Rnase R处理都能在20min内得到纯度较高的环状RNA。

Claims (10)

  1. 一种用于制备环状RNA的载体，其包含能够转录为环状RNA前体分子的DNA序列，所述环状RNA前体分子的3’端带有多聚A尾。
  2. 根据权利要求1所述的用于制备环状RNA的载体，其中，所述的多聚A尾，长度为5～150个A。
  3. 根据权利要求1或2所述的用于制备环状RNA的载体，所述载体包含以下顺序排列的元件：
    Anabaena pre-tRNA 3’I型内含子片段；
    内部核糖体进入位点(IRES)；
    蛋白质编码区或非编码区；
    Anabaena pre-tRNA 5’I型内含子片段；
    能转录为所述多聚A尾的DNA序列。
  4. 根据权利要求3所述的用于制备环状RNA的载体，所述的载体还包含以下元件中的一种或多种：
    5’同源臂；插入在所述的Anabaena pre-tRNA 3’I型内含子片段中；
    5’间隔子序列；在所述的Anabaena pre-tRNA 3’I型内含子片段与所述的内部核糖体进入位点之间；
    3’间隔子序列；在所述的蛋白质编码区或非编码区与所述的Anabaena pre-tRNA 5’I型内含子片段之间；
    3’同源臂；插入在所述的Anabaena pre-tRNA 5’I型内含子片段中。
  5. 根据权利要求1所述的用于制备环状RNA的载体，其中，所述环状RNA可编码hFIX、SP-B、VEGF-A、人甲基丙二酰辅酶A变异酶、CFTR、癌症自身抗原、基因编辑酶、HIV抗体、CD19抗体、CD22抗体、CD3抗体、CLDN6抗体、Luc2、GLuc、Fluc、eGFP、hEPO、Cas9核酸内切酶、新型冠状病毒S蛋白或肿瘤抑制因子。
  6. 根据权利要求1所述的用于制备环状RNA的载体，其具有SEQ ID NO.1所示序列的第1～1017位核苷酸序列和/或第1576～1781位核苷酸序列。
  7. 一种环状RNA前体分子，其是通过权利要求1-6任一项所述的载体进行体外转录得到的。
  8. 权利要求1至6任一项所述的载体在制备环状RNA中的应用。
  9. 一种环状RNA前体分子的制备方法，其包括步骤：
    a.将权利要求1-6任一项所述的用于制备环状RNA的载体进行PCR扩增，获得带有多聚A尾的体外表达RNA的DNA模板；
    b.构建包括a所述的DNA模板在内的RNA体外合成体系，进行RNA的体外合成获得可以环化的RNA前体分子。
  10. 一种环状RNA的制备方法，其包括步骤：
    以权利要求1-6任一项所述的用于制备环状RNA的载体制备环状RNA前体分子；
    对环状RNA前体分子进行环化，得到环状RNA。
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