Compositions for preventing repeated addition and non-specific primer extension of conversion oligonucleotides during cDNA synthesis and methods of use thereof

Abstract

The present invention provides optimized template conversion oligonucleotides, methods and kits for performing reverse transcriptions. The optimized template conversion oligonucleotides include modifications at the 5'end and the 3 'end to prevent the formation of a concatemer and enhance the specificity of reverse transcription.

Description

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. HG011868 awarded by the National Institutes of Health. The U.S. Government may have certain rights in this invention.

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Application No. 63 / 375,592, filed on September 14, 2022, which is hereby incorporated by reference in its entirety. Background Art

[0005] RNA sequencing has become the standard method of transcriptome analysis. Compared with other methods, it provides the level of transcript and its isomer and the measurement of diversity much more accurately. When the terminal sequence of transcript is unknown, or when sequencing library is prepared by the RNA material (for example, total RNA from unicellular) of very limited amount, robust full-length cDNA synthesis and adapter addition are crucial for producing representative unbiased nucleic acid material source from the transcriptome studied. In this case, reverse transcriptase (RT) template switching reaction has been utilized to simultaneously enrich full-length cDNA and add two different adapters to the 5 ' end and 3 ' end of cDNA molecule in the first chain building-up process, which overcomes many shortcomings of the method based on connection. The unicellular application utilizing template switching depends on the efficiency and specificity of reverse transcription and template switching reaction. Because RNA input is extremely low, the product of non-specific reverse transcription from template switching oligonucleotide (template switching oligo) and template switching oligonucleotide multiplexing (concatemerization) leading sequencing library, thereby limit the application of this method.

[0006] Therefore, there is a need in the art for improved compositions to increase the specificity and yield of RNA-seq library preparation based on template switching. The present invention addresses this unmet need. Summary of the Invention

[0007] The present disclosure features template switching oligonucleotides (TSOs) and compositions comprising the same, and methods of use thereof. In one embodiment, the template switching oligonucleotide (TSO) comprises a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence, or a hybrid DNA-RNA sequence. In embodiments, the TSO can anneal by base pairing with a non-templated nucleotide, such as a non-templated nucleotide added to the 5' end of the target nucleic acid molecule. In embodiments, the non-templated nucleotide is added to the target nucleic acid by a reverse transcriptase. In embodiments, the TSO further comprises at least one of a 3' end modification and a 5' end modification.

[0008] In an embodiment, TSO comprises a 3' end modification. In an embodiment, the 3' end modification may be a modification that removes the 3' hydroxyl group or a modification that blocks the 3' hydroxyl group. In one embodiment, TSO comprises a 3' end modification selected from the group consisting of: 3' ddT, 3' ddU, 3' reversed dT, 3' C3 spacer, 3' amino, 3' rU oxidized by periodate, 3' phosphorylation, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' alkene, 3' (CH2) nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n ≥ 1) and 3' (CH2CH2O) n (n ≥ 1).

[0009] In an embodiment, the TSO comprises a 5' end modification. In one embodiment, the 5' end is modified with a chemical group to reduce or substantially block multiplexing. In one embodiment, the 5' end is modified with a chemical group selected from the group consisting of a trityl group, a trebbler, a dendrimer, biotin, a fluorescent dye, a ROX NHS ester, a (CH2)n (n≥1) long spacer, a palmitate phosphoramidite, a 3-cyanovinylcarbazole phosphoramidite, a cholesterol group, and a psoralen.

[0010] In one embodiment, the 5' end comprises at least 10, 9, 8, 7, 6, 5, 4, 3 or 2 continuous abasic sites. In one embodiment, the 5' end comprises at least 10 continuous abasic sites. In one embodiment, the 5' end comprises at least 8 continuous abasic sites. In one embodiment, the 5' end comprises at least 6 continuous abasic sites. In one embodiment, the 5' end comprises at least 5 continuous abasic sites. In one embodiment, the 5' end comprises at least 4 continuous abasic sites. In one embodiment, the 5' end comprises at least 3 continuous abasic sites. In one embodiment, the 5' end comprises at least 2 continuous abasic sites.

[0011] In one embodiment, the 5' end comprises at least one non-natural nucleotide or nucleotide analog. In one embodiment, the nucleotide at the 5' end is a non-natural nucleotide or nucleotide analog. In one embodiment, the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

[0012] In one embodiment, the TSO comprises at least one 3' end modification and at least one 5' end modification.

[0013] In one embodiment, the present disclosure relates to a reverse transcription (RT) primer comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence or a hybrid DNA-RNA sequence, wherein the RT primer comprises a 5' end modification. In one embodiment, the 5' end is modified with a chemical group to block multiplexing. In one embodiment, the 5' end is modified with a chemical group selected from the group consisting of 5'trebler and 5'trityl. In one embodiment, the 5' end comprises at least 3 continuous abasic sites. In one embodiment, the nucleotides at the 5' end are non-natural nucleotides or nucleotide analogs. In one embodiment, the RT primer comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG) or a combination of iso-dC and iso-dG at the 5' end.

[0014] In one embodiment, the present disclosure relates to a method of generating a cDNA molecule from an RNA template, the method comprising contacting the RNA template with a TSO comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence, or a hybrid DNA-RNA sequence, wherein the TSO is capable of annealing by base pairing with a non-templated nucleotide that has been added to the 5' end of a target nucleic acid molecule in a non-templated addition process by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3' end modification and a 5' end modification, a reverse transcription (RT) primer, and a reverse transcriptase.

[0015] In one embodiment, the RT primer comprises a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence, or a hybrid DNA-RNA sequence, wherein the RT primer comprises a 5' end modification. In one embodiment, the 5' end is modified with a trityl group, a trebbler, a dendrimer, biotin, a fluorescent dye, a ROX NHS ester, a (CH2)n (n≥1) long spacer, a palmitic acid phosphoramidite, a 3-cyanovinylcarbazole phosphoramidite, a cholesterol group, or a psoralen.

[0016] In one embodiment, the reverse transcriptase is MarathonRT or a variant thereof, Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT) or a variant thereof, Avian Myeloblastosis Virus reverse transcriptase (AMV RT) or a variant thereof, HIV reverse transcriptase (HIV RT) or a variant thereof, Bombyx mori R2 RNA element reverse transcriptase (R2RT) or a variant thereof, or TGIRT TM or variations thereof.

[0017] In one embodiment, the method comprises RT-PCR, qRT-PCR, capillary electrophoresis (CE) for RNA structure mapping, transcriptome analysis, in-cell sequencing, next-generation RNA sequencing (RNA-seq), nanopore sequencing, PacBio sequencing, zero-mode waveguide sequencing, cDNA library synthesis or cDNA synthesis assay, or any combination thereof.

[0018] In one embodiment, the present invention relates to a reverse transcription assay for generating a cDNA molecule from an RNA template, the method comprising contacting the RNA template with a TSO comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence, or a hybrid DNA-RNA sequence, wherein the TSO is capable of annealing by base pairing with a non-templated nucleotide that has been added to the 5' end of a target nucleic acid molecule in a non-templated addition process by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3' end modification and a 5' end modification, a reverse transcription (RT) primer, and a reverse transcriptase.

[0019] In one embodiment, the RT primer is selected from the group consisting of a DNA primer, an RNA primer, and a primer comprising at least one modified oligonucleotide.

[0020] In one embodiment, the reverse transcriptase is MarathonRT or a variant thereof, Moloney murine leukemia virus reverse transcriptase (MMLV RT) or a variant thereof, avian myeloblastosis virus reverse transcriptase (AMV RT) or a variant thereof, HIV reverse transcriptase (HIV RT) or a variant thereof, Bombyx mori R2 RNA element reverse transcriptase (R2RT) or a variant thereof, or TGIRT TM or variations thereof.

[0021] In one embodiment, the reverse transcription is performed in a buffer comprising PEG 8000. In one embodiment, the reverse transcription is performed in a buffer comprising LiCl.

[0022] In one embodiment, the present invention relates to a kit for performing an assay for generating cDNA molecules from an RNA template, the method comprising contacting the RNA template with a TSO comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence, or a hybrid DNA-RNA sequence, wherein the TSO is capable of annealing by base pairing with non-templated nucleotides that have been added to the 5' end of a target nucleic acid molecule in a non-templated addition process by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3' end modification and a 5' end modification, a reverse transcription (RT) primer, and a reverse transcriptase.

[0023] In one embodiment, the RT primer is a DNA primer, an RNA primer, or a primer comprising at least one modified oligonucleotide.

[0024] In one embodiment, the reverse transcriptase is MarathonRT or a variant thereof, Moloney murine leukemia virus reverse transcriptase (MMLV RT) or a variant thereof, avian myeloblastosis virus reverse transcriptase (AMV RT) or a variant thereof, HIV reverse transcriptase (HIV RT) or a variant thereof, Bombyx mori R2 RNA element reverse transcriptase (R2RT) or a variant thereof, and TGIRT TM or variations thereof.

[0025] In one embodiment, the kit comprises a buffer comprising PEG 8000. In one embodiment, the kit comprises a buffer comprising LiCl.

[0026] In one embodiment, the present invention relates to a template switching oligonucleotide (TSO) comprising:

[0027] (i) a DNA nucleotide sequence, an RNA nucleotide sequence, or a hybrid DNA-RNA sequence; and

[0028] (ii) at least one of a 3'-end modification and a 5'-end modification.

[0029] In one embodiment, the TSO comprises a 3' terminal modification. In one embodiment, the TSO comprises a 5' terminal modification. In one embodiment, the TSO comprises both a 3' terminal modification and a 5' terminal modification.

[0030] In one embodiment, the 3' end modification is a nucleotide sugar modification or a nucleobase modification.

[0031] In one embodiment, the 3' end modification is a modification that removes the 3' hydroxyl group or a modification that blocks the 3' hydroxyl group.

[0032] In one embodiment, the 3' end modification is 3'ddT, 3'ddU, 3' reversed dT, 3'C3 spacer, 3' amino, 3'rU oxidized by periodate, 3' phosphorylated, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' alkene, 3'(CH2)nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n≥1) or 3'(CH2CH2O)n (n≥1).

[0033] In one embodiment, the 5' end modification is a nucleotide sugar modification or a nucleobase modification.

[0034] In one embodiment, the 5' end modification comprises a trityl group, a trebbler, a dendrimer, biotin, a fluorescent dye, a ROX NHS ester, a (CH2)n (n≥1) long spacer, a palmitic acid phosphoramidite, a 3-cyanovinylcarbazole phosphoramidite, a cholesterol group, or a psoralen.

[0035] In one embodiment, the 3' end modification is a modification that removes the 3' hydroxyl group or a modification that blocks the 3' hydroxyl group.

[0036] In one embodiment, the 5' end comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive abasic sites. In one embodiment, the 5' end comprises at least 5 consecutive abasic sites. In one embodiment, the 5' end comprises at least 3 consecutive abasic sites.

[0037] In one embodiment, the 5' end comprises at least one non-natural nucleotide or nucleotide analogue.

[0038] In one embodiment, the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

[0039] In one embodiment, the present invention relates to a method for reducing multiplexing of a template switching oligonucleotide (TSO), the method comprising providing a reaction mixture comprising a TSO, a reverse transcription (RT) primer, and a reverse transcriptase,

[0040] TSO includes:

[0041] (i) 3' end modification; and / or

[0042] (ii) 5' end modification.

[0043] In one embodiment, the multiplexing is reduced, e.g., by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% based on a reference standard.

[0044] In one embodiment, the 3' end modification is 3'ddT, 3'ddU, 3' reversed dT, 3'C3 spacer, 3' amino, 3'rU oxidized by periodate, 3' phosphorylated, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' alkene, 3'(CH2)nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n≥1) or 3'(CH2CH2O)n (n≥1).

[0045] In one embodiment, the TSO comprises a 5'-terminal modification of a trityl group, a trebbler, a dendrimer, biotin, a fluorescent dye, a ROX NHS ester, a (CH2)n (n≥1) long spacer, a palmitic acid phosphoramidite, a 3-cyanovinylcarbazole phosphoramidite, a cholesteryl group, or psoralen.

[0046] In one embodiment, the 5' end of the TSO comprises at least 3 consecutive abasic sites.

[0047] In one embodiment, the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

[0048] In one embodiment, the TSO comprises SEQ ID NO:3.

[0049] In one embodiment, the RT primer comprises SEQ ID NO:2.

[0050] In one embodiment, the present invention relates to a method for reducing non-specific reverse transcription from a template switching oligonucleotide (TSO), the method comprising providing a reaction mixture comprising a TSO, a reverse transcription (RT) primer, and a reverse transcriptase,

[0051] TSO includes:

[0052] (i) 3' end modification; and / or

[0053] (ii) 5' end modification.

[0054] In one embodiment, the multiplexing is reduced, e.g., by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% based on a reference standard.

[0055] In one embodiment, the 3' end modification is 3'ddT, 3'ddU, 3' reversed dT, 3'C3 spacer, 3' amino, 3'rU oxidized by periodate, 3' phosphorylated, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' alkene, 3'(CH2)nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n≥1) or 3'(CH2CH2O)n (n≥1).

[0056] In one embodiment, the TSO comprises a 5'-terminal modification of a trityl group, a trebbler, a dendrimer, biotin, a fluorescent dye, a ROX NHS ester, a (CH2)n (n≥1) long spacer, a palmitic acid phosphoramidite, a 3-cyanovinylcarbazole phosphoramidite, a cholesteryl group, or psoralen.

[0057] In one embodiment, the 5' end of the TSO comprises at least 3 consecutive abasic sites.

[0058] In one embodiment, the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

[0059] In one embodiment, the TSO comprises SEQ ID NO:3.

[0060] In one embodiment, the RT primer comprises SEQ ID NO:2.

[0061] In one embodiment, the present invention relates to a method for increasing the yield of a target polynucleotide sequence in an RNA-seq library, the method comprising providing a reaction mixture comprising TSO, a reverse transcription (RT) primer, and a reverse transcriptase,

[0062] TSO includes:

[0063] (i) 3' end modification; and / or

[0064] (ii) 5' end modification.

[0065] In one embodiment, the multiplexing is reduced, e.g., by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% based on a reference standard.

[0066] In one embodiment, the 3' end modification is 3'ddT, 3'ddU, 3' reversed dT, 3'C3 spacer, 3' amino, 3'rU oxidized by periodate, 3' phosphorylated, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' alkene, 3'(CH2)nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n≥1) or 3'(CH2CH2O)n (n≥1).

[0067] In one embodiment, the TSO comprises a 5'-terminal modification of a trityl group, a trebbler, a dendrimer, biotin, a fluorescent dye, a ROX NHS ester, a (CH2)n (n≥1) long spacer, a palmitic acid phosphoramidite, a 3-cyanovinylcarbazole phosphoramidite, a cholesteryl group, or psoralen.

[0068] In one embodiment, the 5' end of the TSO comprises at least 3 consecutive abasic sites.

[0069] In one embodiment, the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

[0070] In one embodiment, the TSO comprises SEQ ID NO:3.

[0071] In one embodiment, the RT primer comprises SEQ ID NO:2.

[0072] In one embodiment, the present invention relates to a method for increasing the specificity of an RNA-seq library, the method comprising providing a reaction mixture comprising TSO, a reverse transcription (RT) primer, and a reverse transcriptase,

[0073] TSO includes:

[0074] (i) 3' end modification; and / or

[0075] (ii) 5' end modification.

[0076] In one embodiment, the multiplexing is reduced, e.g., by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% based on a reference standard.

[0077] In one embodiment, the 3' end modification is 3'ddT, 3'ddU, 3' reversed dT, 3'C3 spacer, 3' amino, 3'rU oxidized by periodate, 3' phosphorylated, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' alkene, 3'(CH2)nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n≥1) or 3'(CH2CH2O)n (n≥1).

[0078] In one embodiment, the TSO comprises a 5'-terminal modification of a trityl group, a trebbler, a dendrimer, biotin, a fluorescent dye, a ROX NHS ester, a (CH2)n (n≥1) long spacer, a palmitic acid phosphoramidite, a 3-cyanovinylcarbazole phosphoramidite, a cholesteryl group, or psoralen.

[0079] In one embodiment, the 5' end of the TSO comprises at least 3 consecutive abasic sites.

[0080] In one embodiment, the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

[0081] In one embodiment, the TSO comprises SEQ ID NO:3.

[0082] In one embodiment, the RT primer comprises SEQ ID NO:2. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] The following detailed description of the preferred embodiments of the present invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the accompanying drawings presently preferred embodiments. However, it should be understood that the invention is not limited to the precise arrangements and means of the embodiments shown in the accompanying drawings.

[0084] Figure 1 A schematic diagram of the design of template-switching oligonucleotides (TSOs) is provided. 5' and 3' modifications are intended to prevent unwanted reactions during RNA sequencing library preparation.

[0085] Figure 2 An overview of template switching reactions in which TSO concatemers are formed without TSO 5' modification is provided.

[0086] Figure 3 An overview of a template switching reaction with a TSO 5' modification in which TSO multiplexing is reduced is provided. The 5' modification (*) prevents the second TSO from annealing to the "AAA" overhang.

[0087] Figure 4 Various modifications that have been shown to be effective in reducing TSO polyadenylation are depicted. These include five consecutive abasic sites (5'-5 abasic), three consecutive abasic sites (5'-3 abasic), a trebler group (5'-trebler), and a trityl group (5'-trityl).

[0088] Figures 5A and 5B depict data for the quantification of TSO multiplexing products using TSO with a 5' modification. Figure 5A depicts electrophoretic analysis of template switching products using TSO without a 5' modification (lane 1) or with a 5' modification (lanes 2-5). The number of multiplexed TSOs per template switching product is indicated. Figure 5B depicts quantification of the efficiency of multiple template switching reactions (≥2, orange) using TSO without a 5' modification (lane 1) or with a 5' modification (lanes 2-5).

[0089] Figure 6 Schematic depicting the TSO 3' modification, which prevents TSO from acting as a primer. When preparing RNA-seq libraries using very low RNA input (e.g., total RNA from a single cell), cDNA products derived from TSO priming will dominate the RNA-seq library. Blocking 3' modifications to the 3'-OH of TSO (e.g., dideoxynucleotides) effectively prevents unwanted reactions.

[0090] 7A to 7C Depicted are the nucleotide sequence specificity of non-templated nucleotide addition (NTA) by different reverse transcriptases. Figure 7A The nucleotide sequence specificity of MarathonRT is depicted. Figure 7B The nucleotide sequence specificity of MMLV RT is depicted. Figure 7C The nucleotide sequence specificity of TGIRT is depicted. The sequence of the TSO can be selected based on standard NTA by reverse transcriptase. MarathonRT specifically adds a triadenosine overhang to the 3' end of the cDNA. It requires a TSO with three uridines (RNA) or three thymidines (DNA) at the 3' end for efficient template switching. Moloney murine leukemia virus (MMLV) RT adds a tricytidine overhang to the 3' end of the cDNA. It requires a TSO with three guanosines at the 3' end for efficient template switching. TGIRT TM Single nucleotide overhangs (a mixture of A, G, C, and T) are most efficiently added to the 3' end of the cDNA. TSO with any nucleotide at the 3' end can mediate template switching. DETAILED DESCRIPTION

[0091] The present invention provides a composition and method for controlling non-specific reverse transcription and template switching products while maintaining efficient full-length cDNA synthesis. The key components for reverse transcription and template switching reactions have been optimized to increase the yield of reverse transcription reactions. Specifically, a high-processive RT encoded by a class II intron of Eubacterium rectale is used instead of a retroviral RT to increase full-length cDNA yield and reduce deviations during reverse transcription and template switching; reaction buffer components are optimized to increase template switching efficiency for higher full-length cDNA yield; the nucleotide sequences of RT primers and template switching oligonucleotides are optimized, and chemical modifications are performed on the 3' end of the template switching oligonucleotide to reduce non-specific reverse transcription; the 5' end of the template switching oligonucleotide is chemically modified and non-standard nucleotides are included at the 5' end to prevent multiplexing during template switching.

[0092] In one aspect, the present invention provides a composition comprising an optimized template switching oligonucleotide (TSO) comprising at least one 3' end modification, at least one 5' end modification, or a combination of at least one 3' end modification and at least one 5' end modification.

[0093] In one embodiment, TSO comprises a 3' end modification. Exemplary 3' end modifications include, but are not limited to, 3'ddT, 3'ddU, 3' reversed dT, 3' C3 spacer, 3' amino, 3' rU oxidized by periodate, 3' phosphorylation, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' olefin, 3' (CH2) nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n ≥ 1) and 3' (CH2CH2O) n (n ≥ 1). In one embodiment, TSO comprises at least one chemical group that blocks the 3' hydroxyl group. In one embodiment, TSO comprises at least one modification that removes the 3' hydroxyl group.

[0094] In one embodiment, the TSO comprises at least one 5' end modification. In some embodiments, the TSO comprises a combination of 5' end modifications.

[0095] In one embodiment, the TSO comprises at least one non-standard nucleotide. In some embodiments, the at least one non-standard nucleotide is at the 5' end of the TSO. In one embodiment, the non-standard nucleotide is isodeoxycytidine.

[0096] In one embodiment, the TSO comprises at least one chemical group that blocks the 5' end. In some embodiments, the chemical group comprises a bulky adduct that prevents the reverse transcriptase and additional TSO molecules from binding to the single-stranded overhang on the template molecule. Exemplary chemical groups that can be added to the TSO to block the 5' end to prevent multiplexing include, but are not limited to, trityl, dendrimers (e.g., trebblers), biotin, fluorescent dyes, ROX NHS esters, (CH2)n (n≥1) long spacers (e.g., spacer C12), palmitic acid phosphoramidite, 3-cyanovinylcarbazole phosphoramidite, cholesterol groups, and psoralens (e.g., psoralen C2 phosphoramidite and psoralen C6 phosphoramidite).

[0097] In one embodiment, the TSO comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 consecutive abasic sites at the 5' end. In some embodiments, the abasic sites prevent binding to additional nucleic acid molecules and prevent binding by the reverse transcriptase, thereby preventing further extension of the sequence through repeated rounds of reverse transcription and template switching. Exemplary abasic sites include, but are not limited to, apurinic and apyrimidinic sites. In some embodiments, the TSO comprises 3 consecutive abasic sites at the 5' end.

[0098] In one embodiment, the TSO comprises a nucleotide sequence to minimize base pairing between any two TSO molecules and between the TSO and RT primers.

[0099] In one aspect, the present invention provides a composition comprising an optimized RT primer. In one embodiment, the RT primer comprises at least one 5' end modification. In some embodiments, the RT primer comprises a combination of 5' end modifications. In some embodiments, the 5' end modification comprises a bulky adduct that prevents additional nucleic acid molecules from binding to the single-stranded overhang on the template molecule.

[0100] In one embodiment, the RT primer comprises at least one non-standard nucleotide. In some embodiments, at least one non-standard nucleotide is at the 5' end of the RT primer. In one embodiment, the non-standard nucleotide is isodeoxycytidine.

[0101] In one embodiment, the RT primer comprises at least one chemical group that blocks the 5' end. In some embodiments, the chemical group comprises a bulky adduct that prevents additional nucleic acid molecules from binding to the single-stranded overhang on the template molecule. Exemplary chemical groups that can be added to the RT primer to block the 5' end to prevent multiplexing include, but are not limited to, 5' trebler and 5' trityl.

[0102] In one embodiment, the RT primer comprises at least 2,3,4,5,6,7,8,9,10 or more than 10 continuous abasic sites at the 5 ' end.In some embodiments, the abasic sites prevent being combined with extra nucleic acid molecules and prevent being combined by reverse transcriptase, so the abasic sites prevent further extending the sequence by repeating multiple rounds of reverse transcription.Exemplary abasic sites include but are not limited to apurinic and apyrimidinic sites.In some embodiments, the RT primer comprises 3 continuous abasic sites at the 5 ' end.

[0103] In one embodiment, the TSO and RT primers comprise nucleotide sequences designed to minimize base pairing between any two RT primer molecules, between any two TSO molecules, and between the RT primer and TSO. In some embodiments, the sequences of the TSO and RT primers comprise only cytosine and thymidine nucleotides, which prevent base pair formation between the TSO and RT primers. For example, in one embodiment, the nucleotide sequence of the optimized RT primer is 5'-CCTTCTCCTTCTCCTCCTTTCTCCTTTTTTTT TTTTTTTTTT-3' (SEQ ID NO: 2); the nucleotide sequence of the optimized template-switching oligonucleotide is 5'-CCCTCTCTCTCTCTTTCCTCTCTCTTTT-3' (SEQ ID NO: 3). However, the present invention is not limited to the exemplary RT primers and TSO.

[0104] In one aspect, the present invention provides a composition comprising an optimized RT reaction buffer.

[0105] In one aspect, the present invention provides an optimized reaction buffer that enhances the activity of a reverse transcriptase. In some embodiments, the optimized reaction buffer comprises one or more of the following: PEG8000 at a concentration of about 1% to about 20%, Tris at a concentration of about 10mM to about 100mM; LiCl at a concentration of about 20mM to about 500mM, MgCl2 at a concentration of about 0.5mM to about 5mM, and DTT at a concentration of about 1mM to about 10mM. In one embodiment, the pH of the optimized reaction buffer is about 7.5 to 8.5.

[0106] In some embodiments, the optimized reaction buffer further comprises a protein stabilizer. Exemplary protein stabilizers include, but are not limited to, osmotic stabilizers such as glycerol, erythritol, arabitol, sorbitol, mannitol, xylitol, mannisdomannitol, glucosylglycerol, glucose, fructose, sucrose, trehalose, isofluorosid, dextran, fructan, and polyethylene glycol; amino acids and their derivatives such as glycine, alanine, proline, taurine, betaine, octopine, glutamic acid, sarcosine, γ-aminobutyric acid, trimethylamine, N-oxide (TMAO); ion stabilizers such as citrate, sulfate, acetate, phosphate, and quaternary amine; and proteins such as bovine serum albumin (BSA).

[0107] In one embodiment, the invention provides a method of performing RT, the method comprising contacting an RNA sample with optimized RT primers and optimized TSO in an optimized RT reaction buffer.

[0108] In some embodiments, the reverse transcriptase is a class II intron RT. In some embodiments, the reverse transcriptase is a retroviral RT. Exemplary reverse transcriptases that can be used in the assays of the present invention include, but are not limited to, MarathonRT, MMLV RT, AMV RT, HIV RT, R2RT, and TGIRT. TM , or variants thereof. In some embodiments, the reverse transcription reaction is effective to create a full-length DNA product. In another embodiment, the reverse transcription reaction requires less of at least one of a TSO oligonucleotide, an RT primer, an RNA template, a reverse transcriptase, or a combination thereof relative to the amount of reverse transcriptase required for a reverse transcription reaction using another TSO or RT primer. In one embodiment, the method comprises amplifying RNA in a single reaction.

[0109] definition

[0110] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to be limiting.

[0111] As used herein, each of the following terms has the meaning associated with it in this section.

[0112] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical object of the article. For example, "an element" refers to one element or more than one element.

[0113] As used herein, "about" when referring to a measurable value, such as an amount and a time duration, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are suitable for performing the disclosed methods.

[0114] As used herein, "autologous" refers to biological material derived from the same individual into which the material will subsequently be reintroduced.

[0115] As used herein, "allogeneic" refers to biological material derived from a genetically different individual of the same species as the individual into which the material is being introduced.

[0116] The terms "cell" and "cell population" are used interchangeably and generally refer to a plurality of cells, i.e., more than one cell. A population can be a pure population comprising one cell type. Alternatively, a population can comprise more than one cell type. In the present invention, there is no limit to the number of cell types that a cell population can comprise.

[0117] The term "complementary" as used herein with respect to a polynucleotide is defined as a polynucleotide having a sequence that comprises the reverse complement of another polynucleotide sequence, e.g., a polynucleotide having a sequence capable of base pairing (e.g., Watson-Crick base pairing) with another polynucleotide to form an antiparallel double-stranded polynucleotide duplex.

[0118] As used herein, the term "end" with respect to a polynucleotide refers to any sequence located within the first 20 nucleotides (e.g., the 5' end) or the last 20 nucleotides (e.g., the 3' end) of a polynucleotide. In some embodiments, the template switching oligonucleotide (TSO) comprises a modification within the 5' end of the TSO or within the 3' end of the TSO.

[0119] As used herein, "terminus" refers to the first nucleotide (e.g., the 5' terminus) or the last nucleotide (e.g., the 3' terminus) of a polynucleotide sequence. In some embodiments, the template switching oligonucleotide (TSO) comprises a modification at the 5' terminus of the TSO and / or at the 3' terminus of the TSO.

[0120] "Encoding" refers to the inherent properties of a specific nucleotide sequence in a polynucleotide (such as a gene, DNA, or RNA) that serves as a template for synthesizing other polymers and macromolecules with a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, as well as the resulting biological properties, in a biological process. Thus, if transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, the gene encodes the protein. Both the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is typically provided in a sequence listing) and the non-coding strand (used as a template for transcription of a gene or cDNA) may be referred to as encoding a protein or other product of the gene or cDNA.

[0121] "Expression vector" refers to a vector containing a recombinant polynucleotide containing an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or an in vitro expression system. Expression vectors include all vectors known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides.

[0122] "Homologous" refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example, if a position in each of the two DNA molecules is occupied by adenine, then the two molecules are homologous at that position. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared x 100. For example, if 6 out of 10 positions in the two sequences are matched or homologous, then the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Typically, comparisons are made when the two sequences are aligned to provide maximum homology.

[0123] "Isolated" means altered or removed from its native state. For example, a nucleic acid or peptide naturally present in a living organism is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials in its native state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form or in a non-native environment, such as, for example, a host cell.

[0124] In the context of the present invention, the following abbreviations for common nucleic acid bases are used: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0125] Unless otherwise indicated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. The phrase "nucleotide sequence encoding a protein or RNA" may also include introns, to the extent that a nucleotide sequence encoding a protein may contain introns in certain versions. The term "polynucleotide" as used herein is defined as a chain of nucleotides. In addition, a nucleic acid is a polymer of nucleotides. Therefore, nucleic acids and polynucleotides as used herein are interchangeable. Those skilled in the art have the following general knowledge: nucleic acids are polynucleotides, which can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. Polynucleotides as used herein include, but are not limited to, all nucleic acid sequences obtained by any available means in the art, including, but not limited to, recombinant means, i.e., cloning nucleic acid sequences from a recombinant library or a cell genome, using conventional cloning techniques and PCR, etc., as well as by synthetic means. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably.

[0126] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit on the maximum number of amino acids that can constitute a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids interconnected by peptide bonds. As used herein, the term refers to both short chains, such as are also commonly referred to in the art as peptides, oligopeptides, and oligomers, and long chains, such as are commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include naturally occurring peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0127] The term "promoter" as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.

[0128] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product that is operably linked to the promoter / regulatory sequence. In some cases, the sequence may be a core promoter sequence, while in other cases, the sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. For example, the promoter / regulatory sequence may be a promoter / regulatory sequence that conditionally expresses a gene product.

[0129] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0130] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0131] The term "template" as used herein with respect to a polynucleotide refers to a single-stranded polynucleotide substrate for a nucleic acid polymerase such as a reverse transcriptase. For example, a nucleic acid polymerase such as a reverse transcriptase can synthesize a polynucleotide strand complementary to the template strand. In some embodiments, a single-stranded RNA polynucleotide can be a template for a reverse transcriptase.

[0132] The term "product" as used herein with respect to a polynucleotide refers to a polynucleotide chain synthesized by a nucleotide polymerase. In some embodiments, the nucleotide polymerase is a reverse transcriptase. In some embodiments, the product polynucleotide is a deoxyribonucleic acid (DNA) polynucleotide synthesized by a reverse transcriptase using a ribonucleic acid (RNA) polynucleotide as a template.

[0133] The term "reverse transcription" as used herein with respect to a subject molecule (eg, an RNA polynucleotide) refers to the synthesis of a deoxyribonucleic acid (DNA), such as a cDNA polynucleotide, using a ribonucleic acid (RNA) polynucleotide as a template.

[0134] As used herein, the term "reverse transcriptase" refers to a nucleic acid polymerase that is capable of synthesizing a deoxyribonucleic acid (DNA) polynucleotide from a template ribonucleic acid (RNA) polynucleotide. For example, a reverse transcriptase can synthesize a single-stranded complementary DNA (cDNA) polynucleotide product from a messenger RNA (mRNA) expressed in a cell or subject. In some embodiments, the reverse transcriptase can include MarathonRT reverse transcriptase, Moloney murine leukemia virus reverse transcriptase, avian myeloblastosis virus reverse transcriptase, Bombyx mori R2 RNA element reverse transcriptase, or TGIRT reverse transcriptase. TM Reverse transcriptase.

[0135] As used herein, the term "non-templated nucleotide addition" refers to the addition of nucleotides to the 3' end of a product polynucleotide synthesized by a reverse transcriptase, upon reaching the 5' end of the template polynucleotide, e.g., the addition of nucleotides to the product polynucleotide that are not contained in the template polynucleotide. For example, non-templated nucleotide addition can produce a product polynucleotide that comprises a 3' end that extends beyond the 5' end of the template polynucleotide and is not complementary to the template polynucleotide. Typically, non-templated nucleotide addition results in an overhang of 1-3 nucleotides, e.g., a 1, 2, or 3 nucleotide overhang, at the 3' end of the product polynucleotide relative to the template polynucleotide.

[0136] As used herein, the term "template switching" refers to the process by which a reverse transcriptase switches from a first template polynucleotide to a second template polynucleotide while synthesizing a continuous product polynucleotide. Typically, template switching comprises: (i) non-templated nucleotide addition, i.e., adding nucleotides to the 3' end of a product polynucleotide synthesized by a reverse transcriptase upon reaching the 5' end of the template polynucleotide; (ii) base pairing between a template switching oligonucleotide (TSO) and the nucleotide overhang generated by the non-templated addition; and (iii) continuing to synthesize a product polynucleotide by a reverse transcriptase using the TSO as a template polynucleotide. For example.

[0137] As used herein, the term "concatemerization" refers to the tandem connection of multiple identical polynucleotide sequences, such as the connection of multiple template switching oligonucleotide (TSO) sequences. In some embodiments, the concatenation of multiple TSOs can be the result of repeated cycles of non-templated nucleotide addition by reverse transcriptase followed by template switching by reverse transcriptase.

[0138] A "vector" is a composition of matter that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Various vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids, and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted to include non-plasmid and non-viral compounds that promote the transfer of nucleic acids into cells, such as, for example, polylysine compounds and liposomes. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, and retroviral vectors.

[0139] Range: Throughout this disclosure, various aspects of the invention may be presented in range format. It should be understood that descriptions in range format are for convenience and brevity only and should not be construed as inflexible limitations on the scope of the invention. Therefore, descriptions of ranges should be considered to have explicitly disclosed all possible subranges as well as individual numbers within that range. For example, a range description such as from 1 to 6 should be considered to have explicitly disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0140] describe

[0141] In some embodiments, the present invention relates to optimized compositions for performing reverse transcription and methods of use thereof. Compositions of the invention are described herein to improve the yield of reverse transcription assays, reduce multiplexing of template-switching oligonucleotides (TSOs), reduce nonspecific reverse transcription of TSO molecules, and provide sensitive and quantitative detection of RNA transcripts. Thus, the presently described TSOs, RT primers, and optimized reaction conditions provide an enhanced assay system that can be used in a variety of applications, including but not limited to RNA sequencing, RNA amplification, next-generation sequencing, nanopore sequencing, RT-PCR, quantitative PCR, cDNA synthesis, cDNA library synthesis, splice site characterization, viral RNA sequencing, single-cell sequencing, and RNA structure probing.

[0142] In one aspect, the present invention provides a method for reverse transcription. For example, in one embodiment, the method comprises contacting an RNA molecule with one or more TSOs described herein and one or more RT primers described herein, and further contacting the RNA molecule with a high processivity reverse transcriptase.

[0143] Template-switching oligonucleotides

[0144] In one aspect, the invention provides a template switching oligonucleotide (TSO) that has been modified to reduce multiplexing and nonspecific reverse transcription.

[0145] The isolated TSO can be a DNA, RNA, or modified oligonucleotide sequence. The isolated TSO can be a hybrid DNA / RNA oligonucleotide or a modified sequence comprising 8 to 30 DNA nucleotides at the 5' end linked to 3-8 RNA nucleotides at the 3' end, wherein the addition of the RNA nucleotides promotes the binding of the TSO to the DNA molecule. In some embodiments, the TSO is a hybrid DNA / RNA oligonucleotide. For example, the TSO may comprise DNA nucleotides at the 5' end and RNA nucleotides at the 3' end. In some embodiments, the 5' end of the TSO is 8-15 DNA nucleotides in length and the 3' end of the TSO is 3-8 RNA nucleotides in length. In some embodiments, the 5' end of the TSO is 15-20 DNA nucleotides in length and the 3' end of the TSO is 3-8 RNA nucleotides in length. In some embodiments, the 5' end of the TSO is 20-30 DNA nucleotides in length and the 3' end of the TSO is 3-8 RNA nucleotides in length. In some embodiments, the 5' end of the TSO is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 DNA nucleotides in length and the 3' end of the TSO is 3, 4, 5, 6, 7, or 8 RNA nucleotides in length. In some embodiments, the TSO is a DNA oligonucleotide. In some embodiments, the TSO is 8-38 DNA nucleotides in length. In some embodiments, the TSO is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38 DNA nucleotides in length.

[0146] In some embodiments, TSO comprises at least one modified nucleotide or non-natural nucleotide. In one embodiment, the 5' end comprises at least one non-natural nucleotide or nucleotide analog, for example, the 5' end of TSO comprises a non-natural nucleotide or nucleotide analog. In some embodiments, TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end. In some embodiments, the 5' end of TSO comprises isodeoxycytosine (iso-dC). In some embodiments, the 5' end of TSO comprises isodeoxyguanosine (iso-dG). In some embodiments, the 5' end of TSO comprises isodeoxycytosine (iso-dC). In some embodiments, the 5' end of TSO comprises isodeoxyguanosine (iso-dG). In some embodiments, the 5' end of TSO comprises isodeoxycytosine (iso-dC). In some embodiments, the 5' end of TSO comprises isodeoxyguanosine (iso-dG). In some embodiments, the 5' end of TSO comprises both isodeoxycytosine (iso-dC) and isodeoxyguanosine (iso-dG).

[0147] In some embodiments, the TSO comprises at least one 3' end modification, for example, the 3' end of the TSO comprises a chemical modification. Exemplary 3' end modifications include, but are not limited to, 3' ddT, 3' ddU, 3' reversed dT, 3' C3 spacer, 3' amino, 3' rU oxidized by periodate, 3' phosphorylated, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' olefin, 3' (CH2) nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n ≥ 1) and 3' (CH2CH2O) n (n ≥ 1).

[0148] In an embodiment, the 3' end of TSO comprises dideoxythymidine (ddT). In an embodiment, the 3' end of TSO comprises dideoxyuridine (ddU). In an embodiment, the 3' end of TSO comprises reversed deoxythymidine (dT). In an embodiment, the 3' end of TSO comprises a C3 spacer. In an embodiment, the 3' end of TSO comprises an amino group. In an embodiment, the 3' end of TSO comprises uridine (rU) oxidized by periodate. In an embodiment, the 3' end of TSO is phosphorylated. In an embodiment, the 3' end of TSO comprises a fluoro group. In an embodiment, the 3' end of TSO comprises an aldehyde. In an embodiment, the 3' end of TSO comprises a carboxylic acid or a carboxylate. In an embodiment, the 3' end of TSO comprises a thiol group. In an embodiment, the 3' end of TSO comprises an O-methyl group. In an embodiment, the 3' end of TSO comprises an azide group. In an embodiment, the 3' end of TSO comprises an alkyne. In an embodiment, the 3' end of TSO comprises an alkene. In an embodiment, the 3' end of TSO comprises (CH2)nX (X=H, OCH3, CH3, SH, NH2, OH, etc.), wherein n≥1. In an embodiment, the 3' end of TSO comprises (CH2CH2O)n, wherein n≥1.

[0149] In some embodiments, the TSO of the present invention comprises a 5' end modification, for example, the 5' end of the TSO comprises a chemical modification. In one embodiment, the TSO comprises at least one chemical group that blocks the 5' end. Exemplary chemical groups that can be added to the TSO to block the 5' end to prevent multiplexing include, but are not limited to, a 5' AP site (apurinic / apyrimidinic site), a trityl group, a dendrimer (e.g., a trebbler), biotin, a fluorescent dye, a ROX NHS ester, a (CH2)n (n≥1) long spacer (e.g., spacer C12), a palmitic acid phosphoramidite, a 3-cyanovinylcarbazole phosphoramidite, a cholesterol group, and psoralens (e.g., psoralen C2 phosphoramidite and psoralen C6 phosphoramidite).

[0150] In some embodiments, the 5' terminus of TSO comprises a trityl group. In some embodiments, the 5' terminus of TSO comprises a dendrimer. In some embodiments, the 5' terminus of TSO comprises a trebbler. In some embodiments, the 5' terminus of TSO comprises biotin. In some embodiments, the 5' terminus of TSO comprises a fluorescent dye. In some embodiments, the 5' terminus of TSO comprises a ROX NHS ester. In some embodiments, the 5' terminus of TSO comprises a (CH2)n long spacer, where n ≥ 1. In some embodiments, the 5' terminus of TSO comprises a C12 spacer. In some embodiments, the 5' terminus of TSO comprises a palmitic acid phosphoramidite. In some embodiments, the 5' terminus of TSO comprises a 3-cyanovinylcarbazole phosphoramidite. In some embodiments, the 5' terminus of TSO comprises a cholesterol group. In some embodiments, the 5' terminus of TSO comprises psoralen. In some embodiments, the 5' terminus of TSO comprises psoralen C2 phosphoramidite. In some embodiments, the 5' terminus of TSO comprises psoralen C6 phosphoramidite. In some embodiments, the 5' end of TSO comprises an abasic site. In some embodiments, the 5' end of TSO comprises an apurinic site. In some embodiments, the 5' end of TSO comprises an apyrimidinic site. In some embodiments, the 5' end of TSO comprises 1-5 abasic sites. In some embodiments, the 5' end of TSO comprises one abasic site. In some embodiments, the 5' end of TSO comprises two abasic sites. In some embodiments, the 5' end of TSO comprises three abasic sites. In some embodiments, the 5' end of TSO comprises four abasic sites. In some embodiments, the 5' end of TSO comprises five abasic sites. In some embodiments, the 5' end of TSO comprises 1-5 apurinic sites. In some embodiments, the 5' end of TSO comprises one apurinic site. In some embodiments, the 5' end of TSO comprises two apurinic sites. In some embodiments, the 5' end of TSO comprises three apurinic sites. In some embodiments, the 5' end of TSO comprises four apurinic sites. In some embodiments, the 5' end of TSO comprises five apurinic sites. In some embodiments, the 5' end of TSO comprises 1-5 apyrimidinic sites. In some embodiments, the 5' end of TSO comprises one apyrimidinic site. In some embodiments, the 5' end of TSO comprises two apyrimidinic sites. In some embodiments, the 5' end of TSO comprises three apyrimidinic sites. In some embodiments, the 5' end of TSO comprises four apyrimidinic sites. In some embodiments, the 5' end of TSO comprises five apyrimidinic sites. In some embodiments, TSO has the nucleotide sequence of SEQ ID NO: 3. In some embodiments, TSO having SEQ ID NO: 3 comprises a chemical modification.In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a trityl group. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a dendrimer. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a trebbler. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises biotin. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a fluorescent dye. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a ROX NHS ester. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a (CH2)n long spacer, where n ≥ 1. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a spacer C12. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a palmitic acid phosphoramidite. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a 3-cyanovinylcarbazole phosphoramidite. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a cholesterol group. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a psoralen. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a psoralen C2 phosphoramidite. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises a psoralen C6 phosphoramidite. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises an abasic site. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises an apurinic site. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises an apyrimidinic site. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises 1-5 abasic sites. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises one abasic site. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises two abasic sites. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises three abasic sites. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises four abasic sites. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises five abasic sites. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises 1-5 apurinic sites.In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises one apurinic site. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises two apurinic sites. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises three apurinic sites. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises four apurinic sites. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises five apurinic sites. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises 1-5 apyrimidinic sites. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises one apyrimidinic site. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises two apyrimidinic sites. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises three apyrimidinic sites. In some embodiments, the 5′ end of the TSO having SEQ ID NO: 3 comprises four apyrimidinic sites. In some embodiments, the 5' end of the TSO having SEQ ID NO: 3 comprises five apyrimidinic sites.

[0151] Isolated TSO oligonucleotides are obtained using any of a variety of recombinant methods known in the art, such as, for example, TSO can be produced synthetically.

[0152] The nucleic acid molecules of the present invention can be modified to improve binding to the cDNA template, reduce binding to the RT primer and TSO itself, prevent multiplexing, or any combination thereof. Modifications can be added to enhance stability, functionality and / or specificity. In some embodiments, the 3' residue can be modified with a group that blocks the 3' hydroxyl group. Exemplary modifications that block the 3' hydroxyl group include, but are not limited to, 3' dideoxythymidine (ddT), 3' dideoxyuridine (ddU), 3' reverse deoxythymidine (dT), 3' C3 spacer, 3' amino, 3' uridine oxidized by periodate (rU), 3' phosphorylation, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' olefin, 3' (CH2) nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n ≥ 1) and 3' (CH2CH2O) n (n ≥ 1). In an embodiment, the nucleic acid molecule comprises dideoxythymidine (ddT). In embodiments, the nucleic acid molecule comprises dideoxyuridine (ddU). In embodiments, the nucleic acid molecule comprises reverse deoxythymidine (dT). In embodiments, the nucleic acid molecule comprises a C3 spacer. In embodiments, the nucleic acid molecule comprises an amino group. In embodiments, the nucleic acid molecule comprises uridine (rU) oxidized by periodate. In embodiments, the nucleic acid molecule is phosphorylated. In embodiments, the nucleic acid molecule comprises a fluoro group. In embodiments, the nucleic acid molecule comprises an aldehyde. In embodiments, the nucleic acid molecule comprises a carboxylic acid or a carboxylate. In embodiments, the nucleic acid molecule comprises a thiol group. In embodiments, the nucleic acid molecule comprises an O-methyl group. In embodiments, the nucleic acid molecule comprises an azide group. In embodiments, the nucleic acid molecule comprises an alkyne. In embodiments, the nucleic acid molecule comprises an olefin. In embodiments, the nucleic acid molecule comprises (CH2)nX (X=H, OCH3, CH3, SH, NH2, OH, etc.), wherein n≥1. In embodiments, the nucleic acid molecule comprises (CH2CHO)n, wherein n≥1.

[0153] In one embodiment of the present invention, the nucleic acid molecule may include at least one modified nucleotide analog. For example, each end may be stabilized by incorporating a modified nucleotide analog. In some embodiments, the 5'-nucleotide may be substituted or modified with a chemical group to prevent multiplexing. Exemplary substitutions of the 5' group for preventing multiplexing include, but are not limited to, substitutions at the 5' end with at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG. In some embodiments, the 5' end of the nucleic acid molecule includes isodeoxycytosine (iso-dC). In some embodiments, the 5' end of the nucleic acid molecule includes isodeoxyguanosine (iso-dG). In some embodiments, the 5' end of the nucleic acid molecule includes isodeoxycytosine (iso-dC). In some embodiments, the 5' end of the nucleic acid molecule includes isodeoxyguanosine (iso-dG). In some embodiments, the 5' end of the nucleic acid molecule includes both isodeoxycytosine (iso-dC) and isodeoxyguanosine (iso-dG).

[0154] The TSO or RT primers of the present invention may further include one or more additional nucleotide analogs. Non-limiting examples of nucleotide analogs include sugar and / or backbone modified ribonucleotides (i.e., including modifications to the phosphate-sugar backbone). For example, the phosphodiester bond of natural RNA may be modified to include at least one nitrogen or sulfur heteroatom. In exemplary backbone modified ribonucleotides, the phosphate group connected to the adjacent ribonucleotide is replaced by a modifying group (e.g., a phosphorothioate group). In embodiments, TSO includes backbone modifications. In embodiments, TSO includes phosphorothioate modifications. In embodiments, RT primers include backbone modifications. In embodiments, RT primers include phosphorothioate modifications. In some embodiments, sugar-modified ribonucleotides, the 2'OH- group is replaced by a group selected from H, OR, R, halogen, SH, SR, NH2, NHR, NR2, or ON, wherein R is a C1-C6 alkyl, alkenyl, or alkynyl group, and the halogen is F, Cl, Br, or I. In embodiments, TSO includes a 2' sugar modification. In embodiments, TSO includes a 2'H. In an embodiment, TSO comprises 2'O-C1-C6 alkyl. In an embodiment, TSO comprises 2'O-alkenyl. In an embodiment, TSO comprises 2'O-alkynyl. In an embodiment, TSO comprises 2'C1-C6 alkyl. In an embodiment, TSO comprises 2'alkenyl. In an embodiment, TSO comprises 2'alkynyl. In an embodiment, TSO comprises 2'halogen. In an embodiment, TSO comprises 2'F. In an embodiment, TSO comprises 2'Cl. In an embodiment, TSO comprises 2'Br. In an embodiment, TSO comprises 2'I. In an embodiment, TSO comprises 2'SH. In an embodiment, TSO comprises 2'S-C1-C6 alkyl. In an embodiment, TSO comprises 2'S-alkenyl. In an embodiment, TSO comprises 2'S-alkynyl. In an embodiment, TSO comprises 2'NH2. In an embodiment, TSO comprises 2'NH-C1-C6 alkyl. In an embodiment, TSO comprises 2'NH-alkenyl. In an embodiment, TSO comprises 2'NH-alkynyl. In an embodiment, TSO comprises 2'N(C1-C6 alkyl)2. In an embodiment, TSO comprises 2'N(alkenyl)2. In an embodiment, TSO comprises 2'N(alkynyl)2. In an embodiment, TSO comprises 2'ON. In an embodiment, the RT primer comprises a 2' sugar modification. In an embodiment, the RT primer comprises 2'H. In an embodiment, the RT primer comprises 2'O-C1-C6 alkyl. In an embodiment, the RT primer comprises 2'O-alkenyl. In an embodiment, the RT primer comprises 2'O-alkynyl. In an embodiment, the RT primer comprises 2'C1-C6 alkyl. In an embodiment, the RT primer comprises 2'alkenyl. In an embodiment, the RT primer comprises 2'alkynyl. In an embodiment, the RT primer comprises 2'halogen.In embodiments, the RT primer comprises 2'F. In embodiments, the RT primer comprises 2'Cl. In embodiments, the RT primer comprises 2'Br. In embodiments, the RT primer comprises 2'I. In embodiments, the RT primer comprises 2'SH. In embodiments, the RT primer comprises 2'S-C1-C6 alkyl. In embodiments, the RT primer comprises 2'S-alkenyl. In embodiments, the RT primer comprises 2'S-alkynyl. In embodiments, the RT primer comprises 2'NH2. In embodiments, the RT primer comprises 2'NH-C1-C6 alkyl. In embodiments, the RT primer comprises 2'NH-alkenyl. In embodiments, the RT primer comprises 2'NH-alkynyl. In embodiments, the RT primer comprises 2'N(C1-C6 alkyl)2. In embodiments, the RT primer comprises 2'N(alkenyl)2. In embodiments, the RT primer comprises 2'N(alkynyl)2. In embodiments, the RT primer comprises 2'ON.

[0155] Other examples of modifications are nucleobase-modified ribonucleotides, i.e., ribonucleotides containing at least one non-naturally occurring nucleobase instead of a naturally occurring nucleobase. The bases can be modified to block the activity of adenosine deaminase. Exemplary modified nucleobases include, but are not limited to, uridine and / or cytidine modified at position 5, such as 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at position 8, such as 8-bromoguanosine; deazanucleotides, such as 7-deazaadenosine; O- and N-alkylated nucleotides, such as N6-methyladenosine are suitable. The above modifications can be combined.

[0156] In some cases, the nucleic acid molecule comprises at least one of the following chemical modifications: 2'-H, 2'-O-methyl or 2'-OH modifications of one or more nucleotides. In certain embodiments, the nucleic acid molecules of the present invention may have enhanced nuclease resistance. In order to increase nuclease resistance, the nucleic acid molecule may include, for example, a 2'-modified ribose unit and / or a thiophosphate bond. For example, the 2' hydroxyl (OH) may be modified or replaced with many different "oxygen" or "deoxy" substituents. In order to increase nuclease resistance, the nucleic acid molecule of the present invention may include 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, 2'-O-aminopropyl, 2'-amino and / or thiophosphate bonds. Comprising locked nucleic acid (LNA), ethylene nucleic acid (ENA), such as 2'-4'-ethylene bridged nucleic acid, and certain core base modifications, such as 2-amino-A, 2-thio (e.g., 2-thio-U), G-clamp (G-clamp) modifications, can also increase binding affinity to the target.

[0157] In one embodiment, the nucleic acid molecule includes 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA). In one embodiment, the nucleic acid molecule includes at least one 2'-O-methyl modified nucleotide, and in some embodiments, all nucleotides of the nucleic acid molecule include a 2'-O-methyl modification.

[0158] Nucleic acid agents discussed herein include other unmodified RNA and DNA and modified RNA and DNA.Unmodified RNA refers to molecules in which the components of nucleic acid (i.e., sugar, base and phosphate moieties) are identical or substantially identical to the components present in nature. Rare or uncommon but naturally occurring RNA has been referred to as modified RNA in the art, see, for example, Limbach et al. (Nucleic Acids Res., 1994, 22: 2183-2196). Such rare or uncommon RNA, commonly referred to as modified RNA, is typically the result of post-transcriptional modification and belongs to the term unmodified RNA as used herein. Modified RNA as used herein refers to molecules in which one or more components of nucleic acid (i.e., sugar, base and phosphate moieties) are different from those components present in nature. Although they are referred to as "modified RNA", due to modification, they certainly include molecules that are not RNA in the strict sense. Nucleoside surrogates are molecules in which the ribose phosphate backbone is replaced with a non-ribose phosphate construct that allows the bases to be presented in the correct spatial relationship such that hybridization is substantially similar to that seen with the ribose phosphate backbone, such as a non-charged mimetic of the ribose phosphate backbone.

[0159] Modifications of the nucleic acids of the invention may occur at one or more of the phosphate group, sugar group, backbone, N-terminus, C-terminus, or nucleobases.

[0160] Reverse transcription (RT) primers

[0161] In one aspect, the present invention provides an RT primer that has been optimized to minimize duplex formation between TSO and the RT primer and between two RT primer molecules while retaining the ability to specifically hybridize to an RNA template molecule.

[0162] In some embodiments, the RT primers of the present invention contain the same 5' modification as TSO to prevent multiplexing of the RT primer when the RT primer is used as a template switching oligonucleotide.

[0163] Reverse transcriptase

[0164] In one embodiment, the present invention provides compositions and methods for performing reverse transcription. Reverse transcription generally refers to the process of generating a DNA molecule from an RNA template molecule and is performed by a reverse transcriptase (RT). In some embodiments, the reverse transcriptase is a class II intron RT. In some embodiments, the reverse transcriptase is a retroviral RT. Exemplary reverse transcriptases that can be used for assays of the present invention include, but are not limited to, MarathonRT, MMLV RT, AMV RT, HIV RT, R2RT, and TGIRT. TM .

[0165] In one embodiment, the reverse transcriptase is derived from MarathonRT. For example, in certain embodiments, the reverse transcriptase comprises MarathonRT or a variant thereof. In one embodiment, MarathonRT is modified relative to unmodified MarathonRT. For example, in certain embodiments, the variant comprises one or more point mutations, insertion mutations, or deletion mutations relative to wild-type MarathonRT. In certain embodiments, the variant comprises a fusion protein comprising MarathonRT, a MarathonRT mutant, or a MarathonRT domain.

[0166] In one embodiment, the composition comprises wild-type MarathonRT. The amino acid sequence of wild-type MarathonRT is shown below and represented as SEQ ID NO: 1:

[0167] MDTSNLMEQILSSDNLNRAYLQVVRNKGAEGVDGMKYTELKEHLAKNGETIKGQLRTRKYKPQPARRVEIPKPDGGVRNLGVPTVTDRFIQQAIAQVLTPIYEEQFHD HSYGFRPNRCAQQAILTALNIMNDGNDWIVDIDLEKFFDTVNHDKLMTLIGRTIKDGDVISIVRKYLVSGIMIDDEYEDSIVGTPQGGNLSPLLANIMLNELDKEMEKR GLNFVRYADDCIIMVGSEMSANRVMRNISRFIEEKLGLKVNMTKSKVDRPSGLKYLGFGFYFDPRAHQFKAKPHAKSVAKFKKRMKELTCRSWGVSNSYKVEKLNQLIR GWINYFKIGSMKTLCKELDSRIRYRLRMCIWKQWKTPQNQEKNLVKLGIDRNTARRVAYTGKRIAYVCNKGAVNVAISNKRLASFGLISMLDYYIEKCVTC (Er Maturase).

[0168] Full-length MarathonRT includes a "secondary" RNA binding site and a DNA binding domain that can affect the stability, specificity and efficiency of reverse transcription of the RNA template. In one embodiment, the reverse transcriptase comprises a MarathonRT variant in which one or more secondary RNA binding sites on the protein surface are mutated to reduce the non-specific binding of the reverse transcriptase protein to the RNA template, thereby promoting binding at the polymerase cleft and promoting enzyme conversion. In one such embodiment, the variant of MarathonRT includes at least one point mutation selected from the group of R58X, K59X, K61X, K163X, K216X, R217X, K338X, K342X and R353X, wherein X represents any amino acid. In another such embodiment, the variant of MarathonRT includes at least one point mutation selected from the group of R58A, K59A, K61A, K163A, K216A, R217A, K338A, K342A and R353A. Exemplary variants of MarathonRT that can be used in the reverse transcription assays of the present invention include, but are not limited to, those described in detail in International Patent Publication No. WO2019005955A1, which is incorporated herein by reference in its entirety.

[0169] Compared with other reverse transcriptases, MarathonRT can be reverse transcribed at lower temperatures, and the engineering of more thermostable MarathonRT enables RNA templates to be amplified in a single reaction (i.e., without the need to use DNA → DNA amplification reaction). Analysis of thermophilic protein structure and function shows that they tend to have a large number of side chain hydrogen bonds and salt bridges in the rigid part of the tertiary structure. Therefore, in one embodiment, the reverse transcriptase of the present invention includes MarathonRT variants (Zhao C et al., 2016, Nature structural & molecular biology, 23 (6): 558-65) having Lys-Glu pairs at the position of the proximal end in 3-D space according to the structure of the enzyme. In one such embodiment, the variant includes at least one point mutation selected from the group consisting of L11E (can form a salt bridge with R56), L21E (can form a salt bridge with K41) and S13E (can form a salt bridge with K52).

[0170] In one embodiment, the reverse transcriptase of the present invention comprises a MarathonRT variant engineered to include a proofreading (e.g., 3'-5' exonuclease) domain to enhance fidelity. In one such embodiment, the proofreading domain comprises an exonuclease domain. In another such embodiment, the proofreading domain is attached to the C-terminus of the MarathonRT variant. In another such embodiment, the proofreading domain is attached to the C-terminus of the MarathonRT variant by a linker molecule or sequence (see, e.g., Ellefson, JW et al., 2016, Science, 352(6293):1590-3).

[0171] Class II intron-encoded reverse transcriptases are generally conserved between species, but some may have additional beneficial properties compared to other reverse transcriptases. Therefore, in one embodiment, the reverse transcriptase of the present invention comprises a MarathonRT variant, wherein at least one fragment or domain of MarathonRT is replaced by a fragment or domain of a class II intron-encoded reverse transcriptase from a species other than Eubacterium rectum. For example, in one embodiment, the RT domains (finger-like and palm-like) of the MarathonRT reverse transcriptase are replaced by RT domains from a thermophilic class II intron-encoded reverse transcriptase to enhance thermal stability. In another embodiment, the α-ring of MarathonRT is replaced by a longer α-ring from another class II intron-encoded reverse transcriptase to enhance processivity. In one embodiment, one or more amino acids are substituted by hydrophobic amino acids or charged amino acids to improve thermal stability.

[0172] In one embodiment, the reverse transcriptase of the present invention comprises a MarathonRT variant in which one or more residues are replaced by one or more residues derived from a class II intron-encoded reverse transcriptase from an organism other than Eubacterium rectum. For example, in some embodiments, the MarathonRT variant may comprise one or more point mutations based on conserved residues in a thermophilic class II intron-encoded reverse transcriptase. In one embodiment, the variant comprises at least one mutation selected from the following group: A29X, V82X, E104X, I129X, I137X, T161X, I168X, I170X, V171X, and M337X, wherein X represents any amino acid. In one embodiment, the mutation is at least one selected from the following group: A29X, V82X, E104X, I129X, I137X, T161X, I168X, I170X, V171X, and M337X, wherein X represents any amino acid. In one embodiment, the variant comprises at least one mutation selected from the group consisting of A29S, V82I, E104P, I129Y, I137V, T161R, I168L, I170L, V171I, and M337T. In one embodiment, the variant comprises a triple point mutation of A29S / V82I / E104P. In some cases, these mutations further improve the thermostability of the enzyme.

[0173] In one embodiment, the reverse transcriptase of the invention comprises a MarathonRT variant comprising one or more mutations in the thumb domain relative to wild-type MarathonRT.

[0174] In one embodiment, the variant comprises at least one point mutation selected from the group consisting of K338X, K342X, and R353X, wherein X represents any amino acid. In another such embodiment, the variant comprises at least one point mutation selected from the group consisting of K338A, K342A, and R353A.

[0175] In one such embodiment, one or more mutations are incorporated on the surface of the thumb domain to optimize its ability to latch onto a template. In one such embodiment, the variant comprises at least one point mutation selected from the group consisting of S315X, E319X, and Q323X, wherein X represents any amino acid. In another such embodiment, the variant comprises at least one point mutation selected from the group consisting of S315K, E319K, and Q323K.

[0176] In one embodiment, the reverse transcriptase comprises one or more mutations in the catalytic active site to reduce the fidelity of the enzyme, which will enhance its value for RNA structure mapping because structure-specific damage used to probe RNA structure is marked by misincorporation events. Similarly, mutations that increase the error rate of the enzyme can be used in certain RNA and transcriptome mapping experiments. Thus, in some embodiments, the polypeptide comprises at least one mutation selected from the following group: A225X, R114X, Y224X, I179X, M180X, I181X, E143X, K65X, L201X, where X represents any amino acid. Specifically, mutations at A225 (such as A225V, A225S, A225M or A225V), mutations at R114 (such as R114K, R114A), mutations at Y224 (such as Y224F), mutations at I179 (such as I179F), mutations at M180 (such as M180V), mutations at I181 (such as I181W), mutations at E143 (such as E143A or E143K), mutations at K65 (such as K65A), mutations at L201 (such as L201A or L201T), etc. can be used alone or in combination.

[0177] In one embodiment, the compositions of the invention comprise a polypeptide comprising a Roseburia intestinalis (Ri) maturase, or a variant or fragment thereof. In one such embodiment, the Ri maturase comprises one or more mutations corresponding to one or more mutations described herein.

[0178] Compared with other reverse transcriptases, reverse transcriptase of the present invention can produce more products (such as full-length products) at a specific temperature. In one aspect, while keeping all other reaction conditions similar or identical, full-length products are synthesized under different temperatures (such as a temperature is lower, such as between 37 ℃ and 50 ℃, and a temperature is higher, such as between 50 ℃ and 78 ℃). The amount of the full-length product produced can be determined using technology well known in the art, for example, at a first temperature (such as 37 ℃, 38 ℃, 39 ℃, 40 ℃ etc.), reverse transcription reaction is carried out and the amount of the full-length transcript produced is determined, at a temperature (such as 45 ℃, 50 ℃, 52.5 ℃, 55 ℃ etc.) higher than the first temperature, a second reverse transcription reaction is carried out and the amount of the full-length product produced is determined, and the amount produced at two temperatures is compared. A convenient comparative form is to determine that the amount of the full-length product at the first temperature accounts for the percentage of the amount of the full-length product produced at the second (i.e., rising) temperature. The reaction conditions used for the two reactions (e.g., salt concentration, buffer concentration, pH, divalent metal ion concentration, protein stabilizer concentration, macromolecular crowding agent concentration, nucleoside triphosphate concentration, template concentration, reverse transcriptase concentration, primer concentration, length of time for the reaction to proceed, etc.) can be the same for the two reactions. Those skilled in the art can determine appropriate reaction conditions using routine techniques, and examples of such conditions are provided herein.

[0179] The reverse transcription assays of the present invention can produce at least about 5%, at least 10%, at least 15%, at least 25%, at least 50%, at least 75%, at least 100%, or at least 200% more product or full-length product compared to reverse transcription assays performed using non-optimized TSO, RT primers, or performed under different reaction conditions.

[0180] Compared to a reverse transcription assay performed under the same or different reaction conditions using a TSO having an available 3' hydroxyl group, the reverse transcription assay of the present invention can produce from about 2-fold to more than 100-fold more product or full-length product. Compared to a reverse transcription assay performed under the same or different reaction conditions using a TSO without a 3' end modification, the reverse transcription assay of the present invention can produce from about 2-fold to more than 100-fold more product or full-length product. Compared to a reverse transcription assay performed under the same or different reaction conditions using a TSO without a non-natural nucleotide at the 5' end, the reverse transcription assay of the present invention can produce from about 2-fold to more than 100-fold more product or full-length product. Compared to a reverse transcription assay performed under the same or different reaction conditions using a TSO without a large blocking group at the 5' end, the reverse transcription assay of the present invention can produce from about 2-fold to more than 100-fold more product or full-length product.

[0181] The reverse transcription assays of the present invention can produce at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 75-fold, at least 100-fold, at least 150-fold, at least 200-fold, at least 300-fold, at least 400-fold, at least 500-fold, at least 1000-fold, at least 5,000-fold, or at least 10,000-fold more product than a reverse transcription assay performed under the same or different reaction conditions using a TSO with an available 3' hydroxyl group, lacking a non-natural nucleotide at the 5' end, lacking a bulky chemical group at the 5' end, or any combination thereof.

[0182] In one embodiment, the invention provides a full-length cDNA derived from a full-length RNA produced by reverse transcription assays as described herein. In one embodiment, the RNA has significant secondary or tertiary structure, and / or is long (length is greater than or equal to 5,000 bases). For example, MarathonRT and MarathonRT derivative peptides are high-permanence reverse transcriptases. In one embodiment, the length of the RNA reversely transcribed into DNA is at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000 or at least about 10000 bases. In one embodiment, the DNA so reverse transcribed is at least about 100, at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1000, at least about 2000, at least about 3000, at least about 4000, at least about 5000, at least about 6000, at least about 7000, at least about 8000, at least about 9000, or at least about 10,000 bases in length.

[0183] The present invention also includes reaction solutions for reverse transcribing nucleic acid molecules, as well as reverse transcription methods using such reaction solutions and product nucleic acid molecules produced using such methods. In many cases, the reaction solution of the present invention will contain one or more of the following components: (1) one or more buffering agents (e.g., sodium phosphate, sodium acetate, 2-(N-morpholino)-ethanesulfonic acid (MES), tris-(hydroxymethyl)aminomethane (Tris), 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPS), citrate, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), acetate, 3-(N-morpholino)propanesulfonic acid (MOPS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), etc.), (2) one or more monovalent cation salts (e.g., LiCl, NaCl, KCl, NH4Cl, etc.), (3) one or more divalent cation salts (e.g., MnCl2, MgCl2, MgSO4, CaCl2, etc.), (4) one or more reducing agents (e.g., dithiothreitol, 2-mercaptoethanol, etc.), (5) one or more ionic or nonionic detergents (e.g., TRITON X-100 TM 、NONIDET P40 TM , sodium lauryl sulfate, etc.), (6) one or more stabilizers (e.g., trehalose, betaine, BSA, glycerol, PEG8000), (7) one or more DNA polymerase inhibitors (e.g., actinomycin D, etc.), (8) nucleotides (e.g., dNTPs, such as dGTP, dATP, dCTP, dTTP, etc.), (9) RNA to be reverse transcribed and / or amplified, (10) one or more RNase inhibitors (e.g., RNASEOUT TM , Invitrogen Corporation, Carlsbad, Calif., etc.), (11) reverse transcriptase, and / or (12) one or more diluents (e.g., water). Other components and / or constituents (e.g., the RT primer of the present invention and the TSO oligonucleotide of the present invention) may also be present in the reaction solution.

[0184] In some embodiments, the present invention includes an optimized reaction buffer that enhances the RT activity of MarathonRT. In one embodiment, the optimized reaction buffer comprises PEG8000 at a concentration of about 1% to 20%, Tris at a concentration of about 10mM to about 100mM; LiCl at a concentration of about 20mM to about 500mM, MgCl2 at a concentration of about 0.5mM to about 5mM, and DTT at a concentration of about 1mM to about 10mM, wherein the pH of the reaction buffer is about 7.5 to 8.5. In one embodiment, the optimized reaction buffer comprises about 10% PEG8000, about 50mM Tris, about 100mM LiCl, about 2mM MgCl2, and about 5mM DTT; and the pH is about 8.3.

[0185] In one embodiment, the optimized reaction buffer further comprises a protein stabilizer. Exemplary protein stabilizers include, but are not limited to, osmotic stabilizers such as glycerol, erythritol, arabitol, sorbitol, mannitol, xylitol, mannisdomannitol, glucosylglycerol, glucose, fructose, sucrose, trehalose, isofluoroglycosides, dextran, fructan, and polyethylene glycol; amino acids and derivatives thereof such as glycine, alanine, proline, taurine, betaine, octopine, glutamic acid, sarcosine, γ-aminobutyric acid, trimethylamine, N-oxide (TMAO); ion stabilizers such as citrate, sulfate, acetate, phosphate, and quaternary amine; and proteins such as bovine serum albumin (BSA).

[0186] In one embodiment, the optimized reaction buffer comprises trehalose at a concentration of about 0.1 M to about 1 M. In one embodiment, the optimized reaction buffer comprises betaine at a concentration of about 0.1 M to about 10 M. In one embodiment, the optimized reaction buffer comprises BSA at a concentration of about 0.5 mg / mL to about 2 mg / mL. In one embodiment, the optimized reaction buffer comprises glycerol at a concentration of about 1% to about 50%.

[0187] The concentration of the buffer in the reaction solution of the present invention will vary depending on the specific buffer used. Typically, the working concentration of the buffer (i.e., the concentration in the reaction mixture) will be from about 5 mM to about 500 mM (e.g., about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, about 100 mM, about 5 mM to about 500 mM, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, about 800 mM, about 850 mM, about 900 mM, about 950 mM, about 1000 mM, about 500 mM, about 1000 mM, about

[0014] The working concentration of Tris is typically about 0 mM to about 500 mM, about 20 mM to about 500 mM, about 25 mM to about 500 mM, about 30 mM to about 500 mM, about 40 mM to about 500 mM, about 50 mM to about 500 mM, about 75 mM to about 500 mM, about 100 mM to about 500 mM, about 25 mM to about 50 mM, about 25 mM to about 75 mM, about 25 mM to about 100 mM, about 25 mM to about 200 mM, about 25 mM to about 300 mM, etc. When using Tris (e.g., Tris-HCl), the working concentration of Tris will typically be about 5 mM to about 100 mM, about 5 mM to about 75 mM, about 10 mM to about 75 mM, about 10 mM to about 60 mM, about 10 mM to about 50 mM, about 25 mM to about 50 mM, etc.

[0188] The final pH of the solutions of the present invention will typically be set and maintained by the buffering agents present in the reaction solutions of the present invention. The pH of the reaction solutions of the present invention, and therefore the pH of the reaction mixtures of the present invention, will vary depending on the specific application and the buffering agents present, but will typically be from about pH 5.5 to about pH 9.0 (e.g., about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, about pH 7.4, about pH 7.5, about pH 7.6, about pH 7.7, about pH 7.8, about pH 7.9, about pH 8.0, about pH 8.1, about pH 8.2, about pH 8.3, about pH 8.4, about pH 8.5, about pH 8.6, about pH 8.7, about pH 8.8, about pH 8.9, about pH 9.0, about pH 6.0 to about pH 8.5, about pH 6.5 to about pH 8.5, about pH 7.0 to about pH 8.5, about pH 7.5 to about pH 8 8.5, about pH 7.9 to about pH 8.5, about pH 8.0 to about pH 8.5, about pH 8.2 to about pH 8.5, about pH 8.3 to about pH 8.5, about pH 8.4 to about pH 8.5, about pH 8.4 to about pH 9.0, about pH 8.5 to about pH 9.0, etc.).

[0189] As shown, one or more monovalent cation salts (e.g., LiCl, NaCl, KCl, NH4Cl, etc.) may be included in the reaction solution of the present invention. In many cases, the salts used in the reaction solution of the present invention will dissociate in solution to generate at least one monovalent species (e.g., Li + 、Na + , K + NH4 +When included in the reaction solutions of the present invention, the salts will typically be present at a concentration of about 0.5 mM to about 500 mM, alone or in combination (e.g., about 1 mM, about 2 mM, about 3 mM, about 5 mM, about 10 mM, about 12 mM, about 15 mM, about 17 mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 64 mM, about 65 mM, about 66 mM, about 67 mM, about 68 mM, about 69 mM, about 70 mM, about 71 mM, about 72 mM, about 73 mM, about 74 mM, about 75 mM, about 76 mM, about 77 mM, about 78 mM, about 79 mM, about 80 mM, about 81 mM, about 82 mM, about 83 mM, about 84 mM, about 85 mM, about 86 mM, about 87 mM, about 88 mM, about 89 mM, about 90 mM, about 91 mM, about 92 mM mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 120mM, about 140mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, about 300mM, about 325mM, about 350mM, about 375mM, about 400mM, about 1mM to about 500mM, about 5mM to about 500mM, about 10mM to about 500mM, about 20mM to about 500mM, about 30mM to about 500mM, about 40mM to about 500mM, about 50mM to about 500mM, about 60mM to about 500mM, about 65mM to about 500mM, about 75mM to about 500mM, about 85mM to about 500mM, about 90mM to about 500mM, about 100mM to about 500mM, about 125mM to about 500mM, about 150mM to about 500mM, about 200mM to about 500mM, about 10mM to about 100 mM, about 10 mM to about 75 mM, about 10 mM to about 50 mM, about 20 mM to about 200 mM, about 20 mM to about 150 mM, about 20 mM to about 125 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 75 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 30 mM to about 500 mM, about 30 mM to about 100 mM, about 30 mM to about 70 mM, about 30 mM to about 50 mM, etc.).

[0190] As shown, one or more divalent cation salts (e.g., MnCl2, MgCl2, MgSO4, CaCl2, etc.) may be included in the reaction solution of the present invention. In many cases, the salts used in the reaction solution of the present invention will dissociate in solution to produce at least one divalent species (e.g., MgCl2, MgSO4, CaCl2, etc.). ++ 、Mn ++ , Ca ++When included in the reaction solutions of the present invention, the salts will typically be present at a concentration of about 0.5 mM to about 500 mM, alone or in combination (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 15 mM, about 17 mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 5 5mM, about 60mM, about 64mM, about 65mM, about 70mM, about 75mM, about 80mM, about 85mM, about 90mM, about 95mM, about 100mM, about 120mM, about 140mM, about 150mM, about 175mM, about 200mM, about 225mM, about 250mM, about 275mM, about 300mM, about 325mM, about 350mM, about 375mM, about 400mM, about 1mM to about 500mM, about 5mM to about 500mM, about 10mM to about 500mM, about 20mM to about 500mM, about 30mM to about 500mM, about 40mM to about 500mM, about 50mM to about 500mM, about 60mM to about 500mM, about 65mM to about 500mM, about 75mM to about 500mM, about 85mM to about 500mM, about 90mM to about 500mM, about 100mM to about 500mM, about 125mM to about 500mM, about 150mM to about 500mM, about 200mM to about 500mM, about 10mM to about 500mM mM, about 10 mM to about 50 mM, about 20 mM to about 200 mM, about 20 mM to about 150 mM, about 20 mM to about 125 mM, about 20 mM to about 100 mM, about 20 mM to about 80 mM, about 20 mM to about 75 mM, about 20 mM to about 60 mM, about 20 mM to about 50 mM, about 30 mM to about 500 mM, about 30 mM to about 100 mM, about 30 mM to about 70 mM, about 30 mM to about 50 mM, etc.).

[0191] When included in the reaction solutions of the present invention, reducing agents (e.g., dithiothreitol, β-mercaptoethanol, etc.) will typically be present at a concentration of about 0.1 mM to about 50 mM, alone or in combination (e.g., about 0.2 mM, about 0.3 mM, about 0.5 mM, about 0.7 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 10 mM, about 12 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 63 mM, about 64 mM, about 65 mM, 17mM, about 20mM, about 22mM, about 23mM, about 24mM, about 25mM, about 27mM, about 30mM, about 35mM, about 40mM, about 45mM, about 50mM, about 0.1mM to about 50mM, about 0.5mM to about 50mM, about 1mM to about 50mM, about 2mM to about 50mM, about 3mM to about 50mM, about 0.5mM to about 20mM, about 0.5mM to about 50mM about 10 mM, about 0.5 mM to about 5 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 20 mM, about 1 mM to about 10 mM, about 1 mM to about 5 mM, about 1 mM to about 3.4 mM, about 0.5 mM to about 3.0 mM, about 1 mM to about 3.0 mM, about 1.5 mM to about 3.0 mM, about 2 mM to about 3.0 mM, about 0.5 mM to about 2.5 mM, about 1 mM to about 2 .5mM, about 1.5mM to about 2.5mM, about 2mM to about 3.0mM, about 2.5mM to about 3.0mM, about 0.5mM to about 2mM, about 0.5mM to about 1.5mM, about 0.5mM to about 1.1mM, about 5.0mM to about 10mM, about 5.0mM to about 15mM, about 5.0mM to about 20mM, about 10mM to about 15mM, about 10mM to about 20mM, etc.).

[0192] The reaction solution of the present invention may also include one or more ionic or nonionic detergents (such as TRITON X-100 TM 、NONIDET P40 TM, sodium lauryl sulfate, etc.). When included in the reaction solutions of the present invention, detergents will typically be present at concentrations ranging from about 0.01% to about 5.0%, either singly or in combination (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 0.01% to about 5.0%, about 0.01% to about 4.0%, about 0.01% to about 3.0%, about 0.01% to about 2.0%, about 0.01% to about 1.0%, about 0.05% to about 5.0%, about 0.05% to about 3.0%, about 0.05% to about 2.0%, about 0.05% to about 1.0%, about 0.1% to about 5.0%, about 0.1% to about 4.0%, about 0.1% to about 3.0%, about 0.1% to about 2.0%, about 0.1% to about 1.0%, about 0.1% to about 0.5%, etc.). For example, the reaction solution of the present invention may include TRITON X-100 at a concentration of about 0.01% to about 2.0%, about 0.03% to about 1.0%, about 0.04% to about 1.0%, about 0.05% to about 0.5%, about 0.04% to about 0.6%, about 0.04% to about 0.3%, etc. TM .

[0193] The reaction solutions of the present invention may also include one or more stabilizers (e.g., PEG8000, trehalose, betaine, BSA, glycerol). In some embodiments, when included in the reaction solutions of the present invention, the stabilizer may be present at a concentration of 0.01 M to about 50 M alone or in combination (e.g., about 0.05 M, about 0.1 M, 0.2 M, about 0.3 M, about 0.5 M, about 0.6 M, about 0.7 M, about 0.9 M, about 1 M, about 2 M, about 3 M, about 4 M, about 5 M, about 6 M, about 10 M, about 12 M, about 15 M, about 17 M, about 20 M, about 22 M, about 23 M, about 24 M, about 26 M, about 27 M, about 28 M, about 29 M, about 30 M, about 31 M, about 32 M, about 33 M, about 34 M, about 35 M, about 36 M, about 37 M, about 38 M, about 39 M, about 40 M, about 41 M, about 42 M, about 43 M, about 44 M, about 45 M, about 46 M, about 47 M, about 48 M, about 49 M, about 50 M, about 51 M, about 52 M, about 53 M, about 54 M, about 55 M, about 56 M, about 57 M, about 58 M, about 59 M, about 60 M, about 61 M, about 62 M, about 63 M, about 64 M, about 65 M, about 66 M, about 67 M, about 68 M, about 69 M, about 70 M, about 71 M, about 72 M, about 73 , about 24M, about 25M, about 27M, about 30M, about 35M, about 40M, about 45M, about 50M, about 0.1M to about 1M, about 0.5M to about 5M, about 0.2M to about 2M, about 0.3M to about 3M, about 0.4M to about 4M, about 0.5M to about 5M, about 0.2M to about 0.8M, about 0.5M to about 1M, about 0.05M to about 1M, about 0.05M to about 10M, about 0.05M to about 20M, etc.). In some embodiments, when included in the reaction solutions of the present invention, such stabilizers may be present at a concentration of from about 0.01 mg / ml to about 100 mg / ml, alone or in combination (e.g., about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, about 0.05 mg / ml, about 0.06 mg / ml, about 0.07 mg / ml, about 0.08 mg / ml, about 0.09 mg / ml, about 0.1 mg / ml, about 0.11 mg / ml, about 0.12 mg / ml, about 0.15 mg / ml, about 0.17 mg / ml, about 0.2 mg / ml, about 0.2 5 mg / ml, about 0.35 mg / ml, about 0.5 mg / ml, about 0.75 mg / ml, about 1.0 mg / ml, about 1.5 mg / ml, about 2.0 mg / ml, about 2.5 mg / ml, about 3.0 mg / ml, about 3.5 mg / ml, about 4.0 mg / ml, about 5.0 mg / ml, about 6.0 mg / ml, about 7.0 mg / ml, about 8.0 mg / ml, about 9.0 mg / ml, about 10.0 mg / ml, about 0.05 mg / ml to about 3.0 mg / ml, about 0.1 mg / ml to about 5.0 mg / ml, about 0.2 mg / ml to about 2.0 mg / ml, etc.).In some embodiments, when included in the reaction solutions of the present invention, such stabilizers may be present individually or in combined concentrations ranging from about 0.1% to about 50% (e.g., about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.5%, about 2.0%, about 3.0%, about 5.0%, about 7.0%, about 8.0%, about 9.0%, about 10.0%, about 11.0%, about 12.0%, about 13.0%, about 14.0%, about 15.0%, about 16.0%, about 17.0%, about 18.0%, about 19.0%, about 20.0%, about 21.0%, about 22.0%, about 23.0%, about 24.0%, about .0%, about 9.0%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 20%, about 22%, about 25%, about 27%, about 30%, about 35%, about 40%, about 45%, about 50%, about 0.1% to about 50%, about 0.1% to about 40%, about 0.1% to about 30%, about 0.0% to about 20%, about 0.1% to about 10%, etc.

[0194] The reaction solutions of the present invention may also include one or more DNA polymerase inhibitors (e.g., actinomycin D, etc.). When included in the reaction solutions of the present invention, such inhibitors will typically be present at a concentration of about 0.1 μg / ml to about 100 μg / ml, alone or in combination (e.g., about 0.1 μg / ml, about 0.2 μg / ml, about 0.3 μg / ml, about 0.4 μg / ml, about 0.5 μg / ml, about 0.6 μg / ml, about 0.7 μg / ml, about 0.8 μg / ml, about 0.9 μg / ml, about 1.0 μg / ml, about 1.1 μg / ml, about 1.3 μg / ml, about 1.5 μg / ml, about 1.7 μg / ml, about 2.0 μg / ml, about 2.5 μg / ml). l, about 3.5 μg / ml, about 5.0 μg / ml, about 7.5 μg / ml, about 10 μg / ml, about 15 μg / ml, about 20 μg / ml, about 25 μg / ml, about 30 μg / ml, about 35 μg / ml, about 40 μg / ml, about 50 μg / ml, about 60 μg / ml, about 70 μg / ml, about 80 μg / ml, about 90 μg / ml, about 100 μg / ml, about 0.5 μg / ml to about 30 μg / ml, about 0.75 μg / ml to about 30 μg / ml, about 1.0 μg / ml to about 30 μg / ml, about 2.0 μg / ml l to about 30 μg / ml, about 3.0 μg / ml to about 30 μg / ml, about 4.0 μg / ml to about 30 μg / ml, about 5.0 μg / ml to about 30 μg / ml, about 7.5 μg / ml to about 30 μg / ml, about 10 μg / ml to about 30 μg / ml, about 15 μg / ml to about 30 μg / ml, about 0.5 μg / ml to about 20 μg / ml, about 0.5 μg / ml to about 10 μg / ml, about 0.5 μg / ml to about 5 μg / ml, about 0.5 μg / ml to about 2 μg / ml, about 0.5 μg / ml to about 1 μg / ml, about 1 μg / ml to about 10 μg / ml, about 1 μg / ml to about 5 μg / ml, about 1 μg / ml to about 2 μg / ml, about 1 μg / ml to about 100 μg / ml, about 10 μg / ml to about 100 μg / ml, about 20 μg / ml to about 100 μg / ml, about 40 μg / ml to about 100 μg / ml, about 30 μg / ml to about 80 μg / ml, about 30 μg / ml to about 70 μg / ml, about 40 μg / ml to about 60 μg / ml, about 40 μg / ml to about 70 μg / ml, about 40 μg / ml to about 80 μg / ml, etc.).

[0195] The reaction solution of the present invention may also include one or more additional additives to improve RT activity, including agents that improve primer utilization efficiency and increase product yield. In one embodiment, the reaction solution includes an agent that reduces non-specific binding of primers to the MarathonRT surface. The agent may include any protein, nucleic acid molecule, or small molecule that prevents or reduces non-specific binding. In certain embodiments, the agent includes D4A or a variant thereof. D4A and variants of D4A that may be included in the reverse transcription assay of the present invention include, but are not limited to, those described in detail in International Patent Publication WO2019005955A1, which is incorporated herein by reference in its entirety.

[0196] When included in the reaction solutions of the present invention, D4A or a variant thereof can be present in a ratio of D4A (or a variant thereof) to MarathonRT concentration of about 0.1:1 to about 100:1. For example, in some embodiments, D4A or a variant thereof can be present in a ratio of about 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1 The D4A (or variant thereof) concentration to the MarathonRT concentration is present in a ratio of 5:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1 or 100:1.

[0197] In many cases, nucleotides (e.g., dNTPs, such as dGTP, dATP, dCTP, dTTP, etc.) will be present in the reaction mixtures of the invention. Typically, a single nucleotide will be present at a concentration of about 0.05 mM to about 50 mM (e.g., about 0.07 mM, about 0.1 mM, about 0.15 mM, about 0.18 mM, about 0.2 mM, about 0.3 mM, about 0.5 mM, about 0.7 mM, about 0.9 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 10 mM, about 12 mM, about 15 mM, about 17 mM, about 18 mM, about 20 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM, about 38 mM, about 39 mM, about 40 mM, about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, about 50 mM, about 51 mM, about 52 mM, about 53 mM, about 54 mM, about 55 mM, about 56 mM, about 57 mM, about 58 mM, about 59 mM, about 60 mM, about 61 mM, about 62 mM, about 6 mM, about 20 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 0.1 mM to about 50 mM, about 0.5 mM to about 50 mM, about 1 mM to about 50 mM, about 2 mM to about 50 mM, about 3 mM to about 50 mM, about 0.5 mM to about 20 mM, about 0.5 mM to about 10mM, about 0.5mM to about 5mM, about 0.5mM to about 2.5mM, about 1mM to about 20mM, about 1mM to about 10mM, about 1mM to about 5mM, about 1mM to about 3.4mM, about 0.5mM to about 3.0mM, about 1mM to about 3.0mM, about 1.5mM to about 3.0mM, about 2mM to about 3.0mM, about 0.5mM to about 2.5mM, about 1mM to about 2. In some embodiments, the present invention provides a nucleotide concentration that is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide. In some embodiments, the nucleotide concentration of the nucleotide is greater than or equal to 1 nucleotide.

[0198] RNA will typically be present in the reaction solution of the present invention. In most cases, RNA will be added to the reaction solution shortly before reverse transcription. Therefore, a reaction solution that does not contain RNA can be provided. This will typically be the case when the reaction solution is provided in a kit. RNA, when present in the reaction solution, will typically be present at a concentration of 0.01 picograms to 100 μg / 20 μl reaction mixture (e.g., about 0.01 picograms / 20 μl, about 0.1 picograms / 20 μl, about 0.5 picograms / 20 μl, about 1 picogram / 20 μl, about 10 picograms / 20 μl, about 50 picograms / 20 μl, about 100 picograms / 20 μl, about 200 picograms / 20 μl, about 10 picograms / 20 μl, about 500 picograms / 20 μl, about 800 picograms / 20 μl). l, about 1.0 ng / 20 μl, about 5.0 ng / 20 μl, about 10 ng / 20 μl, about 25 ng / 20 μl, about 50 ng / 20 μl, about 75 ng / 20 μl, about 100 ng / 20 μl, about 150 ng / 20 μl, about 250 ng / 20 μl, about 400 ng / 20 μl, about 500 ng / 20 μl, about 750 ng / 20 μl, about 1.0 μg / 20 μl, about 5.0 μg / 20 μl, about 10 μg / 20 μl l, about 20 μg / 20 μl, about 30 μg / 20 μl, about 40 μg / 20 μl, about 50 μg / 20 μl, about 70 μg / 20 μl, about 85 μg / 20 μl, about 100 μg / 20 μl, about 10 picograms / 20 μl to about 100 μg / 20 μl, about 10 picograms / 20 μl to about 100 μg / 20 μl, about 100 picograms / 20 μl to about 100 μg / 20 μl, about 100 picograms / 20 μl to about 100 μg / 20 μl, about 1.0 ng / 20 μl to about 100 μg / 20 μl, about 1 μg / 20 μl, about 100 ng / 20 μl to about 100 μg / 20 μl, about 10 picograms / 20 μl to about 10 μg / 20 μl, about 10 picograms / 20 μl to about 5 μg / 20 μl, about 100 ng / 20 μl to about 5 μg / 20 μl, about 1 μg / 20 μl to about 10 μg / 20 μl, about 1 μg / 20 μl to about 5 μg / 20 μl, about 100 ng / 20 μl to about 1 μg / 20 μl, about 500 ng / 20 μl to about 5 μg / 20 μl, etc.). As will be appreciated by those skilled in the art, different reverse transcription reactions can be performed in volumes other than 20 μl. In such cases, the total amount of RNA present will vary with the volume used. Therefore, the above amounts are provided as examples of the amount of RNA per 20 μl reaction solution.

[0199] Reverse transcriptase may also be present in the reaction solution. When present, the reverse transcriptase will typically be present at a concentration resulting in about 0.01 to about 1,000 units of reverse transcriptase activity per μl (e.g., about 0.01 units / μl, about 0.05 units / μl, about 0.1 units / μl, about 0.2 units / μl, about 0.3 units / μl, about 0.4 units / μl, about 0.5 units / μl, about 0.7 units / μl, about 1.0 units / μl, about 1.5 units / μl, about 2.0 units / μl, about 2.5 units / μl, about 5.0 units / μl, about 7.5 units / μl, about 1 0 units / μl, about 20 units / μl, about 25 units / μl, about 50 units / μl, about 100 units / μl, about 150 units / μl, about 200 units / μl, about 250 units / μl, about 350 units / μl, about 500 units / μl, about 750 units / μl, about 1,000 units / μl, about 0.1 units / μl to about 1,000 units / μl, about 0.2 units / μl to about 1,000 units / μl, about 1.0 units / μl to about 1,000 units / μl, about 5.0 units / μl, about 1,000 units / μl, about 2.0 units / μl to about 1,000 units / μl, about 3.0 units / μl, about 4.0 units / μl, about 5.0 units / μl, about 6.0 units / μl, about 7.5 units / μl, about 8.0 units / μl, about 9.0 units / μl, about 10.0 units / μl, about 11.0 units / μl, about 12.0 units / μl, about 13.0 units / μl, about 14.0 units / μl, about 15.0 units / μl, about 16.0 units / μl, about 17.0 units / μl, about 18.0 units / μl, about 19.0 units / μl, about l to about 1,000 units / μl, about 10 units / μl to about 1,000 units / μl, about 20 units / μl to about 1,000 units / μl, about 50 units / μl to about 1,000 units / μl, about 100 units / μl to about 1,000 units / μl, about 200 units / μl to about 1,000 units / μl, about 400 units / μl to about 1,000 units / μl, about 500 units / μl to about 1,000 units / μl, about 0.1 units / μl to about 300 units / μl, about 0.1 units / μl to about 1,000 units / μl, about In some embodiments, the reaction solution includes a reverse transcriptase described herein at a lower concentration than that required to produce an equivalent product from other reverse transcriptases.

[0200] The reaction solution of the present invention can be prepared into concentrated solution (such as 5 × solution), which is diluted to the working concentration of final use. For 5 × reaction solution, 5: 1 dilution is needed so that such 5 × solution reaches working concentration. The reaction solution of the present invention can be prepared into solutions such as 2 ×, 3 ×, 4 ×, 5 ×, 6 ×, 7 ×, 8 ×, 9 ×, 10 ×. A major limitation of the multiple concentration of such solution is that precipitation occurs when compound reaches specific concentration in solution. Therefore, concentrated reaction solution will generally be prepared into the concentration of various components sufficiently low, thereby the precipitation of buffer components will not occur. As will be appreciated by those skilled in the art, the feasible upper concentration limit of each solution will vary with specific solution and the component present.

[0201] In many cases, the reaction solutions of the present invention will be provided in a sterile form. The individual components of the reaction solution may be sterilized prior to mixing, or the reaction solution may be sterilized after preparation. Sterilization of such solutions may be performed by any suitable means including autoclaving or ultrafiltration.

[0202] method

[0203] In various embodiments, the present invention includes methods for using TSO, RT primers, or a combination of TSO and RT primers for reverse transcription reactions. For example, in one embodiment, the method includes contacting an RNA template with TSO, RT primers, and a reverse transcriptase under suitable conditions to produce a transcribed DNA molecule from the RNA template.

[0204] In various embodiments, the present invention includes methods for performing a reverse transcription reaction using MarathonRT or a variant thereof, or a nucleic acid encoding MarathonRT or a variant thereof, in combination with a TSO and an RT primer of the present invention. For example, in some embodiments, the method includes using MarathonRT or a variant thereof, or a nucleic acid encoding MarathonRT or a variant thereof, in combination with a TSO and an RT primer, wherein the TSO comprises at least one modification at the 5' end, at least one modification at the 3' end, or modifications at both the 5' end and the 3' end to prevent multiplexing and nonspecific reverse transcription during template switching.

[0205] For example, in one embodiment, the method comprises mixing TSO, an RT primer, and a reverse transcriptase (comprising MarathonRT or a variant thereof) under appropriate conditions; and contacting the mixture with an RNA template to produce a transcribed DNA molecule from the RNA template.

[0206] In various embodiments, the present invention includes methods for reverse transcription in an optimized reaction buffer. For example, in one embodiment, the method includes adding TSO, RT primers, and a reverse transcriptase (e.g., MarathonRT or a variant thereof) to an optimized reaction buffer; and contacting the mixture with an RNA template to produce a transcribed DNA molecule from the RNA template. In one embodiment, the optimized reaction buffer comprises PEG8000 at a concentration of about 1% to about 20%, Tris at a concentration of about 10mM to about 100mM; LiCl at a concentration of about 20mM to about 500mM, MgCl2 at a concentration of about 0.5mM to about 5mM, DTT at a concentration of about 1mM to about 10mM, wherein the pH of the reaction buffer is about 7.5 to 8.5. In one embodiment, the optimized reaction buffer comprises about 10% PEG8000, about 50mM Tris, about 100mM LiCl, about 2mM MgCl2, about 5mM DTT; and a pH of about 8.3.

[0207] In one embodiment, the optimized reaction buffer comprises a protein stabilizer. Exemplary protein stabilizers include, but are not limited to, osmotic stabilizers such as glycerol, erythritol, arabitol, sorbitol, mannitol, xylitol, mannisdomannitol, glucosylglycerol, glucose, fructose, sucrose, trehalose, isofluoroglycosides, dextran, fructan, and polyethylene glycol; amino acids and derivatives thereof such as glycine, alanine, proline, taurine, betaine, octopine, glutamic acid, sarcosine, γ-aminobutyric acid, trimethylamine, N-oxide (TMAO); ion stabilizers such as citrate, sulfate, acetate, phosphate, and quaternary amine; and proteins such as bovine serum albumin (BSA).

[0208] In one embodiment, the optimized reaction buffer comprises trehalose at a concentration of about 0.1 M to about 1 M. In one embodiment, the optimized reaction buffer comprises betaine at a concentration of about 0.1 M to about 10 M. In one embodiment, the optimized reaction buffer comprises BSA at a concentration of about 0.5 mg / mL to about 2 mg / mL. In one embodiment, the optimized reaction buffer comprises glycerol at a concentration of about 1% to about 50%.

[0209] Using TSO and RT primers

[0210] Any technology that uses reverse transcription as a method or step can utilize the TSO, RT primers, or combinations thereof of the present invention. In various embodiments, improved TSO, RT primers, or combinations thereof are used to perform reverse transcription as part of an assay. In various embodiments, the assay can be at least one selected from the group consisting of RT-PCR, qRT-PCR, capillary electrophoresis (CE) for RNA structure mapping (such as SHAPE-seq or SHAPE-MaP, DMS-seq), transcriptome analysis, in-cell sequencing, next-generation RNA sequencing (RNA-seq), nanopore sequencing, PacBio sequencing, zero-mode waveguide sequencing, cDNA library synthesis, cDNA synthesis, and combinations thereof.

[0211] In some aspects, the methods provide for reverse transcription at physiological temperature, or at a lower temperature relative to the temperature required when using a non-MarathonRT derivative reverse transcriptase. In some cases, the lower temperature of the reverse transcription reaction provides a reduced rate of degradation of the RNA molecule during the reaction relative to the rate of degradation of the RNA molecule in a reverse transcription reaction using a non-MarathonRT derivative reverse transcriptase.

[0212] In one embodiment, the method involves reverse transcription of long and / or complex RNA molecules.

[0213] In one embodiment, the method comprises formulating a reaction solution comprising a low concentration of a TSO or RT primer described herein (compared to the concentration required for a reaction using a different TSO or RT primer).

[0214] In one embodiment, the method comprises formulating a reaction solution comprising a high concentration of a TSO or RT primer described herein (compared to the concentration required for a reaction using a different TSO or RT primer).

[0215] In one embodiment, the method comprises a single reaction amplification of RNA, which is made possible by the true thermal cycling capability of the reverse transcriptases described herein. For example, the thermal cycling capability of the reverse transcriptases described herein allows RNA amplification without the need for DNA replication.

[0216] In one embodiment, the improved TSO, RT primers, or a combination thereof are used in a quantitative RT-PCR (qRT-PCR) procedure. In qRT-PCR, the formation of PCR products is monitored in each cycle of PCR. Amplification is typically measured in a thermal cycler having an additional device for measuring a fluorescent signal during the amplification reaction. See, for example, U.S. Patent No. 6,174,670 and U.S. Patent No. 8,137,616. In one embodiment, a thermostable improved MarathonRT enzyme is used instead of a DNA→DNA polymerase for the qRT-PCR procedure.

[0217] In one embodiment, the improved TSO, RT primers, or a combination thereof are used for isothermal DNA amplification using an engineered reverse transcriptase with improved strand displacement activity on a DNA template.

[0218] In one embodiment, improved TSO, RT primer or its combination are used for capillary electrophoresis (CE) to carry out RNA structure mapping program. It is an important step to increase RNA structure data throughput to apply capillary electrophoresis to RNA structure detection. Gel electrophoresis usually resolves about one hundred RNA bases at a time, so it may be necessary to run tens to hundreds of gels to detect RNA that is several thousand bases long. Capillary electrophoresis allows to resolve 300-650 bases from structure detection experiments, and multiple channels can be run simultaneously to increase the flux of RNA structure detection. The readout of the detection experiment is usually a DNA primer by reverse transcription 5' fluorescent labeling, which specifically anneals with the RNA of interest. If RNA is several thousand bases long, multiple primers are designed to anneal along the length of the transcript. Modification or cracking of the RNA template causes the primer extension reaction to terminate prematurely, resulting in different lengths of the cDNA products resolved by capillary electrophoresis. Software tools such as CAFA and Shapefinder can automate data acquisition from capillary electrophoresis, further improving speed and accuracy (see, eg, Wan, Y. et al., 2011, Nat Rev Genet., 12(9): 1-26).

[0219] In one embodiment, improved TSO, RT primers, or a combination thereof are used for next generation RNA sequencing (RNA-seq) procedures. High throughput RNA sequencing (RNA-Seq) technology, made possible by the latest developments in next generation sequencing, has become a powerful tool for analyzing gene expression profiles, detecting transcript variants, and understanding the functions of non-coding regulatory RNAs. Standard RNA-Seq libraries are generated by connecting sequencing adapters to double-stranded DNA. There are two main methods for preparing chain-specific RNA-Seq libraries. The first method includes connecting different adapters to the 3' and 5' ends of the RNA molecule (see, for example, the IonTotal RNA-Seq Kit v2 of Life Technologies). Another more widely used method includes adding dUTP in addition to dNTP in the second strand DNA synthesis. After linker ligation, the second-strand DNA can be specifically digested by the enzyme uracil-N-glycosylase (UNG), so that only the strand of the library containing the first-strand cDNA will be sequenced and information about the orientation of the transcript can therefore be obtained (see M. Sultan et al., Biochemical and Biophysical Research Communications 422 (2012) 643-646; see also PCT patent application No. PCT / EP2016 / 069997).

[0220] The present invention also relates to a method for preparing one or more nucleic acid molecules and / or labeled nucleic acid molecules, comprising mixing one or more nucleic acid templates (e.g., one or more RNA templates or messenger RNA templates) with TSO, RT primers, or a combination thereof, and one or more polypeptides having reverse transcriptase activity, and incubating the mixture under conditions sufficient to synthesize one or more first nucleic acid molecules complementary to all or part of the one or more nucleic acid templates, wherein at least one of the synthesized molecules is optionally labeled and / or comprises one or more labeled nucleotides and / or wherein the synthesized molecules can be optionally modified to contain one or more labels. In one embodiment, the one or more first nucleic acid molecules are single-stranded cDNA molecules. According to this aspect of the invention, nucleic acid templates suitable for reverse transcription include any nucleic acid molecule or population of nucleic acid molecules (e.g., RNA, mRNA), particularly those derived from cells or tissues. In one aspect, according to the present invention, a population of mRNA molecules (a large number of different mRNA molecules, typically obtained from cells or tissues) is used to prepare a labeled cDNA library. Exemplary sources of nucleic acid templates include viruses, virus-infected cells, bacterial cells, fungal cells, plant cells, and animal cells.

[0221] The present invention also relates to methods for preparing one or more double-stranded nucleic acid molecules (which may optionally be labeled). Such methods comprise (a) mixing one or more nucleic acid templates (e.g., RNA or mRNA, or a population of mRNA templates) with a TSO, RT primer, or a combination thereof, and one or more polypeptides having reverse transcriptase activity; (b) incubating the mixture under conditions sufficient to produce one or more first nucleic acid molecules complementary to all or part of the one or more templates; and (c) incubating the one or more first nucleic acid molecules under conditions sufficient to produce one or more second nucleic acid molecules complementary to all or part of the one or more first nucleic acid molecules, thereby forming one or more double-stranded nucleic acid molecules comprising the first and second nucleic acid molecules. According to the present invention, the first and / or second nucleic acid molecules may be labeled (e.g., may comprise one or more identical or different labeled nucleotides and / or may be modified to comprise one or more identical or different labels). Thus, labeled nucleotides may be used in one or both synthesis steps. Such methods may comprise using one or more DNA polymerases as part of the process for preparing the one or more double-stranded nucleic acid molecules. The present invention also relates to compositions that can be used to prepare such double-stranded nucleic acid molecules. Such compositions include TSO, RT primers, or a combination thereof, one or more reverse transcriptases and optionally one or more DNA polymerases, a suitable buffer, and / or one or more nucleotides (eg, including labeled nucleotides).

[0222] The present invention also relates to nucleic acid molecules and / or labeled nucleic acid molecules (particularly single-stranded or double-stranded cDNA molecules) produced according to the above methods, as well as kits comprising these nucleic acid molecules. Such molecules or kits can be used for detecting nucleic acid molecules (e.g., by hybridization) or for diagnostic purposes.

[0223] Reagent test kit

[0224] The present invention also relates to a kit for the reverse transcription method of the present invention. Such a kit can be used to prepare nucleic acid molecules and / or labeled nucleic acid molecules (single-stranded or double-stranded). The kit of the present invention may include a carrier, such as a box or a case, wherein one or more containers, such as vials, tubes and bottles, are sealed. In the kit of the present invention, the first container may contain one or more reverse transcriptases of the present invention or one or more compositions of the present invention. The kit of the present invention may also contain at least one component selected from one or more TSOs, one or more RT primers and reverse transcriptases in the same or different containers. In one embodiment, the kit of the present invention may also contain an agent that reduces non-specific binding of primers to reverse transcriptases in the same or different containers. In one embodiment, the kit of the present invention may also contain an optimized reaction buffer as described elsewhere herein or a component for producing an optimized reaction buffer in the same or different containers. Alternatively, the components of the kit can be subpackaged into separate containers.

[0225] The present invention further relates to a test kit for the method of the present invention. Such test kits can be used for preparing, sequencing or amplifying nucleic acid molecules (single-stranded or double-stranded), for example, at a specific temperature as described herein. The test kit of the present invention may comprise a carrier, such as a box or a case, wherein one or more (e.g., one, two, three, four, five, ten, twelve, fifteen, etc.) containers, such as vials, tubes and bottles, are enclosed. In the test kit of the present invention, the first container comprises one or more reverse transcriptases of the present invention. The test kit of the present invention may also comprise one or more DNA polymerases (e.g., thermostable DNA polymerases), one or more (e.g., one, two, three, four, five, ten, twelve, fifteen, etc.) suitable buffers for nucleic acid synthesis, one or more nucleotides and one or more (e.g., one, two, three, four, five, ten, twelve, fifteen, etc.) oligonucleotide primers in the same or different containers. The test kit of the present invention may also comprise instructions or schemes for implementing the method of the present invention.

[0226] In various embodiments, the present invention provides kits for performing a reverse transcription reaction. In one embodiment, the kit comprises at least one TSO oligonucleotide, at least one RT primer, and a reverse transcription polypeptide or variant thereof. In one embodiment, the kit comprises instructional materials describing the use of the kit for performing a reverse transcription reaction, wherein the instructional materials establish an increased functional relationship between the kit components and the individual using the kit. In one embodiment, the kit is used by one person or entity. In another embodiment, the kit is used by more than one person or entity. In one embodiment, the kit is used without any additional composition or method. In another embodiment, the kit is used in conjunction with at least one additional composition or method.

[0227] Experimental Examples

[0228] The present invention will be described in further detail with reference to the following experimental examples. These examples are provided for illustration only and, unless otherwise specified, are not intended to be limiting. Therefore, the present invention should in no way be construed as being limited to the following examples, but rather should be construed as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0229] Without further description, it is believed that one of ordinary skill in the art can use the foregoing description and the following illustrative examples to make and utilize the present invention and practice the claimed methods. Therefore, the following working examples should not be construed as limiting the remainder of the disclosure in any way.

[0230] Example 1: Optimization of Template Switching Oligonucleotides (TSO)

[0231] In RNA-seq experiments, accurate quantification of single transcripts, identification of new transcripts, or identification of unknown transcription start sites requires an effective method to convert mRNA molecules into full-length cDNA. At the same time, the 5' and 3' ends of the cDNA molecules need to be effectively connected to two different universal sequence adapters for library preparation. These steps can be achieved simultaneously by combining the high processivity reverse transcriptase activity and template switching activity of MarathonRT (or Er maturation enzyme) in a one-pot reaction, which is particularly useful when the RNA input amount (such as the RNA input amount from a single cell) is very low. Template switching oligonucleotides (TSOs) containing selected universal sequences are designed to be connected to the 3' end of the cDNA molecule during the template switching process.

[0232] Specifically, during the first strand synthesis process, upon reaching the 5' end of an RNA template (such as cellular RNA), the terminal transferase activity of MarathonRT adds some additional nucleotides (primarily deoxyadenosine) to the 3' end of the newly synthesized cDNA chain in a non-templated manner. These bases function as TSO anchor sites during the template switching process. With base pairing between TSO and the additional deoxyadenosine fragment, MarathonRT seamlessly "switches" the template chain from cellular RNA to TSO and continues to extend the primer to the 5' end of TSO. By doing so, the generated cDNA contains the complete 5' end of the transcript and the selected universal sequence is added to the reverse transcription product. Together with labeling the 5' end of the cDNA by oligonucleotide dT primers, this approach allows for the efficient amplification of the entire full-length transcript pool in a completely sequence-independent manner.

[0233] However, TSO and oligonucleotide dT primers can also be amplified during reverse transcription and template switching, which can dominate the resulting sequencing library. First, during reverse transcription, TSO can also serve as a template for reverse transcription by oligonucleotide dT primers, which leads to autonomous amplification of the oligonucleotide. In order to remove unwanted products, the chemical composition and nucleotide sequence of TSO and oligonucleotide dT primers were optimized to reduce the base pairing potential between TSO and oligonucleotide dT primers, thereby avoiding reverse transcription between them (e.g., SEQ ID NO: 3 and SEQ ID NO: 2, respectively). Second, in addition to serving as a template, TSO can also serve as a primer to reverse transcribe itself, so the hydroxyl group at the 3' end of TSO is removed or blocked with a chemical group to prevent extension by MarathonRT ( Figure 1 and Figure 6To remove the hydroxyl group at the 3' end of TSO, dideoxythymidine was used at the 3' end of TSO. Third, template switching by MarathonRT is very efficient, so TSO can be multiplexed multiple times (>100 times) in series due to the recycling of reverse transcriptase and terminal transferase activities ( Figure 2 To prevent TSO concatemers, one or more bulky chemical groups are added to the 5' end of TSO, which effectively prevents multiple template switching cycles through MarathonRT ( Figure 1 and 3 -5). These chemical groups include multiple (3-5) 5'AP sites (apurinic / apyrimidinic sites), 5'trebler and 5'trityl ( Figure 4 and Figure 5). Therefore, the chemical composition of this specific TSO and its application are novel. In summary, by systematically optimizing TSO, we utilized the template switching reaction of MarathonRT to prepare RNA-seq libraries from ultra-low RNA input.

[0234] The RT (MarathonRT or Er maturation enzyme) of the class II intron encoding used in this system is highly persistent, and its activity is not affected by the sequence and structure of the RNA template during reverse transcription and template switching. Using this enzyme for reverse transcription and template switching effectively improves full-length cDNA yield and reduces the deviation caused by the heterogeneity of template sequence and structure. By optimizing reaction conditions (such as using LiCl instead of KCl and adding PEG8000, template switching efficiency increases from 2% to 50%), this significantly improves cDNA yield. Effective template switching causes the multiplexing of template switching oligonucleotides, which takes a large amount of sequencing reads. Adding chemical modifications to the 5' end of the template switching oligonucleotide or using non-standard nucleotides at the 5' end of the template switching oligonucleotide prevents template switching oligonucleotide concatemers, which are almost undetectable in sequencing reads. When using a very small amount of RNA template, the non-specific reverse transcription of RT primers and template switching oligonucleotides leads to cDNA products. The nucleotide sequences of primers and template switching oligonucleotides have been optimized to minimize the base pairing potential between them. Through system optimization, the optimized method provides sensitive and quantitative detection of RNA transcripts in RNA sequencing experiments.

[0235] Figures 7A to 7CModifications of TSOs that can be used for reverse transcription using various types of reverse transcriptases are shown. Different reverse transcriptases have preferences for different non-templated nucleotide additions (NTA). The sequence of the TSO can be selected based on the standard NTA by the reverse transcriptase. MarathonRT specifically adds a triadenosine overhang to the 3' end of the cDNA. It requires a TSO with three uridines (RNA) or three thymidines (DNA) at the 3' end for efficient template switching. MMLV RT adds a tricytidine overhang to the 3' end of the cDNA. It requires a TSO with three guanosines at the 3' end for efficient template switching. TGIRT TM Single nucleotide overhangs (a mixture of A, G, C, and T) are most efficiently added to the 3' end of the cDNA. TSO with any nucleotide at the 3' end can mediate template switching.

[0236] The disclosures of each patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. Although the present invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the invention may be devised by those skilled in the art without departing from the true spirit and scope of the invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A template switching oligonucleotide (TSO), comprising: (i) a DNA nucleotide sequence, an RNA nucleotide sequence or a hybrid DNA-RNA sequence; and (ii) at least one of a 3'-end modification and a 5'-end modification.

2. The TSO according to claim 1, wherein the TSO comprises a 3' end modification.

3. The TSO of claim 1, wherein the TSO comprises a 5' end modification. The TSO of claim 1 , wherein the TSO comprises both a 3′-end modification and a 5′-end modification.

5. The TSO of claim 1, wherein the 3' end modification is a nucleotide sugar modification or a nucleobase modification. The TSO according to claim 1 , wherein the 3′ end modification is selected from the group consisting of a modification that removes the 3′ hydroxyl group and a modification that blocks the 3′ hydroxyl group.

7. The TSO of claim 1, wherein the 3' end modification is selected from the group consisting of: 3'ddT, 3'ddU, 3' reversed dT, 3'C3 spacer, 3' amino, 3'rU oxidized by periodate, 3' phosphorylation, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' olefin, 3'(CH2)nX (X=H, OCH3, CH3, SH, NH2, OH, etc.; n≥1) and 3'(CH2CH2O)n (n≥1).

8. The TSO of claim 1, wherein the 5' end modification is a nucleotide sugar modification or a nucleobase modification.

9. The TSO of claim 1, wherein the 5'-end modification comprises trityl, trebbler, dendrimer, biotin, fluorescent dye, ROX NHS ester, (CH2)n (n≥1) long spacer, palmitic acid phosphoramidite, 3-cyanovinylcarbazole phosphoramidite, cholesteryl or psoralen.

10. The TSO according to claim 1, wherein the 3' end modification is selected from the group consisting of a modification that removes the 3' hydroxyl group and a modification that blocks the 3' hydroxyl group.

11. The TSO of claim 1, wherein the 5' end comprises at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 consecutive abasic sites.

12. The TSO of claim 1, wherein the 5' end comprises at least 5 consecutive abasic sites.

13. The TSO of claim 1, wherein the 5' end comprises at least 3 consecutive abasic sites.

14. The TSO of claim 1, wherein the 5' end comprises at least one non-natural nucleotide or nucleotide analog.

15. The TSO of claim 1, wherein the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

16. A template switching oligonucleotide (TSO) comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence or a hybrid DNA-RNA sequence, wherein the TSO is capable of annealing by base pairing with a non-templated nucleotide that has been added to the 5' end of a target nucleic acid molecule in a non-templated addition process by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3' end modification and a 5' end modification.

17. The TSO according to claim 16, wherein the 3' end modification is selected from the group consisting of a modification that removes the 3' hydroxyl group and a modification that blocks the 3' hydroxyl group.

18. The TSO of claim 16, wherein the TSO comprises a 3' terminal modification selected from the group consisting of: 3' ddT, 3' ddU, 3' reversed dT, 3' C3 spacer, 3' amino, 3' rU oxidized by periodate, 3' phosphorylated, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' olefin, 3' (CH2) nX (X = H, OCH3, CH3, SH, NH2, OH, etc.; n ≥ 1) and 3' (CH2CH2O) n (n ≥ 1).

19. The TSO of claim 16, wherein the 5' end is modified with a chemical group to block multiplexing.

20. The TSO of claim 16, wherein the 5' end is modified with a chemical group selected from the group consisting of trityl, trebbler, dendrimer, biotin, fluorescent dye, ROX NHS ester, (CH2)n (n≥1) long spacer, palmitic acid phosphoramidite, 3-cyanovinylcarbazole phosphoramidite, cholesteryl and psoralen.

21. The TSO of claim 16, wherein the 5' end comprises at least 3 consecutive abasic sites.

22. The TSO of claim 16, wherein the 5' end comprises at least one non-natural nucleotide or nucleotide analog.

23. The TSO of claim 16, wherein the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

24. The TSO of claim 16, wherein the TSO comprises at least one 3' end modification and at least one 5' end modification.

25. A reverse transcription (RT) primer comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence or a hybrid DNA-RNA sequence, wherein the RT primer comprises a 5' end modification.

26. The RT primer of claim 25, wherein the 5' end is modified with a chemical group to block multiplexing.

27. The RT primer of claim 25, wherein the 5' end is modified with a chemical group selected from the group consisting of trityl, trebbler, dendrimer, biotin, fluorescent dye, ROX NHS ester, (CH2)n (n≥1) long spacer, palmitic acid phosphoramidite, 3-cyanovinylcarbazole phosphoramidite, cholesterol group and psoralen.

28. The RT primer of claim 25, wherein the 5' end comprises at least 3 consecutive abasic sites.

29. The RT primer of claim 25, wherein the 5' end comprises at least one non-natural nucleotide or nucleotide analog.

30. The RT primer of claim 25, wherein the RT primer comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

31. A method of generating a cDNA molecule from an RNA template, the method comprising contacting the RNA template with the TSO of claim 25, a reverse transcription (RT) primer, and a reverse transcriptase.

32. The method of claim 31, wherein the RT primer comprises a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence, or a hybrid DNA-RNA sequence, wherein the RT primer comprises a 5' end modification.

33. The method of claim 31, wherein the reverse transcriptase is selected from the group consisting of MarathonRT or variants thereof, Moloney murine leukemia virus reverse transcriptase (MMLV RT) or variants thereof, avian myeloblastic leukemia virus reverse transcriptase (AMV RT) or variants thereof, HIV reverse transcriptase (HIV RT) or variants thereof, Bombyx mori R2 RNA element reverse transcriptase (R2 RT) or variants thereof, and TGIRT TM or variations thereof.

34. The method of claim 31, wherein the method is included in an assay selected from the group consisting of RT-PCR, qRT-PCR, capillary electrophoresis (CE) for RNA structure mapping, transcriptome analysis, in-cell sequencing, next generation RNA sequencing (RNA-seq), nanopore sequencing, PacBio sequencing, zero mode waveguide sequencing, cDNA library synthesis, cDNA synthesis, or any combination thereof.

35. A reverse transcription assay for generating a cDNA molecule from an RNA template, the method comprising contacting the RNA template with a TSO comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence, or a hybrid DNA-RNA sequence, wherein the TSO is capable of annealing by base pairing with a non-templated nucleotide that has been added to the 5' end of a target nucleic acid molecule during a non-templated addition process by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3' end modification and a 5' end modification, a reverse transcription (RT) primer, and a reverse transcriptase.

36. The method of claim 35, wherein the RT primer is selected from the group consisting of a DNA primer, an RNA primer, a primer comprising at least one modified oligonucleotide, and the RT primer according to any one of claims 10-15.

37. The assay of claim 35, wherein the reverse transcriptase is selected from the group consisting of MarathonRT or variants thereof, Moloney murine leukemia virus reverse transcriptase (MMLV RT) or variants thereof, avian myeloblastic leukemia virus reverse transcriptase (AMV RT) or variants thereof, HIV reverse transcriptase (HIV RT) or variants thereof, Bombyx mori R2 RNA element reverse transcriptase (R2 RT) or variants thereof, and TGIRT TM or variations thereof.

38. The assay of claim 35, wherein the reverse transcription is performed in a buffer comprising PEG8000.

39. The assay of claim 35, wherein the reverse transcription is performed in a buffer comprising LiCl.

40. A kit for performing an assay for generating cDNA molecules from an RNA template, the method comprising contacting the RNA template with a TSO comprising a DNA nucleotide sequence, an RNA nucleotide sequence, a modified nucleotide sequence, or a hybrid DNA-RNA sequence, wherein the TSO is capable of annealing by base pairing with a non-templated nucleotide that has been added to the 5' end of a target nucleic acid molecule during a non-templated addition process by a reverse transcriptase, and wherein the TSO further comprises at least one of a 3' end modification and a 5' end modification, a reverse transcription (RT) primer, and a reverse transcriptase.

41. The kit of claim 40, wherein the RT primer is selected from the group consisting of a DNA primer, an RNA primer, a primer comprising at least one modified oligonucleotide, and the RT primer according to any one of claims 10-15.

42. The kit of claim 40, wherein the reverse transcriptase is selected from the group consisting of MarathonRT or variants thereof, Moloney murine leukemia virus reverse transcriptase (MMLV RT) or variants thereof, avian myeloblastic leukemia virus reverse transcriptase (AMV RT) or variants thereof, HIV reverse transcriptase (HIV RT) or variants thereof, Bombyx mori R2 RNA element reverse transcriptase (R2 RT) or variants thereof, and TGIRT TM or variations thereof.

43. The kit of claim 40, wherein the kit comprises a buffer containing PEG8000.

44. The kit of claim 40, wherein the kit comprises a buffer comprising LiCl.

45. A method of reducing multiplexing of a template switching oligonucleotide (TSO), the method comprising providing a reaction mixture comprising a TSO, a reverse transcription (RT) primer, and a reverse transcriptase, Wherein the TSO comprises: (i) 3' end modification; and / or (ii) 5' end modification.

46. ​​The method of claim 45, wherein the multiplexing is reduced, for example, by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% based on a reference standard.

47. The method of claim 45, wherein the 3' end modification is selected from the group consisting of 3'ddT, 3'ddU, 3' reversed dT, 3'C3 spacer, 3' amino, 3'rU oxidized by periodate, 3' phosphorylation, 3' fluoro, 3' aldehyde, 3' carboxylic acid or carboxylate, 3' thiol, 3' O-methyl, 3' azido, 3' alkyne, 3' olefin, 3'(CH2)nX (X=H, OCH3, CH3, SH, NH2, OH, etc.; n≥1) and 3'(CH2CH2O)n (n≥1).

48. The method of claim 45, wherein the TSO comprises a 5' end modification selected from the group consisting of trityl, trebbler, dendrimer, biotin, fluorescent dye, ROX NHS ester, (CH2)n (n≥1) long spacer, palmitic acid phosphoramidite, 3-cyanovinylcarbazole phosphoramidite, cholesteryl, and psoralen.

49. The method of claim 45, wherein the 5' end of the TSO comprises at least 3 consecutive abasic sites.

50. The method of claim 45, wherein the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

51. The method of claim 45, wherein the TSO comprises SEQ ID NO:

3.

52. The method of claim 45, wherein the RT primer comprises SEQ ID NO:

2.

53. A method for reducing non-specific reverse transcription from a template switching oligonucleotide (TSO), the method comprising providing a reaction mixture comprising TSO, a reverse transcription (RT) primer and a reverse transcriptase, Wherein the TSO comprises: (i) 3' end modification; and / or (ii) 5' end modification.

54. The method of claim 49, wherein the non-specific reverse transcription is reduced, for example, by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% based on a reference standard.

55. The method of claim 53, wherein the TSO comprises a 5' end modification selected from the group consisting of trityl, trebbler, dendrimer, biotin, fluorescent dye, ROX NHS ester, (CH2)n (n≥1) long spacer, palmitic acid phosphoramidite, 3-cyanovinylcarbazole phosphoramidite, cholesteryl and psoralen.

56. The method of claim 53, wherein the 5' end of the TSO comprises at least 3 consecutive abasic sites.

57. The method of claim 53, wherein the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

58. The method of claim 53, wherein the TSO comprises SEQ ID NO:

3.

59. The method of claim 53, wherein the RT primer comprises SEQ ID NO:

2.

60. A method for increasing the yield of a target polynucleotide sequence in an RNA-seq library, the method comprising providing a reaction mixture comprising TSO, a reverse transcription (RT) primer, and a reverse transcriptase, Wherein the TSO comprises: (i) 3' end modification; and / or (ii) 5' end modification.

61. The method of claim 60, wherein the yield of the target polynucleotide is increased, for example, by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% based on a reference standard.

62. The method of claim 60, wherein the TSO comprises a 5' end modification selected from the group consisting of trityl, trebbler, dendrimer, biotin, fluorescent dye, ROX NHS ester, (CH2)n (n≥1) long spacer, palmitic acid phosphoramidite, 3-cyanovinylcarbazole phosphoramidite, cholesteryl, and psoralen.

63. The method of claim 60, wherein the 5' end of the TSO comprises at least 3 consecutive abasic sites.

64. The method of claim 60, wherein the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

65. The method of claim 60, wherein the TSO comprises SEQ ID NO:

3.

66. The method of claim 60, wherein the RT primer comprises SEQ ID NO:

2.

67. A method for increasing the specificity of an RNA-seq library, the method comprising providing a reaction mixture comprising TSO, a reverse transcription (RT) primer, and a reverse transcriptase, Wherein the TSO comprises: (i) 3' end modification; and / or (ii) 5' end modification; The RNA-seq library is prepared using template switching.

68. The method of claim 67, wherein the specificity of the RNA-seq library is increased, for example, by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% based on a reference standard.

69. The method of claim 67, wherein the TSO comprises a 5' end modification selected from the group consisting of trityl, trebbler, dendrimer, biotin, fluorescent dye, ROX NHS ester, (CH2)n (n≥1) long spacer, palmitic acid phosphoramidite, 3-cyanovinylcarbazole phosphoramidite, cholesteryl and psoralen.

70. The method of claim 67, wherein the 5' end of the TSO comprises at least 3 consecutive abasic sites.

71. The method of claim 67, wherein the TSO comprises at least one isodeoxycytosine (iso-dC), isodeoxyguanosine (iso-dG), or a combination of iso-dC and iso-dG at the 5' end.

72. The method of claim 67, wherein the TSO comprises SEQ ID NO:

3.

73. The method of claim 67, wherein the RT primer comprises SEQ ID NO:2.

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