In vitro enzymatic RNA synthesis

Abstract

The present invention relates to nucleic acid constructs capable of producing RNA products, methods and kits for producing RNA products using the nucleic acid constructs.

Description

[0001] The present invention relates to nucleic acid constructs capable of producing RNA products, methods for producing RNA products using the nucleic acid constructs, and kits. Background Art

[0002] With the development of biotechnology, it is important to be able to efficiently synthesize different types of RNA products such as CRISPR guide RNAs, small RNAs, RNA aptamers, or mRNAs, which are widely used for gene expression or its regulation. Among the in vitro RNA synthesis methods that have been developed, one way to produce RNA is based on in vitro transcription using RNA polymerase and a DNA structure comprising a double-stranded promoter region and an RNA transcription template. Most methods of this approach require an intact double-stranded or at least partially double-stranded DNA structure (Milligan et al., Nucleic Acids Res. 1987 Nov 11; 15(21): 8783-98). Preparation of such double-stranded DNA structures is quite time-consuming, especially since two independent DNA strands need to be synthesized separately. In addition, none of these template structures can precisely and efficiently produce RNA.

[0003] Therefore, there is still a need for new DNA constructs that are easy to synthesize and more efficient for in vitro RNA transcription for producing RNA products. Summary of the Invention

[0004] After studying this problem, the present inventors found that in vitro RNA transcription can be efficiently carried out by using a nucleic acid structure comprising a hairpin-forming oligonucleotide, which in turn comprises a reverse complement of a promoter, a loop region, and a promoter. Due to the loop region, the promoter and its reverse complement are linked in the same oligonucleotide. The nucleic acid structure of the present invention comprises, in sequence from the 5'-end to the 3'-end, a single-stranded nucleic acid, a reverse complement of a promoter, a loop region, and a promoter, and the single-stranded nucleic acid comprises a template portion for producing the RNA to be synthesized. Therefore, only one single-stranded nucleic acid needs to be synthesized by an enzymatic or chemical synthesis process. Surprisingly, compared with the in vitro transcription efficiency of using intact double-stranded or partially double-stranded DNA structures described in the prior art, the nucleic acid constructs of the present invention allow an improvement in the in vitro transcription efficiency of the template.

[0005] Surprisingly, the present inventors also found that the presence of a termination motif at the 5'-end of the template portion improves the termination of RNA polymerase, thereby increasing the RNA synthesis yield. More surprisingly, using a termination motif comprising one or more 2'-O modified nucleotides further reduces the non-template activity of RNA polymerase.

[0006] In one aspect, the present invention relates to a nucleic acid construct capable of producing at least one RNA product, which comprises, from 5' to 3': (i) a single-stranded nucleic acid comprising at least one template portion for producing the RNA product, and (ii) a hairpin-forming oligonucleotide, which in turn comprises a reverse complement of a promoter, a loop region, and a promoter, wherein the reverse complement of the promoter and the promoter are capable of forming a double-stranded oligonucleotide.

[0007] In some embodiments, the single-stranded nucleic acid of the nucleic acid construct comprises at least one termination motif at the 5' end of the template portion of the single-stranded nucleic acid.

[0008] In some embodiments, the termination motif is selected from at least one of a modified nucleotide, an abasic site, a hairpin structure, a protein binding site, a chemical linker, or a combination thereof, preferably at least two. In some embodiments, the termination motif comprises one or two 2'-O modified nucleotides. For example, the 2'-O modified nucleotide comprises a 2'-O modification group which is 2'-O-(2-methoxyethyl); in such an instance, the 2'-O modified nucleotide may be interchangeably named 2'-O-(2-methoxyethyl) nucleotide, 2'-O-(2-methoxyethyl) modified nucleotide, 2'-O-methoxyethyl modified nucleotide, or 2'-O-methoxy-ethyl modified nucleotide.

[0009] In some embodiments, the nucleic acid construct is a free nucleic acid in solution.

[0010] In some embodiments, the nucleic acid construct is attached to a solid support via its 5' end.

[0011] Another aspect of the present invention relates to a kit for producing an RNA product, which comprises:

[0012] - an RNA polymerase, ribonucleotides, optionally one or more modified nucleotides, and one or more reaction buffers, and

[0013] - (i) a nucleic acid construct as described in the present invention, or

[0014] - (ii) an initiator having a 3' terminal nucleotide with a free 3' hydroxyl group, a template-free DNA polymerase, a variety of 3'-O blocked nucleoside triphosphates, and a deblocking agent.

[0015] In some embodiments, the kit further comprises a solid support, wherein the nucleic acid construct or the initiator is attached to the solid support via their 5' ends.

[0016] In some embodiments, the kit of the present invention is a kit for generating single guide RNA (sgRNA), and the kit comprises an initiator having a deoxyribonucleic acid sequence complementary to the nucleic acid sequence of the constant part of the sgRNA.

[0017] In some embodiments, the kit for generating sgRNA comprises an RNA polymerase, several ribonucleotides, and the nucleic acid construct of the present invention, and the nucleic acid construct comprises a template part for generating sgRNA.

[0018] Another aspect of the present invention relates to a method for generating an RNA product, which comprises the following steps: (a) providing the nucleic acid construct as described in the present invention,

[0019] (b) contacting the nucleic acid construct with an RNA polymerase and ribonucleotides to generate the RNA product,

[0020] (c) optionally repeating step (b),

[0021] (d) optionally performing at least one post-transcriptional modification,

[0022] (e) optionally recovering the RNA product generated in steps (b), (c), and / or (d).

[0023] Brief Description of the Drawings

[0024] Figure 1A Illustrates an embodiment of the nucleic acid construct of the present invention, which comprises a single-stranded nucleic acid, a reverse complement (RC) of a promoter, a loop region, and the promoter from the 5'-end to the 3'-end, and the single-stranded nucleic acid comprises a template part for generating an RNA product.

[0025] Figure 1B Illustrates another embodiment of the nucleic acid construct of the present invention, which is attached to a solid support through its 5'-end, and comprises a single-stranded nucleic acid, a reverse complement (RC) of a promoter, a loop region, and the promoter from the 5'-end to the 3'-end, and the single-stranded nucleic acid comprises a termination motif and a template part for generating an RNA product.

[0026] Figure 1C Illustrates according to Figure 1B an embodiment of the nucleic acid construct of the present invention, wherein the template part corresponds to a sequence for generating sgRNA.

[0027] Figure 1D Illustrates Figure 1C the generation of an sgRNA product by in vitro transcription of the nucleic acid construct of the present invention shown.

[0028] Figure 2Shows the fluorescence levels generated by Broccoli fluorescent RNA, which was in vitro transcribed from 5 free nucleic acid constructs respectively, namely, two constructs of the present invention named "6-nt loop" and "9-nt loop", two constructs of the prior art named "dsDNA" and "ssDNA+promoter" as positive controls, and a single-stranded construct named "ssDNA" as a negative control. Fluorescence was measured during 12 hours of in vitro transcription.

[0029] Figure 3A Shows the fluorescence levels generated by Broccoli fluorescent RNA, which was in vitro transcribed from 6 nucleic acid constructs respectively, namely, two free constructs of the present invention (named "6-nt loop" and "9-nt loop") and two constructs of the present invention immobilized on resin (named "resin 6-nt loop" and "resin 9-nt loop"), the prior art construct "dsDNA" as a positive control and the single-stranded construct "ssDNA" as a negative control. Fluorescence was measured during 18 hours of in vitro transcription.

[0030] Figure 3B Shows the electrophoresis data of Broccoli fluorescent RNA generated by in vitro transcription of 4 constructs of the present invention, namely, "6-nt loop", "9-nt loop", "resin 6-nt loop", "resin 9-nt loop", and the construct "dsDNA" as a positive control.

[0031] Figure 4Electrophoresis data of RNA generated by in vitro transcription of six constructs of the present invention are shown. The constructs comprise single-stranded nucleic acids that consist, from 5' to 3', of a 15-nucleotide sequence, a termination motif, and a 21-nucleotide sequence that serves as the template portion for generating the RNA product. The termination motif in these constructs is located at the 5'-end of the 21-nucleotide sequence. The termination motifs tested are AP2 (two adjacent abasic sites), AP3 (three adjacent abasic sites), APiAP (two abasic sites separated by deoxyinosine), APTAP (two abasic sites separated by deoxythymidine), APT3 (three abasic sites separated by deoxythymidine, respectively), or C9S (or C9SP, also known as spacer 9, which is named 9-O-dimethoxytrityl-triethylene glycol, 1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, CAS#146668-73-7). Thus, C9S is a triethylene glycol spacer. Constructs "IVT termination" and "no termination" are two constructs used as controls. The "IVT termination" construct comprises a 21-nucleotide single-stranded nucleic acid as a template. The "no termination" construct comprises a single-stranded nucleic acid that comprises a 21-nucleotide sequence immediately following the 15-nucleotide sequence (i.e., there is no termination motif between the two sequences).

[0032] Figure 5A The percentage of the best sgRNA sequences generated by the constructs of the present invention is shown. The constructs comprise a template portion for generating sgRNA and a termination motif at the 5'-end of the template portion. The termination motifs tested are AP2, C9S, dU2 (deoxyuridine, which is converted to an abasic site after treatment with UDG enzyme), or AP2 / C9S conjugated with 2'-O-methoxy-ethyl modified nucleotides ("C9S-Methox" and "AP2-Methox"). For each sgRNA, the percentages of the best sequence (Best, black), the best sequence with 1 additional dT (Perfect T, light gray), and the best sequence with 2 additional dT (Perfect TT, dark gray) are indicated.

[0033] Figure 5B Electrophoresis data of RNA generated by in vitro transcription of four constructs of the present invention are shown. The constructs comprise single-stranded nucleic acids that consist, from 5' to 3', of a 15-nucleotide sequence, a termination motif, and a 21-nucleotide sequence that serves as the template portion for generating the RNA product. The termination motif in these constructs is at the 5'-end of the 21-nucleotide sequence. The termination motifs tested are C9SP alone (no Met), or C9SP conjugated with one (1Met) or two (2Met) 2'-O-methoxy-ethyl modified nucleotides. As Figure 4 described, the constructs "IVT termination" and "no termination" are used as controls. Detailed implementation manners

[0034] Definition

[0035] The present disclosure will be best understood by reference to the following definitions.

[0036] In the context of the present invention, the term "template portion" refers to a portion of a single-stranded nucleic acid that can be transcribed by an RNA polymerase to produce an RNA product through base pairing.

[0037] As used herein, the term "promoter" refers to a single-stranded DNA that contains a nucleotide sequence involved in the recognition and binding of an RNA polymerase and / or other proteins (such as transcription factors necessary for initiating gene transcription). A promoter is the DNA region where an RNA polymerase begins to transcribe a gene. Normally, the promoter sequence is usually located immediately upstream or at the 5' end of the transcription start site (Lin et al., 2018 Molecular Cell, Volume 70, issue 1, P60 - 71). Both the promoter and the transcription start site are bound by an RNA polymerase and the necessary transcription factors. The promoter sequence describes the direction of transcription and indicates which DNA strand will be transcribed.

[0038] As used herein, the term "reverse complement" refers to a nucleotide sequence within a nucleic acid strand that is complementary to a given nucleotide sequence within the same nucleic acid strand but in the reverse order. Thus, when the single-stranded nucleic acid folds back on itself, the given nucleotide sequence and its reverse complement can hybridize to each other to form a double-stranded region.

[0039] As used herein, the term "hairpin-forming oligonucleotide" refers to an oligonucleotide that contains a first nucleotide sequence, a second nucleotide sequence (which is the reverse complement of the first nucleotide sequence), and a non-self-complementary central region that connects the first nucleotide sequence and the second nucleotide sequence. The first nucleotide sequence and the second nucleotide sequence are capable of hybridizing to form a double-stranded region under appropriate DNA hybridization conditions. In this conformation, the non-self-complementary central region forms a single-stranded loop, and a hairpin containing the loop region is formed.

[0040] As used herein, the term "complementary" in the context of nucleotides refers to base pairing and / or base stacking between two nucleotides. Base pairing can be direct or reverse Hoogsteen base pairing, or direct or reverse Watson & Crick base pairing. Complementary nucleotides can be A and T (or A and U), or C and G. Complementarity can also exist between other natural or unnatural nucleotides if hydrogen bonds can form between the two nucleotides. In the context of nucleic acids, the term "complementary" refers to the ability of two single-stranded nucleic acids to form an antiparallel double-stranded nucleic acid structure. Two single-stranded nucleic acids that are substantially complementary will hybridize to each other under DNA hybridization conditions. The term "substantially complementary" refers to the complete complementarity of two nucleic acids as well as the complementarity sufficient to achieve the desired binding of the two nucleic acids. DNA hybridization conditions are well known in the art and are defined by salt concentration and hybridization temperature.

[0041] Nucleic acid construct

[0042] The present invention relates to a nucleic acid construct that can be used to produce an RNA product. The nucleic acid construct comprises, from 5' to 3', (i) a single-stranded nucleic acid comprising a template portion for producing the RNA product, and (ii) a hairpin-forming oligonucleotide that in turn comprises the reverse complement of a promoter, a loop region, and the promoter.

[0043] The nucleic acid construct of the present invention has a linear primary structure, i.e., the nucleic acid construct is a single-stranded nucleic acid of the first order. However, due to the complementarity between the first nucleotide sequence (promoter) and the second nucleotide sequence (the reverse complement of the promoter), and the non-self-complementary central region that connects the promoter and its reverse complement as the loop region, the nucleic acid construct of the present invention is capable of locally forming a hairpin secondary structure at its 3' end. Thus, under appropriate conditions, the nucleic acid construct of the present invention comprises a single-stranded nucleic acid comprising a template portion for producing the RNA product and a double-stranded region formed by the promoter and its reverse complement.

[0044] Therefore, the nucleic acid construct of the present invention provides a more efficient and easier solution for producing an RNA product.

[0045] Single-stranded nucleic acid

[0046] According to the present invention, the length and sequence of the single-stranded nucleic acid in the nucleic acid construct can be determined by the RNA product to be produced. The single-stranded nucleic acid can comprise any number of nucleotides. According to an embodiment, the single-stranded nucleic acid comprises at least 10 nucleotides. The single-stranded nucleic acid can generally comprise from 10 to 10,000 nucleotides, particularly from 10 to 5,000 nucleotides, more particularly from 10 to 3,000 nucleotides, 10 to 1,000 nucleotides, 10 to 800 nucleotides, 10 to 500 nucleotides, and even more particularly from 10 to 300 nucleotides. Generally, mRNA can comprise up to 10,000 nucleotides, particularly 100 to 5,000 nucleotides. Small RNAs can comprise up to 250 nucleotides. For example, small interfering RNAs (siRNAs) and microRNAs (miRNAs) can comprise 20 to 30 and 20 to 50 nucleotides, respectively. The length of general small nuclear RNAs (snRNAs) is about 150 nucleotides.

[0047] The single-stranded nucleic acid can comprise natural deoxyribonucleotides and / or unnatural nucleotides. Natural deoxyribonucleotides, such as deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. Unnatural nucleotides can include modified bases, sugars, or internucleoside linkages. For example, unnatural nucleotides can include phosphorothioate internucleoside linkages, bases containing linking groups that allow attachment of a label (such as a fluorophore or a hapten), and the like. Examples of unnatural nucleotides can be 2'-O-methyl-modified nucleotides, 2'-F-modified nucleotides, α-thiophosphate-modified nucleotides, or 2'-O-methoxy-ethyl-modified nucleotides.

[0048] In some embodiments, the single-stranded nucleic acid comprises a template portion for producing an RNA product. The template portion is the portion of the single-stranded nucleic acid that is transcribed by an RNA polymerase to produce the RNA product.

[0049] In some embodiments, the sequence of the template portion of the single-stranded nucleic acid is complementary to the sequence of the RNA product to be produced. A person skilled in the art can adjust the sequence of the template portion of the single-stranded nucleic acid according to the sequence of the RNA product to be produced.

[0050] In some embodiments, the single-stranded nucleic acid can comprise a template portion for producing a single guide RNA (sgRNA), a small RNA, an RNA aptamer, or an mRNA.

[0051] In some embodiments, the single-stranded nucleic acid can consist of a template portion for producing a single guide RNA (sgRNA), a small RNA, an RNA aptamer, or an mRNA.

[0052] In some embodiments, the single-stranded nucleic acid can consist of a template portion for generating a single guide RNA (sgRNA), small RNA, RNA aptamer, or mRNA, and at least one termination motif at the 5' end.

[0053] The term "mRNA" refers to single-stranded RNA that can be read by ribosomes during the synthesis of a polypeptide or protein. In some embodiments, the mRNA can contain up to 10,000 nucleotides.

[0054] As used herein, the term "single guide RNA" refers to single-stranded RNA that can be used for CRISPR / Cas9-mediated gene editing. The single guide RNA comprises a variable portion and a constant portion from 5' to 3', the sequence of the variable portion being target-specific, and the sequence of the constant portion being recognized by the Cas 9 nuclease and being constant for the sgRNA.

[0055] As used herein, the term "small RNA" refers to non-coding single-stranded RNA of less than 250 nucleotides. Examples of small RNAs include, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), mitochondrial RNA (mtRNA), small hairpin RNA (shRNA), piwi-interacting RNA (piRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and small rDNA-derived RNA (srRNA).

[0056] The term "RNA aptamer" refers to single-stranded RNA that can selectively bind to a specific target, which includes proteins, peptides, carbohydrates, small molecules, toxins, and even living cells. An example of an RNA aptamer is a fluorescence-turn-on RNA aptamer, which can selectively bind to a fluorophore and then enhance its fluorescence.

[0057] In some embodiments, the single-stranded nucleic acid in the nucleic acid construct of the present invention comprises a template for generating an RNA product selected from sgRNA, miRNA, mtRNA, siRNA, shRNA, and RNA aptamer.

[0058] In some embodiments, the single-stranded nucleic acid in the nucleic acid construct of the present invention consists of a template for generating an RNA product selected from sgRNA, miRNA, mtRNA, siRNA, shRNA, and RNA aptamer, and optionally at least one termination motif at the 5' end.

[0059] In some embodiments, the single-stranded nucleic acid comprises a template for generating an sgRNA and, from 5' to 3', a nucleic acid sequence complementary to the nucleic acid sequence of the constant portion of the sgRNA and a nucleic acid sequence complementary to the nucleic acid sequence of the variable portion of the sgRNA. One skilled in the art can design a sequence complementary to the variable portion of the sgRNA using any conventional method according to the target to be edited.

[0060] In some embodiments, the single-stranded nucleic acid consists of a template for generating an sgRNA and, from 5' to 3', a nucleic acid sequence complementary to the nucleic acid sequence of the constant portion of the sgRNA and a nucleic acid sequence complementary to the nucleic acid sequence of the variable portion of the sgRNA, and optionally at least one termination motif at the 5' end. One skilled in the art can design a sequence complementary to the variable portion of the sgRNA using any conventional method according to the target to be edited.

[0061] According to the present invention, the single-stranded nucleic acid may consist of a template portion for generating an RNA product and optionally at least one termination motif at the 5' end of the template portion of the single-stranded nucleic acid.

[0062] Termination motif

[0063] Surprisingly, the inventors have demonstrated that the presence of a termination motif, particularly a termination motif comprising one or more O-methoxy-ethyl modified nucleotides, at the 5' end of the template portion can improve the release of RNA polymerase from the resulting RNA product, thereby increasing the RNA synthesis yield. In a preferred embodiment, the termination motif comprises at least two, three, four or more modified nucleotides, particularly two, three, four or more O-methoxy-ethyl modified nucleotides.

[0064] Thus, the single-stranded nucleic acid of the present invention may further comprise at least one termination motif at the 5' end of the template portion of the single-stranded nucleic acid, i.e., the single-stranded nucleic acid comprises at least one termination motif and a template portion for generating an RNA product, wherein the termination motif is at the 5' end of the template portion.

[0065] The term "termination motif" refers to a motif that promotes the release of RNA polymerase from a DNA template to terminate transcription. Generally, a termination motif is a nucleic acid having a specific sequence that disrupts the affinity between the DNA template and the RNA polymerase bound to the DNA template. The termination motif can be any naturally occurring prokaryotic, eukaryotic or viral termination motif known in the art, such as a termination motif isolated from or derived from a bacterium or a phage, a variant thereof or any non-natural termination motif described in the art, such as a abasic site or a modified nucleotide.

[0066] In some embodiments, the single-stranded nucleic acid comprises, from 5' to 3', a nucleic acid portion that is not transcribed into RNA, a termination motif, and a template portion for generating an RNA product, wherein the termination motif is at the 5' end of the template portion. The non-transcribed nucleic acid portion can comprise, for example, a cleavable motif.

[0067] In some embodiments, the single-stranded nucleic acid consists of a termination motif and a template portion for generating an RNA product, wherein the termination motif is at the 5' end of the template portion. The single-stranded nucleic acid can comprise more than one termination motif, for example, two termination motifs. In a specific embodiment, the single-stranded nucleic acid comprises at least two termination motifs.

[0068] In some embodiments, the termination motif is selected from at least one of modified nucleotides, abasic sites, hairpin structures, protein binding sites, chemical linkers, and combinations thereof. In a specific embodiment, the termination motif comprises two or more components selected from modified nucleotides, abasic sites, hairpin structures, protein binding sites, chemical linkers, and combinations thereof. For example, the termination motif comprises two or more modified nucleotides, and optionally one or more chemical linkers or spacers. In a specific embodiment, the termination motif comprises at least one modified nucleotide and at least one component selected from at least one of modified nucleotides, abasic sites, modified abasic sites, hairpin structures, protein binding sites, chemical linkers, and combinations thereof. In a specific embodiment, the termination motif comprises at least two modified nucleotides and at least one component selected from abasic sites, modified abasic sites, hairpin structures, protein binding sites, chemical linkers, and combinations thereof.

[0069] Examples of modified nucleotides that can be referred to as termination motifs are 5-octadiynyl dU, 6-carboxyfluorescein ddI, 1,2'-dideoxyribose I, 2'-O-methyl modified nucleotides, 2'-O-methoxy-ethyl modified nucleotides, or deoxynucleotides with large side chains.

[0070] In some embodiments, the termination motif comprises 2'-O-methoxy-ethyl modified nucleotides, such as 2'-O-methoxy-ethyl modified AMP, 2'-O-methoxy-ethyl modified CMP, 2'-O-methoxy-ethyl modified GMP, or 2'-O-methoxy-ethyl modified UMP.

[0071] In some embodiments, the termination motif is selected from at least one of AP1, AP2, AP2-Methox, AP3, APiAP, APTAP, APT3, C3S, C9S, C9S-Methox, dU2, and combinations thereof.

[0072] In some embodiments, the termination motif consists of 2'-O-methoxy-ethyl modified nucleotides, such as 2'-O-methoxy-ethyl modified AMP, 2'-O-methoxy-ethyl modified CMP, 2'-O-methoxy-ethyl modified GMP, or 2'-O-methoxy-ethyl modified UMP. In some embodiments, the termination motif consists of two, three, four, or more 2'-O-methoxy-ethyl modified nucleotides.

[0073] As used herein, the term "abasic site", abbreviated as "AP", refers to a deoxyribose in a DNA strand that does not have a nucleobase. Thus, in the context of the present invention, an abasic site is a position in a nucleic acid construct that has a deoxyribose lacking a purine base or a pyrimidine base.

[0074] In some embodiments, the termination motif can comprise one or more adjacent abasic sites or more than one abasic site separated from each other by nucleotides (such as deoxyinosine or deoxythymidine).

[0075] In some embodiments, the termination motif comprises abasic sites. In some embodiments, the termination motif consists of abasic sites.

[0076] In another embodiment, the termination motif comprises two or three adjacent abasic sites. In another embodiment, the termination motif consists of two or three adjacent abasic sites.

[0077] In some embodiments, the termination motif comprises two abasic sites separated by nucleotides. In some embodiments, the termination motif consists of two abasic sites separated by nucleotides.

[0078] In another embodiment, the termination motif comprises three abasic sites separated from each other by nucleotides. In some embodiments, the termination motif consists of three abasic sites separated from each other by nucleotides.

[0079] In some embodiments, the termination motif comprises one or more deoxyuridine (dU), which can be converted into an abasic site, for example, by treatment with UDG enzyme.

[0080] In some embodiments, the termination motif consists of two abasic sites separated by deoxyinosine. In some embodiments, the termination motif consists of two abasic sites separated by deoxythymidine. In some embodiments, the termination motif consists of three abasic sites separated from each other by deoxythymidine.

[0081] In some embodiments, the termination motif comprises or consists of: (i) one or more modified nucleotides and (ii) one or more abasic sites. For example, the termination motif comprises or consists of: (i) two modified nucleotides and (ii) one abasic site, wherein the abasic site is inserted between the two modified nucleotides. In some embodiments, the termination motif can comprise or consist of: (i) one or two 2'-O-methoxy-ethyl modified nucleotides and (ii) two adjacent abasic sites that bind to the 5' end of the modified nucleotide. In some embodiments, the termination motif can comprise or consist of two 2'-O-methoxy-ethyl modified nucleotides separated by one abasic site.

[0082] In some embodiments, the termination motif can also be a chemical linker. In some embodiments, the chemical linker is a triethylene glycol spacer or a phosphoramidite spacer, such as C3S (or C3SP, also known as C3 spacer or C3 spacer phosphoramidite, which is 3-(4,4'-dimethoxytriphenylmethoxy)propyl-1-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite) or C9S.

[0083] In some embodiments, the termination motif comprises or consists of: (i) one or more modified nucleotides and (ii) one or more chemical linkers. Preferably, the termination motif comprises or consists of: (i) at least two modified nucleotides and (ii) one or more chemical linkers. Preferably, the chemical linker is inserted between two modified nucleotides. In some embodiments, the termination motif comprises or consists of: (i) two modified nucleotides and (ii) one chemical linker. In some embodiments, the termination motif consists of two modified nucleotides separated by one chemical linker. In some embodiments, the termination motif can comprise (i) one or two 2'-O-methoxy-ethyl modified nucleotides and (ii) a triethylene glycol spacer that binds to the 5' end of the modified nucleotide. In some embodiments, the termination motif comprises two 2'-O-methoxy-ethyl modified nucleotides separated by the chemical linker C3S. In some embodiments, the termination motif comprises two 2'-O-methoxy-ethyl modified nucleotides separated by the chemical linker C9S.

[0084] When referring to the termination motif, the term "hairpin structure" refers to a nucleotide sequence that can have a secondary hairpin structure. Some bacterial intrinsic terminators can have a hairpin structure. Examples of these types of termination motifs include Rho-independent terminators, more specifically the T7 terminator.

[0085] As used herein, the term "protein binding site" refers to deoxyribonucleic acid, the complementary RNA of which can be recognized by a protein involved in releasing RNA polymerase from the resulting RNA. Examples of such terminator motifs include Rho-dependent terminators, the complementary RNA of which can be recognized by the Rho factor.

[0086] In some embodiments, the terminator motif is a terminator motif isolated from or derived from bacteria or phages, such as the T7 terminator or any variant thereof. Such variants have been described in the art (Mairhofer et al., ACS Synth Biol. 2015 Mar 20;4(3):265-73; Macdonald et al., J Mol Biol. 1994 Apr 29;238(2):145-58.) or can be determined by conventional methods. In some embodiments, the nucleic acid construct does not contain any terminator motif.

[0087] Hairpin-forming oligonucleotide

[0088] In some embodiments, the single-stranded nucleic acid is located near the 5'-end of the hairpin-forming oligonucleotide, i.e., within the boundary of 5, 4, 3, 2, or 1 nucleotide before the 5'-end of the hairpin-forming oligonucleotide, or exactly at the 5'-end of the hairpin-forming oligonucleotide. In a specific embodiment, the 3'-end of the single-stranded nucleic acid is directly linked to the 5'-end of the hairpin-forming oligonucleotide, i.e., directly linked to the 5'-end of the reverse complement of the promoter.

[0089] In some embodiments, the hairpin-forming oligonucleotide sequentially comprises, from 5' to 3', the reverse complement of the promoter, a loop region, and the promoter. In other words, the hairpin-forming oligonucleotide comprises, from 3' to 5', the promoter, the reverse complement of the promoter, and an intervening loop region that connects the promoter and the reverse complement of the promoter.

[0090] The loop region is the connecting portion between the promoter and its reverse complement. The loop region is formed by a single-stranded oligonucleotide composed of natural and / or non-natural deoxyribonucleotides and / or ribonucleotides. Natural deoxyribonucleotides, such as deoxyadenosine, deoxycytidine, deoxyguanosine, and deoxythymidine. Natural ribonucleotides, such as adenosine, cytidine, guanosine, and uridine. Non-natural deoxyribonucleotides and ribonucleotides can include modified bases, sugars, or internucleoside linkages. Advantageously, the sequence of the loop region is not complementary to any other part of the nucleic acid construct of the present invention.

[0091] Surprisingly, the inventors have demonstrated that the presence of a loop region that binds a promoter and its reverse complement in a nucleic acid construct, particularly a loop region having 6 to 9 nucleotides, can increase the transcription efficiency as compared to the transcription efficiency using a double-stranded DNA structure or a partially double-stranded DNA structure of the prior art.

[0092] In some embodiments, the loop region comprises at least 3 nucleotides, particularly 4 to 15 nucleotides, more particularly 5 to 10 nucleotides. In a specific embodiment, the loop region comprises 5, 6, 7, 8, 9 or 10 nucleotides or consists of 5, 6, 7, 8, 9 or 10 nucleotides.

[0093] In a preferred embodiment, the loop region consists of 6, 7 or 9 nucleotides.

[0094] In some embodiments, the promoter of the hairpin-forming oligonucleotide can be any conventional prokaryotic, eukaryotic, viral or synthetic promoter described in the art. Such promoters can be ubiquitous or tissue-specific, homologous or heterologous to the template to be transcribed. The promoter can be a wild-type promoter or a variant having a modified sequence.

[0095] In some embodiments, the promoter is a phage promoter, such as phage T7, T3, Sp6, K11 or gh-1.

[0096] In some embodiments, the promoter is selected from the T7 promoter, the T3 promoter, the Sp6 promoter, the K11 promoter, the gh-1 promoter and any variants thereof.

[0097] As used herein, the term "T7 promoter" refers to the phage T7 promoter having the sequence shown in SEQ ID NO:1. The T7 promoter is specifically recognized by T7 RNA polymerase.

[0098] As used herein, the term "T3 promoter" refers to the phage T3 promoter having the sequence shown in SEQ ID NO:2. The T3 promoter is specifically recognized by T3 RNA polymerase.

[0099] As used herein, the term "Sp6 promoter" refers to the phage Sp6 promoter having the sequence shown in SEQ ID NO:3. The Sp6 promoter is specifically recognized by sp6 RNA polymerase.

[0100] As used herein, the term "K11 promoter" refers to the phage K11 promoter having the sequence shown in SEQ ID NO:4. The K11 promoter is specifically recognized by K11 RNA polymerase.

[0101] As used herein, the term "gh-1 promoter" refers to the bacteriophage gh-1 promoter having the sequence shown in SEQ ID NO:5. The gh-1 promoter is specifically recognized by the gh-1 RNA polymerase.

[0102] As used herein, the term "variant" of a given promoter refers to a promoter derived from a given promoter selected from the group consisting of the T7 promoter, the T3 promoter, the Sp6 promoter, the K11 promoter, and the gh-1 promoter and that can be specifically recognized by the corresponding RNA polymerase to initiate transcription. For example, a variant of the T7 promoter is a promoter derived from the T7 promoter and that can be specifically recognized by the T7 RNA polymerase. Variants of the above promoters are well known in the art, for example, as described in Chen and Schneider, Nucleic Acids Res. 2005; 33(19):6172–6187. Alternatively, they can be determined by a person skilled in the art according to conventional methods.

[0103] In the nucleic acid constructs of the present invention, the sequence of the reverse complement of a promoter is substantially reverse complementary to the promoter sequence.

[0104] In some embodiments, the sequence of the reverse complement of a promoter is completely reverse complementary to the promoter sequence.

[0105] In some embodiments, the sequence of the reverse complement of a promoter contains 1 to 10, particularly 1 to 5, 1 to 4, and more particularly 1 to 3 nucleotide deletions or substitutions compared to the sequence that is completely reverse complementary to the promoter sequence.

[0106] Use of nucleic acid construct

[0107] In some embodiments, the nucleic acid constructs as defined above can be free in solution or attached to a solid support via their 5' end. Such a solid support can be a resin, a bead, such as a magnetic bead, a capture bead, or other material forming a capture bead (e.g., a macroscopic structure and / or an insoluble material), or a planar solid, such as a slide, a membrane, or a plate (e.g., a plate containing a plurality of wells).

[0108] In some embodiments, the nucleic acid construct is covalently attached to a solid support, such as a resin, via its 5'-end. In an embodiment, the nucleic acid construct is attached to the solid support via the 5'-end of a single-stranded nucleic acid. In another embodiment, the nucleic acid construct is attached to the solid support via a cleavable motif, which can be cleaved to release the nucleic acid construct of the present invention. The cleavable motif can be any means known in the prior art, such as a short oligonucleotide containing at least one enzymatically cleavable nucleotide or a chemically cleavable internucleotide bond. Examples of enzymatically cleavable nucleotides include, but are not limited to, deoxyuridine and deoxyinosine. For example, the cleavable motif can be dIT. An example of a chemically cleavable nucleotide is ribouridine (rU), which can be cleaved by KOH.

[0109] Accordingly, an object of the present invention is to provide a nucleic acid construct attached to a solid support via its 5'-end as defined above. In a specific embodiment, the nucleic acid construct is attached to the solid support via a terminator motif at the 5'-end of the nucleic acid construct. In another embodiment, the nucleic acid construct is attached to the solid support via a cleavable motif at the 5'-end of the nucleic acid construct terminator motif.

[0110] Method

[0111] The present invention also provides a method for producing an RNA product by using the nucleic acid construct as defined above. The method comprises the following steps:

[0112] (a) providing the nucleic acid construct as defined above,

[0113] (b) contacting the nucleic acid construct with an RNA polymerase and ribonucleotides to produce the RNA product.

[0114] Step (a)

[0115] In some embodiments, the nucleic acid construct used in step (a) is free in solution.

[0116] In some embodiments, the nucleic acid construct used in step (a) is attached to a solid support via its 5'-end.

[0117] Due to the primary single-stranded structure of the nucleic acid construct of the present invention, the nucleic acid construct of the present invention used in step (a) can be easily synthesized by any conventional method, such as chemical synthesis based on solid-phase phosphoramidite chemistry described by Adams et al. (1983, J. Amer. Chem. Soc., 105, 661) and Froehler et al. (1983, Tetrahedron Lett., 24, 3171), or by enzymatic synthesis. Template-independent enzymatic polynucleotide synthesis methods are described in detail in, for example, WO2015 / 159023, WO2017 / 216472, U.S. Patent 5436143, U.S. Patent 5763594, Jensen et al. (Biochemistry, 57:1821-1832 (2018)) or Mathews et al. (Organic & Biomolecular Chemistry, DOI: 0.1039 / c6ob01371f (2016)); Schmitz et al. (Organic Lett., 1(11):1729-1731 (1999)).

[0118] In some embodiments, the method of the present invention includes a step of synthesizing a nucleic acid construct before step (a).

[0119] In some embodiments, the step of synthesizing the nucleic acid construct is carried out by an enzymatic method, particularly an enzymatic method using a template-free polymerase, such as terminal deoxynucleotidyl transferase (TdT) or variants, such as those described in detail in WO2019 / 135007 for DNA synthesis, or polyA polymerase (PAP) or polyU polymerase (PUP) or variants thereof commonly used for RNA synthesis (e.g., Heinisch et al, WO2021 / 018919). In some embodiments, the nucleic acid construct is synthesized from an initiator having a free 3'-hydroxyl group. The initiator can be attached to a solid support through its 5'-end by any conventional method.

[0120] In some embodiments, the enzymatic method includes the steps of: (i) contacting an initiator having a free 3'-hydroxyl group or an extended fragment thereof with a 3'-O-blocked nucleoside triphosphate and a template-free polymerase to form a 3'-O-blocked extended fragment, (ii) de-blocking the extended fragment to form an extended fragment having a free 3'-hydroxyl group, and (iii) repeating the cycles of (i) and (ii) until a nucleic acid construct is formed.

[0121] Step (b)

[0122] "Contacting" means adding RNA polymerase to a reaction medium containing a nucleic acid construct under reaction conditions suitable for in vitro transcription. These conditions are well known to those skilled in the art (Jani and Fuchs, J Vis Exp. 2012 Mar 26(61):3702). At the end of step (b), the template portion in the nucleic acid construct is transcribed by RNA polymerase, and an RNA product is generated and released into the reaction medium.

[0123] The reaction medium advantageously contains four natural ribonucleotides (rNTPs), such as rATP, rUTP, rGTP, rCTP. The reaction medium can also contain unnatural nucleotides, which can be incorporated into the RNA product by RNA polymerase during in vitro transcription. The unnatural nucleotides can be present in the reaction medium in different ratios relative to the natural rNTPs. The unnatural nucleotides in the reaction medium can be modified nucleotides containing reactive groups for "click" reactions, such as 5-octynyl-rNTP, which can be randomly added to RNA during in vitro transcription. Unnatural nucleotides include, but are not limited to, 2'-O-methyl modified nucleotides, 2'-O-methoxy-ethyl modified nucleotides, 2'-F modified nucleotides, α-thiophosphate modified nucleotides, which increase the stability of the RNA product produced by in vitro transcription (IVT) against RNA degrading enzymes (RNAase), and increase the affinity of the RNA product for in vivo DNA or RNA targets. Other unnatural nucleotides can be 5' caps, such as m7GpppG, m7GpppA, G5ppp5A, G5ppp5G, 3'-O-Me-m7G(5')ppp(5')G and their derivatives, which are selectively incorporated into the 5' end of the RNA product by RNA polymerase. The 5' cap increases the stability against nucleases and reduces the in vivo toxicity side effects of RNA due to the absence of the 5'-triphosphate group. Variants of m7GpppG include, but are not limited to: i) biotinylated m7GpppG, which allows the RNA to be used as a capture probe, such as for target enrichment, especially in NGS target enrichment; ii) fluorescent m7GpppG, which allows the RNA to be used for in vivo imaging applications (such as FISH probes); iii) alkyne-m7GpppG, which allows post-transcriptional chemical modification of RNA by copper-catalyzed click reaction. Similar variants of other 5' caps are also described in the prior art.

[0124] Optionally, the method of the present invention can further include step (c), which consists of repeating step (b) one or more times to produce several copies of the RNA product.

[0125] According to the present invention, the RNA polymerase directly hybridizes with the nucleic acid construct to synthesize an RNA product, i.e., there is no any additional intermediate step in the method, wherein the nucleic acid construct hybridizes with a complementary DNA fragment.

[0126] Thus, in a preferred embodiment, the method consists of the following steps:

[0127] (a) providing a nucleic acid construct as defined above, and

[0128] (b) contacting the nucleic acid construct with an RNA polymerase and ribonucleotides to produce the RNA product, and optionally,

[0129] (c) repeating step (b) to produce several copies of the RNA product. Advantageously, the nucleic acid of step (a) is attached to a solid support.

[0130] The selection of the RNA polymerase is adapted to the promoter in the nucleic acid construct. For example, if the promoter in the nucleic acid construct is a T7 promoter or its variant, T7 RNA polymerase will be used in step (b). Depending on the selected RNA polymerase, those skilled in the art can adjust the reaction conditions (such as pH and temperature) of step (b).

[0131] Optional steps (d) and (e)

[0132] The RNA product produced in step (b) or step (c) can be further subjected to at least one post-transcriptional modification (step (d)).

[0133] The term "post-transcriptional modification" refers to the biological process in which the primary RNA is chemically altered to produce a mature functional RNA molecule. The term "primary RNA" refers to the RNA produced directly after transcription. In the context of the present invention, the RNA produced in step (b) or step (c) is primary RNA. Those skilled in the art can determine whether it is necessary to perform at least one post-transcriptional modification on the RNA product obtained in step (b) or step (c) according to the intended use of the RNA product. For example, if the primary RNA is produced for further protein translation, it may be necessary to perform at least one post-transcriptional modification to convert the primary RNA into mature mRNA. Generally speaking, post-transcriptional modifications include modifications related to the 5' end and / or 3' end of the primary RNA. Examples of post-transcriptional modifications include but are not limited to 5' capping, 3' extension after transcription, and intron splicing.

[0134] In some embodiments, the at least one post-transcriptional modification is 5' capping, 3' extension with polyA polymerase (PAP), ligation of a modified polyA tail, or chemical modification of unnatural nucleotides using a "click" reaction.

[0135] The term "5'-capping" refers to the addition of a 5'-cap as described above to the 5'-end of a primary RNA.

[0136] The term "3'-elongation with polyA polymerase" refers to the addition of adenine to the 3'-end of a primary RNA to form a polyA tail.

[0137] The term "ligating a modified polyA tail" refers to the chemical or enzymatic addition of a modified polyA tail (e.g., containing 2'-O-methyl modified nucleotides or 2'-F modified nucleotides) to the 3'-end of a primary RNA. Chemical ligation can be carried out by cycloaddition reactions (e.g., "click" reactions), nucleophilic substitutions (e.g., NHS ester substitution), or other click reactions. Enzymatic ligation can be carried out by using ligases to add the modified polyA tail.

[0138] Examples of chemical modifications of unnatural nucleotides can be copper-catalyzed or strain-promoted "click reactions", nucleophilic substitution reactions, cycloaddition reactions, or metathesis reactions. For example, a primary RNA containing an unnatural nucleotide containing an alkyne group (e.g., 5-octynyl-rNTP or alkyne-m7GPPPG) can undergo a post-transcriptional chemical copper-catalyzed or strain-promoted "click reaction" with an azide-modified functional molecule. Examples of such functional molecules can be biotin (e.g., PEG4 amide-6-azidohexyl biotin), fluorophore (e.g., 5-FAM-azide), protein label (e.g., antibody-azide), or drug payload (e.g., artemisinin-azide).

[0139] In vitro methods for performing at least one such post-transcriptional modification have been well described in the art (Warren, L., et al. (2010) Cell Stem Cell, 7, 618 - 630). A person skilled in the art is able to implement a dedicated method according to the modification to be carried out.

[0140] The method of the present invention may further include step (e) for recovering the RNA product generated in steps (b), (c), and / or (d). This step allows the separation of the RNA product from the nucleic acid construct and the reaction medium.

[0141] The recovery step can be carried out by any standard nucleic acid purification method, such as by gel purification, affinity column, or any commercially available nucleic acid purification kit.

[0142] In a specific embodiment, the method comprises the following steps or consists of the following steps: (a) providing a nucleic acid construct as defined above, preferably attached to a solid support, and

[0143] (b) contacting the nucleic acid construct with an RNA polymerase and ribonucleotides to produce the RNA product, and optionally,

[0144] (c) Repeat step (b) to produce several copies of said RNA product, and / or optionally,

[0145] (d) Perform at least one post-transcriptional modification, and / or optionally,

[0146] (e) Recover the RNA product produced in steps (b), (c) and / or (d).

[0147] Kit

[0148] The present invention also provides a kit for producing an RNA product. The term "kit" refers to any necessary reagents and optional devices for producing an RNA product by using the nucleic acid construct or method of the present invention.

[0149] In some embodiments, the kit of the present invention comprises an RNA polymerase, several ribonucleotides, one or more reaction buffers and a nucleic acid construct as defined above.

[0150] The RNA polymerase included in the kit can be any conventional prokaryotic, eukaryotic or viral RNA polymerase as described in the art. In a specific embodiment, the RNA polymerase is a bacterial or phage RNA polymerase. In a more specific embodiment, the RNA polymerase of the kit is selected from T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, K11 RNA polymerase and gh-1 RNA polymerase.

[0151] In some embodiments, the kit comprises an RNA polymerase that specifically recognizes the promoter of the nucleic acid construct in the kit. In a specific embodiment, the kit comprises an RNA polymerase homologous to the promoter comprised in the nucleic acid construct in the kit, as described above.

[0152] The ribonucleotides included in the kit are natural ribonucleotides, namely adenosine, cytidine, guanosine and uridine.

[0153] The kit of the present invention may also comprise unnatural nucleotides as described above, which can be incorporated into the RNA product by the RNA polymerase during in vitro transcription.

[0154] The reaction buffer may contain salts and any reagents to optimize the activity of the enzymes included in the kit.

[0155] Alternatively, the kit of the present invention contains any necessary reagents for synthesizing the nucleic acid construct as defined above, and the reagents necessary for generating an RNA product from the nucleic acid construct. In such an embodiment, the kit contains an RNA polymerase, several ribonucleotides and optionally unnatural nucleotides, one or more reaction buffers, an initiator having a 3'-terminal nucleotide with a free 3'-hydroxyl group, a template-free polymerase, a variety of 3'-O-blocked nucleoside triphosphates, and a deblocking agent. In the context of the present invention, the term "initiator" refers to a short single-stranded oligonucleotide having a free 3'-hydroxyl group, which can be further extended by a template-free polymerase (such as TdT). The initiator can contain 3 to 100 nucleotides, particularly 3 to 20 nucleotides. In some embodiments, the initiator can contain a non-nucleic acid compound having a free hydroxyl group, to which TdT can couple a 3'-O-blocked dNTP.

[0156] The template-free polymerase can be any template-free DNA polymerase, such as TdT or its variants commonly used in DNA synthesis (such as Ybert et al, WO2017 / 216472; Champion et al, WO2019 / 135007). Or polyA polymerase (PAP) or polyU polymerase (PUP) or their variants commonly used in RNA synthesis (such as Heinisch et al, WO2021 / 018919).

[0157] The 3'-O-blocked nucleoside triphosphates can be 3'-O-blocked dNTPs and / or 3'-O-blocked rNTPs. These compounds contain a blocking group that prevents chemical changes in the hydroxyl group at the 3'-position during chemical or enzymatic processes. The blocking group can be any blocking group known in the prior art, such as 3'-O-NH2, 3'-O-azidomethyl, 3'-O-allyl or 3'-O-phosphate.

[0158] Therefore, the kit of the present invention can also contain a "deblocking agent", which is a chemical or enzymatic reagent capable of cleaving a specific blocking group. The choice of deblocking agent depends on the type of 3'-nucleotide blocking group used, whether the initiator is attached to a solid support, etc. For example, phosphines, such as tris(2-carboxyethyl)phosphine (TCEP), can be used to cleave 3'O-azidomethyl, palladium complexes can be used to cleave 3'O-allyl, or sodium nitrite can be used to cleave 3'O-amino groups.

[0159] In some embodiments, the kit further contains a solid support, wherein the initiator or the nucleic acid construct is attached to the solid support through its 5'-end. The solid support can be a resin, a bead or a plate.

[0160] The kit of the present invention may also contain pyrophosphatase. The pyrophosphatase can be any conventional pyrophosphatase for in vitro transcription, such as inorganic pyrophosphatase. The enzyme catalyzes the hydrolysis of inorganic pyrophosphate and can be used to enhance RNA / DNA synthesis because inorganic pyrophosphate is a by-product of nucleotide addition and can hinder subsequent nucleotide addition.

[0161] The kit of the present invention may also contain the necessary reagents for performing at least one post-transcriptional modification on the resulting RNA. These reagents can be the necessary chemical compounds and / or enzymes for performing 5'-capping, 3'-extension, and / or ligation-modified polyA tail. For example, these reagents can be polyA polymerase (PAP), T7 ligase, or chemical compounds for performing cycloaddition reactions.

[0162] The kit of the present invention may also contain any delivery system for delivering materials. Such a delivery system includes a system for storing reaction reagents, transporting or delivering reaction reagents from one location to another location, and / or supporting materials (e.g., reaction medium, written instructions for performing assays, etc.). For example, the kit may include one or more outer shells (e.g., boxes) containing the relevant reaction reagents and / or supporting materials.

[0163] The kit of the present invention may also contain a device for recovering the resulting RNA. Such a device can be any conventional polynucleotide recovery system, such as a column for purification by affinity or precipitation.

[0164] In some embodiments, the kit of the present invention is a kit for generating sgRNA. The kit contains an RNA polymerase, several ribonucleotides, an initiator having a 3'-terminal nucleotide with a free 3'-hydroxyl group, a template-free DNA polymerase, a variety of 3'-O-blocked nucleoside triphosphates, and a deblocking agent, wherein the initiator contains a deoxyribonucleic acid having a complementary sequence to the constant part of the sgRNA.

[0165] Alternatively, the kit for generating sgRNA contains an RNA polymerase, several ribonucleotides, and the nucleic acid construct of the present invention, wherein the nucleic acid construct contains a single-stranded nucleic acid, and the single-stranded nucleic acid contains a template part for generating sgRNA.

[0166] The present invention is illustrated in more detail by the following examples.

[0167] Examples

[0168] In vitro transcription

[0169] In vitro transcription was carried out in 30 μL of reaction medium in a microplate, which contained 6.7 mM NTP buffer mixture, 1 pM free nucleic acid construct, 60 μM DFHBI-1T, 2 μL of T7 RNA polymerase mixture (HiScribe from NEB TM T7 High Yield RNA Synthesis Kit) and 14 μL of nuclease-free water. The reaction medium for the immobilized construct was the same, except that the reaction medium contained 7.5 pM of immobilized nucleic acid instead of 1 pM of free nucleic acid construct. Then, the microplate was incubated at 37 °C for 18 h with stirring and fluorescence was read in the GFP channel (excitation at 395 nm and detection at 509 nm).

[0170] Real-time fluorescence quantitative PCR monitoring

[0171] In vitro transcription was carried out according to the above method, except that the microplate was incubated at 37 °C for 12 h without stirring and fluorescence was read in the SYBR channel (excitation at 450 - 490 nm and detection at 515 - 530 nm).

[0172] Gel electrophoresis of transcription products

[0173] After in vitro transcription, 5 μL of reaction medium and 5 μL of RNA loading dye were mixed. The mixture was boiled at 98 °C for 5 min. After cooling on ice, the mixture was loaded onto a TBE-urea 10% gel (Novex TM TBE-urea gel, Invitrogen TM ). The gel was prepared with high-purity reagents, including Tris base, boric acid, EDTA, acrylamide, bisacrylamide, TEMED, APS, and 7 M urea. Gel electrophoresis was run at 180 V for 1 h.

[0174] Example 1: In vitro transcription from free nucleic acid constructs

[0175] Five nucleic acid constructs, including two constructs of the present invention, namely "6-nt loop" and "9-nt loop", two positive control constructs ("dsDNA" and "ssDNA + promoter"), and a negative control "ssDNA", were provided as free nucleic acid constructs in solution.

[0176] The constructs of the present invention sequentially comprise, from the 5' to the 3' end, a single-stranded DNA consisting of the template portion of the Broccoli RNA to be synthesized, the reverse complement of the T7 promoter, a loop of 6 or 9 nucleotides, and the T7 promoter. The constructs "6-nt loop" and "9-nt loop" have the sequences of SEQ ID NO:6 and SEQ ID NO:7, respectively.

[0177] The construct "dsDNA" contains double-stranded DNA encoding Broccoli RNA and a double-stranded functional T7 promoter. The construct is formed by the forward DNA strand of SEQ ID NO:8 and the reverse DNA strand of SEQ ID NO:9.

[0178] The construct "ssDNA+promoter" contains single-stranded DNA that serves as a template for Broccoli RNA and a double-stranded functional T7 promoter. The construct is formed by the forward strand of SEQ ID NO:1 and the reverse strand of SEQ ID NO:9.

[0179] The negative control "ssDNA" is single-stranded DNA that sequentially contains a Broccoli RNA template and a T7 promoter. The construct has the sequence of SEQ ID NO:9.

[0180] Broccoli RNA is a small RNA aptamer that can bind to the small molecule DFHBI-1T and become fluorescent like GFP (Milligan et al., Nucleic Acids Res. 1987 Nov 11; 15(21): 8783-98). Thus, the amount of Broccoli RNA produced from these free nucleic acid constructs by in vitro transcription can be monitored by quantitative fluorescence emission measured by a qPCR instrument.

[0181] In vitro transcription was carried out at 37 °C for 12 hours. The fluorescence intensity is related to the amount of Broccoli RNA produced and was measured every 90 seconds by a qPCR instrument (CFX96 Real-Time System C1000 Touch Thermal Cycler, from Bio-Rad) using the SYBR channel (excitation at 395 nm and detection at 509 nm). The results are shown in Figure 2 .. Both of the free nucleic acid constructs of the present invention tested allowed for more rapid production of Broccoli RNA than the positive control constructs "dsDNA" or "ssDNA+promoter". Thus, the nucleic acid constructs of the present invention are more efficient than the positive control constructs in producing RNA.

[0182] Example 2: In vitro transcription from immobilized nucleic acid constructs

[0183] Two nucleic acid constructs of the present invention are immobilized on a resin ("immobilized nucleic acid constructs"), namely "resin 6-nt loop" and "resin 9-nt loop". The constructs are linked to the resin at the 5' end and sequentially comprise inosine, single-stranded DNA consisting of the template portion of Broccoli RNA, the reverse complement of the T7 promoter, a loop of 6 or 9 nucleotides, and the T7 promoter from the 5' to the 3' end. The constructs "resin 6-nt loop" and "resin 9-nt loop" have the sequences SEQ ID NO:10 and SEQ ID NO:11, respectively.

[0184] The immobilized constructs and the free constructs "9-nt loop", "6-nt loop", "dsDNA", and "ssDNA" described in Example 1 are used for in vitro transcription.

[0185] In vitro transcription is carried out at 37 °C for 17.5 hours. The fluorescence intensity is measured every 90 seconds using a qPCR instrument (CFX96 Real-Time System C1000 Touch Thermal Cycler, from Bio-Rad) with the SYBR channel.

[0186] The results of the fluorescence intensity indicate that the immobilized nucleic acid constructs "resin 6-nt loop" and "resin 9-nt loop" can effectively produce Broccoli RNA ( Figure 3A ). Figure 3B It is also confirmed that the obtained RNA product is Broccoli RNA (51 bp).

[0187] Example 3: Efficiency of termination motif in stopping in vitro transcription

[0188] In vitro transcription (IVT) is carried out using the nucleic acid constructs of the present invention. The constructs comprise short single-stranded nucleic acids which consist of a 15-nucleotide sequence, a termination motif, and a 21-nucleotide sequence serving as the template portion for generating RNA from the 5' to the 3' end. The termination motif is located between the two sequences and at the 5' end of the 21-nucleotide sequence. The termination motifs are two adjacent abasic sites (AP2), three adjacent abasic sites (AP3), two abasic sites separated by deoxyinosine (APiAP), two abasic sites separated by deoxythymidine (APTAP), three abasic sites separated by deoxythymidine (APT3) and a triethylene glycol spacer (C9S) from each other. In vitro transcription is also carried out using the constructs "IVT termination" and "no termination" as controls. The construct "no termination" contains a 36-nucleotide template which has no termination motif between the 21-nucleotide sequence and the 15-nucleotide sequence. The construct "IVT termination" contains a 21-nucleotide template.

[0189] On a 15% TBE-urea gel (Novex TMTBE-Urea Gel, Invitrogen TM ) Analyze the RNA products generated after IVT.

[0190] Figure 4 Show that constructs containing the termination motifs AP2, AP3, APiAP, APTAP, APT3, and C9S all produce 21-nucleotide RNAs, indicating that these termination motifs effectively terminate transcription.

[0191] Example 4: Role of 2'-O-methoxyethyl-modified nucleotides in the nucleic acid constructs of the present invention

[0192] During IVT, RNA polymerase may add unwanted extra dTs to the RNA generated from the template of the RNA. Those extra dTs are the result of the non-template activity of RNA polymerase. To evaluate the effect of 2'-O-methoxy-ethyl groups on RNA polymerase activity, 10 single guide RNAs (sgRNAs) were synthesized from different nucleic acid constructs of the present invention using T7 RNA polymerase. These constructs contain a template portion for generating the sgRNA and a termination motif at the 5' end of the template portion. The termination motifs tested were AP2, C9S, dU2, AP2 bound to 2'-O-methoxy-ethyl modified nucleotides ("AP2-Methox"), or C9S bound to 2'-O-methoxy-ethyl modified nucleotides ("C9S-Methox").

[0193] Before performing IVT, the nucleic acid construct containing dU2 was incubated with UDG enzyme (NEB) at 37 °C for 1 h to convert deoxyuridine (dU) to an abasic site.

[0194] After in vitro transcription, the generated sgRNAs were subjected to RNA sequencing. Library preparation was performed using the SEQuoia Complete Stranded RNA Library Prep Kit (Bio-Rad). Figure 5A Show that termination motifs containing 2'-O-methoxy-ethyl modified nucleotides ("C9S-Methox" and "AP2-Methox") significantly reduce the non-template activity of T7 polymerase compared to AP2, C9S, or dU2.

[0195] In addition, the effect of 2'-O-methoxy-ethyl modified nucleotides on single-stranded nucleic acids was tested. The single-stranded nucleic acids consisted of a 15-nucleotide sequence from 5' to 3', a termination motif, and a 21-nucleotide sequence as the template portion of the single-stranded nucleic acid. The termination motif was C9SP (without Met) or C9SP bound to one (1Met) or two (2Met) 2'-O-methoxy-ethyl modified nucleotides. After IVT, on a 15% TBE-Urea gel (Novex TMTBE-Urea Gel, Invitrogen TM ) Analyze the resulting RNA products above. Figure 5B It shows that the presence of one or two 2'-O-methoxyethyl-modified nucleotides in the termination motif increases the RNA yield of the template.

[0196] Therefore, the termination motif containing one or two 2'-O-methoxyethyl-modified nucleotides in the nucleic acid construct of the present invention reduces the non-template activity of RNA polymerase and increases the yield of RNA produced by in vitro transcription.

Claims

1. A nucleic acid construct capable of producing at least one RNA product, comprising, from 5' to 3': (i) a single-stranded nucleic acid containing at least one template portion for producing the RNA product, and (ii) a hairpin-forming oligonucleotide, which in turn comprises a reverse complement of a promoter, a loop region, and a promoter, wherein the reverse complement of the promoter and the promoter are capable of forming a double-stranded oligonucleotide, and wherein the single-stranded nucleic acid contains at least one termination motif at the 5' end of the template portion of the single-stranded deoxyribonucleic acid, and wherein the termination motif comprises at least two components selected from modified nucleotides, abasic sites, modified abasic sites, hairpin structures, protein binding sites, chemical linkers, and combinations thereof.

2. The nucleic acid construct according to claim 1, wherein the termination motif comprises at least two modified nucleotides, or at least two abasic sites or modified abasic sites.

3. The nucleic acid construct according to claim 2, wherein the termination motif further comprises one or more chemical linkers.

4. The nucleic acid construct according to any one of the preceding claims, wherein the promoter is selected from T7 promoter, T3 promoter, Sp6 promoter, K11 promoter, and gh-1 promoter or variants thereof.

5. The nucleic acid construct according to any one of the preceding claims, wherein the single-stranded nucleic acid contains a template portion for producing sgRNA, small RNA, RNA aptamer, or mRNA.

6. The nucleic acid construct according to any one of the preceding claims, wherein the loop region contains at least 3 nucleotides, particularly 4 to 15 nucleotides, more particularly 5 to 10 nucleotides.

7. The nucleic acid construct according to claim 6, wherein the loop region contains 6, 7, or 9 nucleotides.

8. The nucleic acid construct according to any one of the preceding claims, wherein the single-stranded nucleic acid has a length of at least 10 nucleotides.

9. The nucleic acid construct according to any one of the preceding claims, wherein the nucleic acid construct is attached to a solid support through its 5' end.

10. A kit for producing an RNA product, comprising: - RNA polymerase, ribonucleotides, optionally one or more modified nucleotides, and one or more reaction buffers, and -(i) the nucleic acid construct according to any one of claims 1 to 9, or (ii) an initiator having a 3'-terminal nucleotide with a free 3'-hydroxyl group, a template-free DNA polymerase, a variety of 3'-O-blocked nucleoside triphosphates, and a deblocking agent.

11. The kit according to claim 10, further comprising a solid support, wherein the nucleic acid construct or the initiator is attached to the solid support through its 5' end.

12. The kit for producing sgRNA according to claim 10 or 11, the kit comprising an initiator having a deoxyribonucleic acid sequence complementary to the nucleic acid sequence of the constant portion of the sgRNA.

13. A method for producing an RNA product, comprising the following steps or consisting of the following steps: (a) Provide a nucleic acid construct according to any one of claims 1 to 9, (b) Contact the nucleic acid construct with an RNA polymerase and ribonucleotides to produce the RNA product, (c) Optionally repeat step (b), (d) Optionally perform at least one post-transcriptional modification, (e) Optionally recover the RNA product produced in steps (b), (c) and / or (d).

14. The method according to claim 13, comprising a step of synthesizing the nucleic acid construct before step (a).

15. The method according to claim 14, wherein the step of synthesizing the nucleic acid construct is carried out by an enzymatic method.

16. The method according to any one of claims 13 to 15, wherein the nucleic acid construct of step (a) is attached to a solid support via its 5' end.

17. The method according to any one of claims 13 to 16, wherein the nucleic acid construct comprises a termination motif, the termination motif comprising (i) at least two modified nucleotides, and (ii) one or more chemical linkers.

18. The method according to any one of claims 13 to 17, wherein the nucleic acid construct of step (a) comprises a template portion for generating sgRNA.

Images