Lasso RNA as well as preparation method and application thereof

Abstract

The invention relates to the field of molecular biology, in particular to lasso RNA as well as a preparation method and application thereof. The lasso RNA has a circular RNA formed by connecting 2 '-5' phosphodiester bonds and a poly (A) tail connected to the 3'end of the circular RNA; the circular RNA comprises an exogenous fragment, and the exogenous fragment comprises an internal ribosome entry site element and a target protein coding fragment; the annular RNA has or does not have the following fragments: a 3 '-terminal deoxyribozyme substrate fragment and a 5'-terminal deoxyribozyme substrate fragment. The lasso RNA provided by the invention can be used as RNA of a protein translation template, can be translated into active protein in cells, and can be finely regulated by a series of poly (A) related regulatory factors including polyadenylate polymerase and the like.

Description

Technical Field

[0001] The present application relates to the field of molecular biology, and in particular to a lasso RNA and a preparation method and application thereof. Background Art

[0002] Linear mRNA is usually used as a template for protein expression, and its translation mainly depends on the cap structure at the 5' end and the poly (A) tail at the 3' end. These structures promote the translation and expression of proteins by binding to ribosomes. Circular RNA is a single-stranded RNA molecule with covalent head-to-tail linkage. Compared with linear mRNA, the closed structure of circular RNA makes it less susceptible to nuclease degradation, thus having a longer half-life. For this reason, circular RNA is also used to encode vaccines or medicinal proteins to develop corresponding circular RNA vaccines and drugs. However, circular RNA mainly relies on the internal ribosome entry site (IRES) to initiate translation, so the expression efficiency is low, which limits its application to a certain extent.

[0003] How to provide RNA with both long half-life and high expression efficiency is a technical problem to be solved urgently. In view of this, this application is specially proposed. Summary of the invention

[0004] One or more embodiments of the present application provide a lasso RNA and a preparation method and application thereof, including the following technical solutions:

[0005] One or more embodiments of the present application provide a lariat RNA, wherein the lariat RNA has a circular RNA connected by 2'-5' phosphodiester bonds and a poly(A) tail connected to the 3' end of the circular RNA;

[0006] The circular RNA includes an exogenous segment, and the exogenous segment includes an internal ribosome entry site element and a target protein coding segment;

[0007] The circular RNA has or does not have the following segments: a 3'-end DNAzyme substrate segment and a 5'-end DNAzyme substrate segment.

[0008] In some embodiments of the present application, the sequence of the circular RNA includes a sequence of a second type of intron.

[0009] In some embodiments of the present application, the sequence of the circular RNA includes one or more sequences of the 1st, 2nd, 3rd, 5th and 6th domains of the second type of intron.

[0010] In some embodiments of the present application, relative to the circular RNA in the lariat RNA formed by self-splicing of the second type intron, the fourth domain of the circular RNA is replaced by the exogenous fragment.

[0011] In some embodiments of the present application, the exogenous fragment includes a first internal complementary fragment, a first spacer fragment, an internal ribosome entry site element, a second spacer fragment, a target protein coding fragment, a third spacer fragment, and a second internal complementary fragment connected in sequence, and the first internal complementary fragment and the second internal complementary fragment are complementary and paired.

[0012] In some embodiments of the present application, the length of the target protein coding fragment is 6bp - 100000bp.

[0013] In some embodiments of the present application, the target protein coding fragment encodes one or more of an antigen, an antibody, a polypeptide, an enzyme, a hormone, a growth factor, and a receptor.

[0014] One or more embodiments of the present application provide a precursor RNA, which produces the lariat RNA, and the precursor RNA has or does not have a poly(A) tail sequence.

[0015] In some embodiments of the present application, exon fragments are further connected to the 3' end and the 5' end of the second intron of the precursor RNA, respectively.

[0016] In some embodiments of the present application, a first precursor complementary fragment is further connected to the 3' end of the precursor RNA, and a second precursor complementary fragment is further connected to the 5' end of the precursor RNA, and the first precursor complementary fragment and the second precursor complementary fragment are complementary and paired.

[0017] One or more embodiments of the present application provide a DNA, which produces the precursor RNA.

[0018] One or more embodiments of the present application provide a vector, which includes the DNA.

[0019] One or more embodiments of the present application provide a cell, which includes the lariat RNA, the precursor RNA, the DNA, or the vector.

[0020] One or more embodiments of the present application provide a method for producing the lariat RNA, and the production method includes: cyclizing the precursor RNA, with or without adding the poly(A) tail, to prepare the lariat RNA.

[0021] In some embodiments of the present application, the production method transcribes the vector including the precursor RNA before cyclization to obtain the precursor RNA.

[0022] In some embodiments of the present application, the transcription is carried out in an in vitro transcription system.

[0023] One or more embodiments of the present application provide a method for producing a target protein, the production method comprising:

[0024] Translating the lariat RNA to produce a target protein encoded by the target protein coding fragment in the lariat RNA.

[0025] In some embodiments of the present application, the production method comprises:

[0026] Introducing the lariat RNA, the precursor RNA, the DNA or the vector into a host cell to express the target protein coding fragment in the lariat RNA.

[0027] One or more embodiments of the present application provide an application of the lariat RNA, the precursor RNA, the DNA, the vector or the cell in the preparation of a drug.

[0028] One or more embodiments of the present application provide a drug comprising the lariat RNA, the precursor RNA, the DNA, the vector or the cell.

[0029] Details of one or more embodiments of the present application are set forth in the following description, and other features, objects and advantages of the present application will become apparent from the specification and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application and more completely understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0031] Figure 1 Schematic diagram of the preparation steps of a translatable lariat RNA based on group II intron self-splicing;

[0032] Figure 2 Schematic diagram of the synthesis mechanism of a translatable lariat RNA catalyzed by deoxyribozyme;

[0033] Figure 3 Characterization result diagram of the lariat RNA structure constructed in Example 1;

[0034] Figure 4 Comparison diagram of the expression levels of the lariat RNA constructed in Example 1 and circular RNA without poly(A) tail; DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments, and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and not to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosed content of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various changes or modifications without departing from the connotation of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. In addition, in the following description, a large number of specific details are given to provide a more thorough understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing the embodiments and examples and are not intended to limit this application.

[0037] The term

[0038] Unless otherwise stated or there is a contradiction, the terms or phrases used herein have the following meanings:

[0039] The term "and / or", "or / and", "and / or" as used herein has a selection range that includes any one of two or more related listed items, and also includes any and all combinations of the related listed items. The said any and all combinations include combinations of any two related listed items, any more than two related listed items, or all related listed items. It should be noted that when connecting at least three items with at least two conjunctions selected from "and / or", "or / and", "and / or", it should be understood that in this application, this technical solution undoubtedly includes the technical solution connected by "logical AND", and also undoubtedly includes the technical solution connected by "logical OR". For example, "A and / or B" includes three parallel solutions: A, B, and A + B. Another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, D (that is, the technical solution connected by "logical OR"), and also includes any and all combinations of A, B, C, D, that is, it includes combinations of any two or any three of A, B, C, D, and also includes the combination of the four items A, B, C, D (that is, the technical solution connected by "logical AND").

[0040] In this application, when it comes to "multiple", "multiple types", "multiple times", "multiple elements", etc., unless otherwise specified, it means greater than 2 or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0041] As used herein, "combinations thereof", "any combination thereof", "any combination mode thereof", etc. include all suitable combination modes of any two or more of the listed items.

[0042] In this application, the "suitable" in "suitable combination mode", "suitable mode", "any suitable mode", etc. is subject to being able to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0043] In this application, "preferred", "better", "more preferable", "preferably" are only used to describe the embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0044] In this application, "further", "furthermore", "especially", etc. are used for descriptive purposes, indicating differences in content, but should not be understood as a limitation on the protection scope of this application.

[0045] In this application, "optionally", "optional", "option" mean that it can be either present or absent, that is, it refers to any one of the two alternative schemes of "present" or "absent". If "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0046] In this application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or quantity, nor can it be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive listing and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0047] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open-ended technical solution containing the listed features.

[0048] In this application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the selectable numerical values are considered continuous within the above-mentioned numerical intervals, and include the two numerical endpoints of the numerical range (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as each integer between the two endpoints. In this article, it is equivalent to directly listing each integer. For example, when t is an integer selected from 1 to 10, it means that t is any integer selected from the integer group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0049] The temperature parameter in this application, unless otherwise specified, allows both constant temperature treatment and variation within a certain temperature range. It should be understood that the so-called constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuation within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C is allowed.

[0050] In this application, %(w / w) and wt% both represent weight percentage, %(v / v) refers to volume percentage, and %(w / v) refers to mass-volume percentage.

[0051] All documents mentioned in this application are cited as references in this application, just as if each document was cited separately as a reference. Unless it conflicts with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited for all their contents and all their purposes. When this application involves citing documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When this application involves citing documents, the examples and preferred methods of the relevant technical features cited can also be included as references in this application, but only to the extent that this application can be implemented. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or be amended adaptively according to the description in this application.

[0052] Lariat RNA is a circular RNA with a special branched structure, and natural lariat RNA is an intermediate product formed during RNA splicing. Its circular structure originates from the connection of the GU nucleotides at the 5' end and the adenine (A) at the branch point through a 2'-5' phosphodiester bond during the splicing of precursor mRNA. Lariat RNA combines the dual characteristics of circular RNA and linear RNA. It is not only not easily degraded by nucleases but also has extensibility at the 3' end, showing greater plasticity. Adding a poly(A) tail to the 3' end of lariat RNA can significantly improve the translation efficiency of its encoded protein, making it promising to become a new generation of RNA drug platforms. The RNA provided in this application contains a polyadenylate tail and a circular structure and can be translated into an active protein as a protein translation template in cells (such as eukaryotic cells) and can be finely regulated by a series of poly(A)-related regulatory factors including polyadenylate polymerase.

[0053] In the first aspect of the embodiments of this application, a lariat RNA is provided. The lariat RNA has a circular RNA connected by a 2'-5' phosphodiester bond and a poly(A) tail connected to the 3' end of the circular RNA.

[0054] The circular RNA includes an exogenous fragment, and the exogenous fragment includes an internal ribosome entry site element and a target protein coding fragment.

[0055] The circular RNA has or does not have the following fragments: a 3' end deoxyribozyme substrate fragment and a 5' end deoxyribozyme substrate fragment.

[0056] The lariat RNA provided in this application, which can be used as a protein translation template, can be translated into an active protein in cells and can be finely regulated by a series of poly(A)-related regulatory factors including polyadenylate polymerase.

[0057] In some examples of this application, the sequence of the circular RNA includes the sequence of group II intron. In one example, the

[0058] In some examples of this application, the sequence of the circular RNA includes the sequence of one or more of the first, second, third, fifth, and sixth domains of group II intron.

[0059] In some examples of this application, the fourth domain of the circular RNA is replaced by the exogenous fragment relative to the circular RNA in the lariat RNA formed by the self-splicing of group II intron.

[0060] In some examples of the present application, the exogenous fragment includes a first internal complementary fragment, a first spacer fragment, an internal ribosome entry site element, a second spacer fragment, a target protein coding fragment, a third spacer fragment, and a second internal complementary fragment connected in sequence, and the first internal complementary fragment and the second internal complementary fragment are complementary to each other.

[0061] The present application does not particularly limit the length of the target protein coding fragment. In some examples of the present application, the length of the target protein coding fragment is 6 bp - 100,000 bp, such as 6, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000 bp.

[0062] The present application does not particularly limit the target protein encoded by the target protein coding fragment. In some examples of the present application, the target protein coding fragment encodes one or more of an antigen, an antibody, a polypeptide, an enzyme, a hormone, a growth factor, and a receptor.

[0063] In a second aspect of the embodiments of the present application, a precursor RNA is provided, and the precursor RNA produces the lariat RNA, and the precursor RNA has or does not have a poly(A) tail sequence.

[0064] In some examples of the present application, exon fragments are further connected to the 3' end and 5' end of the second intron of the precursor RNA, respectively.

[0065] In some examples of the present application, a first precursor complementary fragment is further connected to the 3' end of the precursor RNA, and a second precursor complementary fragment is further connected to the 5' end, and the first precursor complementary fragment and the second precursor complementary fragment are complementary to each other.

[0066] In a third aspect of the embodiments of the present application, a DNA is provided, and the DNA produces the precursor RNA.

[0067] The term "DNA" used in the present application may be single-stranded or double-stranded, preferably double-stranded DNA. When a nucleic acid is placed in a functional relationship with another nucleic acid sequence, the nucleic acid is "effectively linked". For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.

[0068] The DNA of the present application mainly refers to isolated DNA. "Isolated" means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to mean the complete absence of these materials or the absence of water, buffer or salt, unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds described herein.

[0069] In the fourth aspect of the embodiments of the present application, a vector is provided, and the vector includes the DNA described above.

[0070] Optionally, the vector is selected from mammalian cell viruses, bacterial plasmids, phages, yeast plasmids, plant cell viruses or combinations thereof.

[0071] The term "vector", also referred to as "expression vector", refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. In another embodiment, the vector is a viral vector, into which additional DNA segments can be ligated into the viral genome. The vectors disclosed herein are capable of autonomous replication in the host cells into which they have been introduced (e.g., bacterial vectors with a bacterial origin of replication and episomal mammalian vectors) or can integrate into the genome of the host cell after introduction into the host cell and thus replicate with the host genome (e.g., non-episomal mammalian vectors). Overall, the vectors can be selected from, but not limited to, mammalian cell viruses, bacterial plasmids, phages, yeast plasmids, plant cell viruses or combinations thereof.

[0072] In the fifth aspect of the embodiments of the present application, a cell is provided, and the cell includes the lariat RNA, the precursor RNA, the DNA or the vector described above.

[0073] The term "cell", also referred to as "host cell", means a cell into which an expression vector has been introduced. Host cells can include bacteria, microorganisms, plant or animal cells. Bacteria that are easily transformable include members of the Enterobacteriaceae, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

[0074] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably, and all such names include progeny. Thus, "transformants" and "transformed cells" include the original test cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that due to deliberate or inadvertent mutations, all progeny may not be precisely identical in DNA content. Include mutant progeny having the same function or biological activity as screened in the original transformed cells. Where different names are intended, it is clear from the context.

[0075] In the sixth aspect of the embodiments of the present application, there is provided a method for producing the lasso RNA, the production method comprising: circularizing the precursor RNA, with or without adding the poly(A) tail, to prepare the lasso RNA.

[0076] In some examples of the present application, the production method transcribes the vector including the above to obtain the precursor RNA before circularization.

[0077] In some examples of the present application, the transcription is carried out in an in vitro transcription system.

[0078] In the seventh aspect of the embodiments of the present application, there is provided a method for producing a target protein, the production method comprising:

[0079] Translating the lasso RNA to produce a target protein encoded by the target protein coding fragment in the lasso RNA.

[0080] In some examples of the present application, the production method comprises:

[0081] Introduce the lasso RNA, the precursor RNA, the DNA or the vector into a host cell to express the target protein coding fragment in the lasso RNA.

[0082] In the eighth aspect of the embodiments of the present application, there is provided an application of the lasso RNA, the precursor RNA, the DNA, the vector or the cell in the preparation of a drug.

[0083] In the ninth aspect of the embodiments of the present application, there is provided a drug, comprising the lasso RNA, the precursor RNA, the DNA, the vector or the cell.

[0084] The RNA with a linear poly(A) tail and a circular coding sequence provided by the present application, namely QRNA. QRNA not only realizes translation in cells, but also can be finely regulated by the poly(A) tail-related regulatory mechanism. The QRNA of the present application can be prepared by, but not limited to, the following methods:

[0085] A. Translatable lasso RNA based on group II intron self-splicing. (The steps are shown in Figure 1 )

[0086] 1. Screen group II introns that self-splice and release lasso RNA as the transformation template.

[0087] Group II introns are a type of ribozyme with self-splicing activity. Natural group II introns have a conserved secondary structure, usually containing 6 domains, namely D1, D2, D3, D4, D5 and D6. Among them, the D4 domain mainly contains a protein-coding sequence, and the correct folding of the remaining domains can trigger the self-splicing of the sequence, connecting the RNA fragments on both sides. Group II introns can be divided into different subtypes such as A, B, C, D, E, etc. Some of these subtypes can release lasso-shaped RNA after splicing.

[0088] Therefore, group II introns can be modified to have the ability to encode proteins and be expressed, and ensure that the modified introns can still be spliced normally.

[0089] 2. Replace the D4 domain of the selected group II intron with the required IRES-CDS sequence.

[0090] In addition, by adding structures such as spacer sequences and terminal complementary sequences to maintain and utilize the RNA secondary structures before and after replacement simulated by online software such as RNAfold and mFold, ensure that the secondary structures of the other five domains of the modified group II intron except the D4 domain are not disturbed.

[0091] In addition to group II intron self-splicing.

[0092] 3. In vitro transcription. To obtain precursor RNA, the synthesized and modified group II intron sequence is used as the in vitro transcription template. The method for obtaining the template can be digesting the plasmid vector with enzymes or performing PCR using primers containing the T7 promoter sequence, and using T7 RNA polymerase to perform in vitro transcription to obtain the spliced precursor RNA.

[0093] 4. In vitro splicing. Since the structure of QRNA includes a circular CDS region and a linear poly(A) tail, it is necessary to obtain lariat RNA through the splicing of group II introns as the substrate for further processing.

[0094] To further improve the splicing efficiency of group II introns, high-salt conditions and appropriate temperature are used to promote the RNA obtained by in vitro transcription. In specific implementation schemes, MgCl2 and NH4Cl are used as metal salts to promote splicing, and the temperature is 37 °C.

[0095] 5. Purification and enrichment

[0096] Since there are differences in molecular weight between the precursor RNA before splicing and the lariat RNA after splicing, the lariat RNA can be purified by chromatography such as size exclusion liquid chromatography, electrophoresis such as agarose gel electrophoresis, etc. In specific implementation schemes, size exclusion high performance liquid chromatography is used to separate the linear precursor and the splicing product to achieve the enrichment of lariat RNA.

[0097] 6. To obtain lariat RNA with a linear tail sequence containing only adenosine, the enriched lariat RNA needs to be further digested with RNAse R to completely remove its own linear tail sequence.

[0098] 7. In vitro enzymatic reaction adds a poly(A) tail to the RNA after RNAse R digestion to prepare complete QRNA. The poly(A) tail can be a polyadenylate tail composed entirely of adenosine, or a sequence incorporated with some non-adenosine such as uridine ribonucleotide, guanosine ribonucleotide, cytidine ribonucleotide or other modified nucleotides, or a partial motif sequence that can recruit polyadenylate polymerase in vivo.

[0099] 8. Transfect cells or experimental animals to test the expression of QRNA. The delivery vector during transfection can be a lipid transfection reagent, LNP or nanoparticle vector, etc.

[0100] 9. Characterize QRNA

[0101] Since QRNA has an atypical circular structure, that is, a circular sequence containing a 2'-5' phosphodiester bond branch site and a linear tail sequence, its linear tail and circular structure can be characterized by various methods respectively.

[0102] In specific embodiments, the following three methods are combined to prove that the splicing product contains both an atypical circular structure and a linear tail sequence.

[0103] Method 1: RNase R digestion. RNase R is a 3'-5' exonuclease that can non-specifically degrade linear RNA or the linear tails of lariat RNAs, but cannot degrade the circular part of lariat RNAs beyond the branch site. Therefore, digesting QRNA with RNase R can effectively identify whether the RNA splicing product contains a circular structure.

[0104] Method 2: Reverse transcribe the splicing product after RNase R digestion and perform PCR on the resulting cDNA using divergent primers. Since only RNAs with circular structures can be specifically amplified by PCR using divergent primers, this method can effectively prove that the splicing product contains a circular structure. Additionally, Sanger sequencing of the PCR product can reveal the specific positions of the RNA branch site and the 5' splicing site.

[0105] Method 3: Perform an in vitro poly(A) tailing experiment on the splicing product after RNase R digestion using polyadenylate polymerase. Ordinary circular RNAs do not have a hydroxyl group at the 3' end and thus cannot be tailed as substrates for polyadenylate polymerase. Different from ordinary circular RNAs, the circular RNA branch site generated by digesting lariat RNAs is connected to the 5' end through a 2'-5' phosphodiester bond and has a naked 3' terminal hydroxyl group, so it can be tailed by polyadenylate polymerase. Compare the length differences of the RNA before and after tailing by agarose gel electrophoresis of the RNA before and after tailing to confirm the formation of complete QRNA containing a poly(A) tail.

[0106] B. Synthesis of translatable lariat RNA catalyzed by deoxyribozyme.

[0107] Deoxyribozymes are a class of in vitro selected single-stranded deoxyribonucleic acids with catalytic functions. Currently reported deoxyribozymes are diverse in types and have various functions such as mediating the formation of phosphodiester bonds by RNA head-to-tail connection and acting as endonucleases for RNA. Deoxyribozymes with RNA as the substrate generally consist of a catalytic core sequence and a substrate complementary sequence. By designing appropriate substrate complementary sequences and using a class of deoxyribozymes with the function of catalyzing the formation of 2'-5' phosphodiester bonds inside RNA to catalyze the ligation of in vitro transcribed RNAs containing IRES and poly(A) tails, translatable lariat RNAs containing both circular coding sequences and linear poly(A) tails can be obtained.

[0108] The technical details of each step in this process are as follows:

[0109] 1. Design appropriate substrate complementary sequences.

[0110] Since the process of deoxyribozyme-catalyzed RNA to form 2'-5' phosphodiester bonds depends on the complementary pairing of two substrate complementary sequences with RNA, and the site where the 2'-5' phosphodiester bond is formed depends on the position of the complementary pairing, generally the 5'-triphosphate of the 5' complementary sequence of RNA and the 2'-hydroxyl of the last nucleotide of the 3' complementary sequence. Therefore, by reasonably designing the complementary sequences, deoxyribozymes can catalyze different substrate RNAs to form intramolecular 2'-5' phosphodiester bonds, that is, lariat structures containing branch sites (see Figure 2 ). At this time, if the substrate RNA is an RNA containing a poly(A) sequence, QRNA can be directly prepared by deoxyribozyme catalysis. If the substrate RNA does not contain a poly(A) sequence, complete QRNA can be prepared by in vitro polyadenylation.

[0111] 2. In vitro transcription of RNA

[0112] To obtain the substrate RNA, the synthesized modified group II intron sequence is used as the in vitro transcription template. The method for obtaining the template can be digesting the plasmid vector with enzymes or performing PCR using primers containing the T7 promoter sequence, and using T7 RNA polymerase to perform in vitro transcription to obtain the substrate RNA. The substrate RNA can be an RNA containing IRES, CDS, and poly(A) sequences, or an RNA without a poly(A) tail.

[0113] 3. Deoxyribozyme catalysis

[0114] The deoxyribozyme used in this protocol is a single-stranded DNA with the function of catalyzing substrate RNA to form 2'-5' phosphodiester bonds. Specifically, the 6BX22 deoxyribozyme reported by the Silverman research group in 2005 (Wang & Silverman, 2005) is used in the protocol, and its catalytic region sequence is:

[0115] 5'-CAGGGGGAGCGAGCACTAATACAAGCGGGTAGGAGGCCC-3'.

[0116] The complementary sequences at both ends of the deoxyribozyme are designed such that the 5'-end is complementary to the 5'-end sequence of the substrate RNA, and the 3'-end is complementary to the sequence of the substrate RNA near poly(A). The catalytic system and steps of the deoxyribozyme are as follows: Mix the substrate RNA and the deoxyribozyme, with the final concentration ratio of substrate RNA:deoxyribozyme being 1:2; then heat at 95°C for 3 min and incubate on ice for 5 min to complete annealing, so that the RNA and the deoxyribozyme are complementary; add HEPES solution with a pH of 7.5, sodium chloride solution, and potassium chloride solution respectively, so that the final concentrations reach 50 mM, 150 mM, and 2 mM respectively; then add manganese chloride solution to a final concentration of 20 mM; incubate at 37°C for 3 h.

[0117] 4. Isolation and purification

[0118] Since there is only a conformational change in RNA before and after the catalysis by the deoxyribozyme and the molecular weight change is extremely small, denaturing polyacrylamide gel electrophoresis or reverse-phase liquid chromatography can be used for separation, recovery, and purification. The steps are as follows: Prepare a polyacrylamide gel with an appropriate concentration according to the molecular weight of the substrate RNA, perform electrophoresis and stain the gel with a nucleic acid dye. Among them, the lariat RNA migrates at a slower rate and should be the top band, and then cut the gel for recovery.

[0119] 5. Characterization of QRNA

[0120] The characterization method is the same as that described in Scheme A.

[0121] The implementation schemes of the present application will be described in detail below in conjunction with the embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specific conditions noted in the following embodiments, the guidance given in the present application is preferably referred to, and it can also be carried out according to the experimental manuals or conventional conditions in the art, or according to the conditions recommended by the manufacturer, or referring to the experimental methods known in the art.

[0122] In the following specific embodiments, for the measurement parameters of the raw material components, if there is no special description, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0123] Example 1: Preparation of QRNA with a poly(A) tail and its expression in cells

[0124] 1. Selection and modification of group II introns

[0125] First, the first intron (C.i.SSU.I1) of the mitochondrial ribosomal small subunit-encoding gene of Coccidioides immitis was selected as the transformation template because it has high in vitro self-splicing activity and the ability to form lariat RNA in vitro (Liu & Pyle, 2021).

[0126] A DNA sequence was directly synthesized according to the natural sequence of group II introns. This DNA sequence is shown in SEQ ID NO.1.

[0127] (1) In addition to the complete sequence of the group II intron, it also contains exon fragment sequences of 60 bp each flanking the intron.

[0128] (2) Meanwhile, to promote the spatial proximity of the 5'-end and 3'-end of the transformed intron, complementary sequences with lengths of 30 bp - 40 bp were added to the 5'-end and 3'-end respectively.

[0129] (3) To construct an in vitro transcription template for RNA capable of expressing the corresponding protein, the Coxsachievirus B3 (CVB3) IRES-EGFP coding sequence, internal complementary sequence, and spacer sequence were inserted into the middle position of the intron, specifically between the 464th bp and the 465th bp;

[0130] Among them, the CVB3 IRES-EGFP sequence can drive the expression of green fluorescent protein, and the internal complementary sequence and spacer sequence can ensure that the group II intron and IRES fold into functional secondary structures respectively.

[0131] 2. Obtaining precursor RNA by in vitro transcription

[0132] a. Template DNA was obtained by PCR amplification using primers containing the T7 promoter sequence. The sequences of the forward primer and the reverse primer are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

[0133] The PCR amplification system is as follows:

[0134] Table 1

[0135] Reagent Volume 2×Taq mix (Vazyme) 10 μL Forward primer (10 μM) 1 μL Reverse primer (10 μM) 1 μL Template DNA 2 ng <![CDATA[ddH2O]]> To 20 μL

[0136] The PCR amplification program is as follows:

[0137] Table 2

[0138]

[0139] b. The DNA obtained by PCR was purified using a DNA purification kit (zymo), and the DNA concentration was measured using a micro-spectrophotometer.

[0140] c. In vitro transcription

[0141] The in vitro transcription system is as follows:

[0142] Table 3

[0143]

[0144]

[0145] d. Incubate at 37 °C for 2 h.

[0146] c. Add 10 μL DNAse I buffer, 70 μL nuclease-free water, and 2 μL DNAse I to the in vitro transcription system, and incubate at 37 °C for 15 min.

[0147] e. Perform column purification using an RNA purification kit (zymo).

[0148] f. Measure the concentration of the obtained RNA using a micro-spectrophotometer.

[0149] 3. In vitro splicing

[0150] a. Dilute 30 μg of in vitro transcribed RNA to 34 μL with nuclease-free water.

[0151] b. Incubate at 90 °C for 1 min and at 37 °C for 2 min.

[0152] c. Prepare the in vitro splicing system as follows:

[0153] Table 4

[0154] Reagent Volume (μL) RNA 34 400 mM HEPES (pH7.0) 5 10X T4 ligase buffer (NEB) 5 <![CDATA[1.5M NH4Cl]]> 5 RNAse inhibitor 1

[0155] d. Incubate at 37 °C for 15 min.

[0156] e. Perform column purification using an RNA purification kit (zymo).

[0157] 4. Enrich the spliced products by HPLC

[0158] a. Use Nuclease-free TE buffer (10 mM Tris, 1 mM EDTA, pH 7.3) as the mobile phase, the chromatographic column is SRT SEC-2000 (sepax), the flow rate of the mobile phase is 0.3 mL / min, and the collected sample is the effluent from 13.75 min to 15 min.

[0159] 5. Digest the enriched spliced products with RNAse R

[0160] a. Dilute 5 μg of RNA to 45 μL with nuclease-free water at 70 °C.

[0161] b. Incubate for 3 min and then cool on ice for 2 min.

[0162] c. Add 0.2 μL of RNAse R (epicentre, RNR07250) and 5 μL of 10× RNAse R buffer (epicentre, RNR07250) to the RNA solution.

[0163] d. Incubate at 37 °C. Take out the reaction system at the 10th minute, add half the amount of RNAse R as in the previous step, mix well and continue to incubate at 37 °C for 10 min.

[0164] e. Perform column purification using an RNA purification kit (zymo).

[0165] 6. Add a poly(A) tail to the RNA digested by RNAse R using polyadenylate polymerase to form QRNA

[0166] a. Prepare the reaction system as follows:

[0167] Table 5

[0168] 10×buffer 2 μL E.coli poly(A)polymerase (5U / μL) 1 μL ATP (10 mM) 2 μL RNAse inhibitor 1 μL RNAse R digested RNA 5 μg Nuclease-free water Make up to 20 μL

[0169] b. Incubate at 37 °C for 5 min.

[0170] c. Perform column purification using an RNA purification kit (zymo).

[0171] 7. Transfect cells with the poly(A)-tailed QRNA to observe

[0172] According to the instructions, transfect QRNA into the 293T cell line using lipofectamine MessengerMax (thermo). Observe the fluorescence after 24 h to determine the expression of EGFP and quantify the GFP fluorescence intensity using ImageJ software.

[0173] 8. Characterization of the QRNA lariat structure

[0174] Since it has been demonstrated in steps 5 and 6 that QRNA resists digestion by RNAse R and can be polyadenylated, the configuration of QRNA is further characterized using divergent primers in combination with Sanger sequencing.

[0175] a. Design a pair of divergent primers near the 5'-end and 3'-end of the group II intron sequence respectively according to the synthetic sequence. The sequences of the forward primer and the reverse primer are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0176] b. Reverse transcribe the RNA digested by RNAse R in step 5. The primer used is the reverse primer among the divergent primers described in step a. The reverse transcription system is as follows:

[0177] Table 6

[0178] RNA 50 ng Divergent primer1 (10 μM) 0.2 μL dNTPs (10 mM Each) 1 μL Nuclease-free water Make up to 14.5 μL

[0179] c. Incubate at 65 °C for 5 min and cool on ice for 2 min.

[0180] d. Subsequently, add the reagents shown in the following table to the system:

[0181] Table 7

[0182] 5×RT buffer 4 μL RNase inhibitor (20U / μL) 0.5 μL Maxima H minus reverse transcriptase (Thermo) 1 μL

[0183] e. Incubate at 65 °C for 30 min and at 85 °C for 5 min.

[0184] f. Perform PCR amplification using the reverse transcription product obtained above. The PCR amplification system is as follows:

[0185] Table 8

[0186] 2×Taq mix (Vazyme) 10 μL Divergent primer1 (10 μM) 1 μL Divergent primer2 (10 μM) 1 μL cDNA 2 μL Ultra-pure water 6 μL

[0187] The PCR amplification program is as follows:

[0188] Table 9

[0189]

[0190] g. Perform agarose gel electrophoresis on the PCR amplification product, and a single clear band can be seen.

[0191] h. Perform Sanger sequencing on the PCR amplification product, and an obvious junction point can be seen, which proves that QRNA contains a circular structure.

[0192] The inventor characterized QRNA using the method in step 8 of Example 1. First, RNAse R was used to digest the RNA obtained after in vitro splicing. It can be seen that all the linear precursor RNAs were digested ( Figure 3 .A), while the spliced QRNA remained ( Figure 3 .B). RT-PCR was performed on the product digested by RNAse R using divergent primers, and an obvious single band was seen ( Figure 3.C), Sanger sequencing of the RT-PCR products showed accurate junction points that met expectations. Figure 3 .D), indicating that QRNA has a circular structure. The RNA obtained after in vitro splicing was polyadenylated in vitro using polyadenylate polymerase, and agarose gel electrophoresis was performed on the RNA treated for different times. It was found that the position of the spliced RNA band below gradually shifted upward on the gel and the migration speed gradually slowed down with the increase in treatment time. Figure 3 .E), indicating that QRNA can be polyadenylated in vitro, that is, QRNA has a 3'-terminal exposed hydroxyl group. Combining the above evidence, it shows that QRNA has a lariat structure.

[0193] To verify that the QRNA obtained in Example 1 has a lariat structure and has a higher translation efficiency compared to ordinary lariat RNAs, the RNAs before and after polyadenylation in step 6 of Example 1 were transfected into 293T cells using lipofectamine MessengerMax respectively, and the fluorescence intensity was quantified. It was found that the translation efficiency of QRNA was significantly improved compared to ordinary lariat RNAs. Figure 4 ).

[0194] SEQ ID NO.1:

[0195]

[0196]

[0197] (1 - 33: 5'-end complementary sequence; 34 - 93: 5'-end exon fragment; 94 - 557: 5'-intron fragment; 558 - 575: 5'-internal complementary sequence; 576 - 596: 5'-spacer sequence; 603 - 1343: IRES sequence; 1356 - 2075: EGFP coding sequence;

[0198] 2086 - 2104: 3'-spacer sequence; 2105 - 2124: 3'-internal complementary sequence; 2152 - 2212: 3'-intron fragment; 2213 - 2272: 3'-end exon fragment; 2273 - 2307: 3'-end complementary sequence).

[0199] SEQ ID NO.2: 5'-taatacgactcactataGGGAGACCCTCGACCGTA-3'.

[0200] SEQ ID NO.3: 5'-CTAGATATAtCCTCaACCGTCGATTGT-3'.

[0201] SEQ ID NO.4: 5’-GCACGTAAATAATGTTTGAGCCGTATGC-3’。

[0202] SEQ ID NO.5: 5’-CCAAGGACACGTTAAATGCC-3’. The technical features of the above-described embodiments and examples can be combined in any suitable manner. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments and examples are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

[0203] The above-described examples only represent several embodiments of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can be made, and these all belong to the protection scope of the present application. In addition, it should be understood that after reading the above teachings of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided in the present application are all within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the content of the appended claims, and the specification and drawings can be used to explain the content of the claims.

Claims

1. A lariat RNA, characterized in that, The lasso RNA has a circular RNA connected by 2'-5' phosphodiester bonds and a poly(A) tail connected to the 3' end of the circular RNA; The circular RNA includes an exogenous fragment, and the exogenous fragment includes an internal ribosome entry site element and a target protein coding fragment; The circular RNA has or does not have the following fragments: a 3'-terminal deoxyribozyme substrate fragment and a 5'-terminal deoxyribozyme substrate fragment.

2. The lasso RNA according to claim 1, wherein The lasso RNA satisfies one or more of the conditions shown in (1) to (3): (1) The sequence of the circular RNA includes the sequence of a group II intron; Optionally, the sequence of the circular RNA includes the sequence of one or more of the first, second, third, fifth, and sixth domains of the group II intron; Optionally, the fourth domain of the circular RNA is replaced by the exogenous fragment relative to the circular RNA in the lasso RNA formed by self-splicing of the group II intron; Optionally, the exogenous fragment includes a first internal complementary fragment, a first spacer fragment, an internal ribosome entry site element, a second spacer fragment, a target protein coding fragment, a third spacer fragment, and a second internal complementary fragment connected in sequence, and the first internal complementary fragment and the second internal complementary fragment are complementary and paired; (2), the length of the target protein coding fragment is 6 bp - 100,000 bp; and, (3), the target protein coding fragment encodes one or more of an antigen, an antibody, a polypeptide, an enzyme, a hormone, a growth factor, and a receptor.

3. A precursor RNA, characterized in that, The precursor RNA produces the lasso RNA according to any one of claims 1 to 2, and the precursor RNA has or does not have a sequence with a poly(A) tail; Optionally, exon fragments are respectively connected to the 3' end and the 5' end of the group II intron of the precursor RNA; Optionally, a first precursor complementary fragment is further connected to the 3' end terminus of the precursor RNA, and a second precursor complementary fragment is further connected to the 5' end terminus of the precursor RNA, and the first precursor complementary fragment and the second precursor complementary fragment are complementary and paired.

4. A DNA, characterized in that, The DNA produces the precursor RNA according to claim 3.

5. Carrier, characterized in that, The vector includes the DNA according to claim 4.

6. A cell, characterized in that, The cell includes the lasso RNA according to any one of claims 1 to 2, the precursor RNA according to claim 3, the DNA according to claim 4, or the vector according to claim 5.

7. The production method of the lasso RNA according to any one of claims 1 to 2, characterized in that, The production method includes: circularizing the precursor RNA according to claim 3, with or without adding the poly(A) tail, to prepare the lasso RNA; Optionally, the production method transcribes the vector according to claim 5 before circularization to obtain the precursor RNA; Optionally, the transcription is carried out in an in vitro transcription system.

8. A method for producing a target protein, characterized in that, The production method includes: Translating the lasso RNA according to any one of claims 1 to 2 to produce a target protein encoded by the target protein coding fragment in the lasso RNA; Optionally, the production method includes: Introduce the lasso RNA according to any one of claims 1 to 2, the precursor RNA according to claim 3, the DNA according to claim 4, or the vector according to claim 5 into a host cell to express the target protein coding fragment in the lasso RNA.

9. Use of the lasso RNA according to any one of claims 1 to 2, the precursor RNA according to claim 3, the DNA according to claim 4, the vector according to claim 5, or the cell according to claim 6 in the preparation of a drug.

10. A drug, characterized in that, Comprising the lasso RNA according to any one of claims 1 to 2, the precursor RNA according to claim 3, the DNA according to claim 4, the vector according to claim 5, or the cell according to claim 6.

Images