Method for adding poly-a sequence

Abstract

The present invention addresses the problem of providing a novel method for suppressing the amount of a by-product derived from an unreacted oligo dT primer remaining after a reverse transcription reaction in cDNA amplification based on a Poly-A tagging method.　Provided is a method for adding a poly-A sequence onto the 3'-end side of a DNA strand, the method comprising: a reverse transcription step for obtaining a DNA strand extended to the 3'-end side by using, as a template, an RNA strand having a poly-A sequence on the 3'-end side thereof, and performing reverse transcription using a first oligo dT primer; and an addition step for adding a poly-A sequence onto the 3'-end side of the DNA strand using a terminal deoxynucleotidyl transferase or an active fragment thereof under conditions in which the intramolecular base pairing between a thymine base and an adenine base added to the 3'-end side of the first oligo dT primer that has not been extended to the 3'-end side in the reverse transcription step is stabilized, wherein the addition step involves adding the poly-A sequence in the presence of an ammonium cation and / or a modified adenine nucleotide, and / or at a temperature of at most 34ºC, and / or the first oligo dT primer used in the reverse transcription step includes a modified thymine nucleoside.

Description

PolyA sequence addition method

[0001] The present invention relates to a method for adding a polyA sequence, a method for determining a base sequence, a method for adding a polyT sequence, and a composition or kit for suppressing the production of by-products.

[0002] Transcriptome analysis is an analytical method for understanding biological processes based on the expression information of all genes. In recent years, single-cell RNA sequencing technology has been developed as a method for transcriptome analysis at the single-cell level, providing a comprehensive understanding of biological processes at the cellular level. Single-cell RNA sequencing technology can also identify the types and states of cells contained in organs without prior knowledge, and is becoming established as an extremely powerful analytical method.

[0003] RNA sequencing techniques targeting minute amounts of RNA, such as RNA derived from a single cell, require a cDNA amplification step prior to sequencing to ensure sufficient detection sensitivity. A typical mammalian cell expresses approximately 100,000 copies of mRNA per cell, but sequence analysis using a next-generation sequencer typically requires 1 to 10 billion copies of sequencing library DNA. Therefore, there is a discrepancy of more than 10,000 times between the copy number of the starting mRNA and the detectable copy number. Therefore, a cDNA amplification step is required to bridge this gap (Non-Patent Document 1).

[0004] PCR reactions are commonly used for cDNA amplification. In PCR-based cDNA amplification, a reverse transcription reaction is performed using an oligo-dT primer containing a specific adapter sequence to synthesize single-stranded cDNA containing the specific adapter sequence at the 5' end. The single-stranded cDNA obtained by such a reverse transcription reaction using an oligo-dT primer cannot be amplified as is by PCR or other methods because the adapter sequence is only attached to the 5' end. Therefore, cDNA conversion is required to convert the cDNA into an amplifiable form with adapter sequences at both ends. Currently, representative cDNA conversion methods include the "poly-A tagging method" (Non-Patent Documents 1, 3, 5) and the "template-switching method" (Non-Patent Documents 1, 10).

[0005] In the "Poly-A tagging method," a poly(A) sequence is added to the 3' end of single-stranded cDNA obtained by reverse transcription using terminal deoxynucleotidyl transferase (often abbreviated as "terminal transferase" or "TdT" herein) in a template-independent manner. Next, double-stranded cDNA is synthesized using an oligo-dT primer containing an adapter sequence at the 5' end, using the single-stranded cDNA obtained by adding the poly(A) sequence as a template. This results in double-stranded cDNA with adapter sequences at both ends, which can be amplified by PCR or other methods (Figure 1). The TdT-mediated addition of poly(A) sequences in the Poly-A tagging method is known to be extremely efficient, nearly 100% (Non-Patent Document 2).

[0006] The present inventors previously developed the Quartz-Seq and Quartz-Seq2 single-cell RNA sequencing technologies based on the poly-A tagging method described above (Non-Patent Documents 3 and 4). The Quartz-Seq2 method achieved a significant improvement in detection sensitivity by improving the efficiency of poly-A tagging by 3.6 times. Furthermore, in an international performance comparison study of single-cell RNA sequencing technologies, the Quartz-Seq2 method ranked first among 13 methods, demonstrating superior gene detection sensitivity of 1.5 to 5 times that of other methods (Non-Patent Document 5). In this performance comparison study, the inventors' Quartz-Seq2 method was the only method that used poly-A tagging, and these results strongly suggest the superiority of poly-A tagging.

[0007] Although the Poly-A tagging method is a cDNA conversion method with superior sensitivity, it still has some issues that need to be addressed. The oligo-dT primer used in the reverse transcription reaction itself is converted into amplifiable double-stranded DNA, resulting in the amplification of large amounts of by-product DNA in addition to the cDNA derived from the template mRNA (Figure 2A). This by-product DNA arises because the poly-A tail is added not only to the target RNA / cDNA hybrid molecule but also to the 3' end of the unreacted oligo-dT primer during the poly-A addition step after the reverse transcription reaction (Non-Patent Documents 3 and 6).

[0008] For example, a single cultured mammalian cell contains approximately 100,000 copies of target RNA. However, because the reverse transcription reaction uses approximately 10,000 to 670,000 times as many oligo-dT primers, a large amount of DNA derived from the oligo-dT primers that was not extended during the reverse transcription reaction is amplified. While it is possible to remove some of the unreacted oligo-dT primers through DNA purification, even if the amount is reduced to 1 / 100, more than 100 times the amount of oligo-dT primers remaining will remain (Non-Patent Documents 4 and 7). Since the DNA amplified from these remaining primers does not contain information derived from the RNA, it interferes with analysis using next-generation sequencers and must therefore be removed as much as possible (Non-Patent Document 8). TdT-induced DNA by-products tend to be short in size, and while this characteristic can sometimes be exploited to remove them using DNA purification techniques, it has also been reported that the size of the by-products is similar to that of cDNA, making them difficult to remove (Non-Patent Document 9).

[0009] To address the above-mentioned problems, the present inventors optimized a method for inhibiting PCR primer binding by placing complementary adapter sequences at both ends (sometimes referred to herein as "Suppression PCR") and demonstrated that it is possible to suppress the amount of by-product synthesis (Non-Patent Document 3). This method utilizes the fact that self-association between adapter sequences occurs preferentially in short nucleic acids, and can suppress amplification in the subsequent PCR reaction even if a poly-A sequence is attached to the reverse transcription primer. However, suppression PCR has the problem of limiting the sequences that can be used as adapters, particularly since both ends must be complementary, hindering its application to various techniques.

[0010] As such, methods have been adopted to suppress the amplification of amplification products derived from by-products of poly-A sequences added to reverse transcription primers, or to remove the amplified products by purification after amplification. However, a fundamental solution requires that the 3' end of the reverse transcription primer itself not be added with a poly-A sequence and thus not be converted into an amplifiable DNA molecule. Previously, a method was developed in which a certain proportion of dideoxynucleotides (ddNTPs) was added to the DNA strand, completely blocking the TdT-mediated elongation reaction when incorporated as the 3'-terminal nucleoside (Patent Document 1, Non-Patent Document 9). However, the ddNTPs, which inhibit PCR, must be removed after the TdT reaction. Therefore, this method is limited to use in reaction systems in which the reverse transcription primer is immobilized, and its application in liquid phase systems is limited. Furthermore, because ddNTPs are incorporated into both by-products and cDNA derived from the target RNA, poly-A and poly-C addition (homopolymerization) to both molecules is equally inhibited. Ideally, a method that only inhibits the addition of a poly-A tail to the 3' end of the reverse transcription primer itself is needed, but no such method has been reported to date.

[0011] WO2021 / 006353

[0012] Sasagawa Y., et al., Adv Exp Med Biol. (2019), 1129:1-17.Mickelsen S., et al., J Immunol. (1999), 163(2):834-43.Sasagawa Y., et al., Genome Biology (2013), 14:R31.Sasagawa Y., et al., Genome Biology (2018), 19:29.Mereu, E., et al., Nature Biotechnology (2020), 38:747-755.Kurimoto, K., et al., Nucleic Acids Res. (2006), 34(5):e42.Mutula K., et al., Adv Biosyst. (2020), 4(1):e1900188.Tang F., et al., Nat Methods. (2009), 6(5):377-82.Shichino S., et al., Commun Biol. (2022), 5(1):602.Wulf, MG, et al., J Biol Chem. (2019), 294(48): 18220-18231.

[0013] The objective of the present invention is to provide a new method for specifically suppressing the amount of by-products derived from unreacted oligo dT primers remaining after reverse transcription in cDNA amplification based on the Poly-A tagging method.

[0014] To solve the above problems, the present inventors focused on the difference in the 3'-terminal sequence between single-stranded cDNA after reverse transcription and unreacted oligo-dT primer. The 3'-terminal of single-stranded cDNA after reverse transcription consists of a sequence complementary to the template RNA and has an RNA / DNA hybrid structure. In contrast, the 3'-terminal of the unreacted oligo-dT primer is single-stranded DNA consisting of a poly-T sequence. When a poly-A sequence is added by terminal transferase, the poly-A sequence is ligated immediately after the poly-T sequence. As a result, intramolecular base pairing between an adenine base (hereinafter sometimes abbreviated as "A base") and a thymine base (hereinafter sometimes abbreviated as "T base") is thought to form an intramolecular double-stranded structure.

[0015] The activity of terminal transferase used for poly(A) addition is known to be significantly affected by the terminal structure of double-stranded DNA. Specifically, it has high activity for adding poly(A) to protruding 3'-ends, but significantly lower activity for recessed 3'-ends (Thomas, KR and Olivera BM, J. Biol. Chem. (1978), 253(2):424-9; Deng G. and Wu R., Nucleic Acids Res. (1981), 9(16):4173-88).

[0016] In general, intramolecular base pairing between A and T bases is weaker than that between G and C bases. Therefore, the inventors suspected that this intramolecular base pairing is insufficient under normal conditions. Therefore, the inventors conceived the idea of ​​performing poly(A) addition by terminal transferase under conditions that stabilize the intramolecular base pairing between A and T bases after reverse transcription. Under these conditions, when terminal transferase adds an A base to the 3' end of an unreacted oligo-dT primer, the added A base base pairs with the poly-T sequence within the same molecule, forming an intramolecular double-stranded structure at the 3' end and stably forming a recessed end. As a result, further poly(A) addition by terminal transferase is suppressed, which in turn suppresses subsequent complementary strand synthesis, thereby suppressing the generation of by-products (Figure 2A vs. Figure 2B, "TdT-mediated poly(A) tailing").

[0017] Based on this idea, the present inventors performed poly(A) addition using terminal transferase in the presence of various modified nucleotides or ammonium cations to promote stabilization of base pairing between A and T bases, or under low-temperature conditions. As a result, under these conditions, poly(A) addition to the 3' end of unreacted oligo(dT) primers was suppressed. Therefore, DNA amplification derived from unreacted oligo(dT) primers was suppressed. Meanwhile, amplification of reverse transcription products derived from RNA templates was not impaired. Unlike conventional suppression PCR methods, the present method does not impose restrictions on adapter sequences and does not inhibit cDNA amplification efficiency. Furthermore, it does not require a purification step to remove the modified nucleotides or chemicals added before PCR, nor does it require immobilization of reverse transcription primers. Therefore, it overcomes the problems of conventional methods.

[0018] The present invention is based on the above findings and provides the following: (1) A method for adding a polyA sequence to the 3'-end of a DNA strand, comprising: a reverse transcription step of performing reverse transcription using an RNA strand having a polyA sequence at its 3'-end as a template with a first oligo-dT primer to obtain a DNA strand extended to the 3'-end; and an addition step of adding the polyA sequence to the 3'-end of the DNA strand using terminal deoxynucleotidyl transferase or an active fragment thereof under conditions that stabilize intramolecular base pairing between an adenine base added to the 3'-end of the first oligo-dT primer that was not extended to the 3'-end in the reverse transcription step and a thymine base, wherein the addition step adds the polyA sequence in the presence of ammonium cations and / or modified adenine nucleotides and / or at a temperature of 34°C or lower, and / or the first oligo-dT primer used in the reverse transcription step contains a modified thymine nucleoside. (2) The method according to (1), wherein the RNA strand is mRNA and / or non-coding RNA having a polyA sequence added to the 3'-end. (3) The method according to (1), wherein the ammonium cation is a tetraalkylammonium cation. (4) The method according to (3), wherein the tetraalkylammonium cation is selected from the group consisting of tetramethylammonium cation, tetraethylammonium cation, tetrapropylammonium cation, tetrabutylammonium cation, tetrapentylammonium cation, and tetrahexylammonium cation. (5) The method according to (1), wherein the ammonium cation is 1 mM to 100 mM. (6) The method according to (1), wherein the modified adenine nucleotide and / or the modified thymine nucleoside comprises a bicyclic sugar moiety, a 2'-modified sugar moiety, and / or a modified nucleobase. (7) The method of (6), wherein the bicyclic sugar moiety comprises a methyleneoxy group bridging the 4' and 2' positions, and / or the 2'-modified sugar moiety comprises a 2'-O-methyl-modified sugar moiety, a 2'-fluoro-modified sugar moiety, a 2'-amine-modified sugar moiety, a 2'-azido-modified sugar moiety, and / or a 2'-O-methoxyethyl-modified sugar moiety.(8) The method according to (1), further comprising an RNA strand degradation step of degrading the RNA strand after the reverse transcription step using RNase H enzyme. (9) The method according to (1), further comprising a complementary strand synthesis step of synthesizing a complementary strand using a second oligo-dT primer, using the DNA strand to which the polyA sequence has been added in the addition step, as a template. (10) The method according to (9), wherein the first oligo-dT primer and the second oligo-dT primer contain the same or different adapter sequences on the 5'-end of the oligo-dT sequence. (11) The method according to (10), wherein the first oligo-dT primer and / or the second oligo-dT primer contain a barcode sequence of 8 to 200 bases in length between the adapter sequence and the oligo-dT sequence. (12) The method according to (10), wherein the first oligo-dT primer and / or the second oligo-dT primer does not contain a barcode sequence. (13) The method according to (10), further comprising an amplification step of amplifying double-stranded DNA composed of the DNA strand and the complementary strand after the complementary strand synthesis step using a pair of nucleic acid primers each containing a nucleotide sequence identical to the same or different adapter sequence at their 3'-ends to obtain an amplification product. (14) A method for determining the nucleotide sequence of an RNA strand having a polyA sequence at its 3'-end, comprising a nucleotide sequencing step of determining the nucleotide sequence of the amplification product obtained according to the method according to (13). (15) The method according to (14), wherein the RNA strand is derived from an isolated or cultured single cell, a cell population composed of multiple cells, a tissue, or an organ. (16) The method according to (15), wherein the method is the Quartz-Seq method or the Quartz-Seq2 method.(17) A method for adding a poly-T sequence to the 3'-end of a DNA strand, comprising: a reverse transcription step of performing reverse transcription using a first oligo dA primer and an RNA strand having a poly-U sequence at its 3'-end as a template to obtain a DNA strand extended at its 3'-end; and an addition step of adding the poly-T sequence to the 3'-end of the DNA strand using terminal deoxynucleotidyl transferase or an active fragment thereof under conditions that stabilize intramolecular base pairing between a thymine base added to the 3'-end of the first oligo dA primer that was not extended at its 3'-end in the reverse transcription step and an adenine base, wherein the addition step adds the poly-T sequence in the presence of ammonium cations and / or modified thymine nucleotides and / or at a temperature of 34°C or lower, and / or the first oligo dA primer used in the reverse transcription step contains a modified adenine nucleoside. (18) A composition or kit for suppressing the production of by-products caused by terminal deoxynucleotidyl transferase, wherein: (i) the by-products are oligo-dT primers that have not been extended to their 3' ends in a reverse transcription reaction, to which a poly-A sequence has been added, the composition or kit comprising a modified oligo-dT primer containing an ammonium cation, a modified adenine nucleotide, and / or a modified thymine nucleoside; and / or (ii) the by-products are oligo-dA primers that have not been extended to their 3' ends in a reverse transcription reaction, to which a poly-T sequence has been added, the composition or kit comprising a modified oligo-dA primer containing an ammonium cation, a modified thymine nucleotide, and / or a modified adenine nucleoside. This specification incorporates the disclosure of Japanese Patent Application No. 2024-118531, from which the present application claims priority.

[0019] According to the present invention, in cDNA amplification based on the Poly-A tagging method, the amount of by-products derived from unreacted oligo dT primers remaining after reverse transcription can be reduced.

[0020] Figure 1 shows the steps from reverse transcription to amplification in the Poly-A tagging method. Figure 2 compares the conventional Poly-A tagging method with the method of the present invention (Suppression tagging method). Figure 2A shows that in the conventional Poly-A tagging method, the polyA tail added to the 3' end of the oligo-dT primer by terminal transferase (TdT) is lengthened, and tagging is performed based on this polyA tail, resulting in amplification of by-products. Figure 2B schematically shows the reduction of by-products in the Suppression tagging method of the present invention. The Suppression tagging method of the present invention uses conditions that stabilize base pairing between A and T bases, thereby suppressing the lengthening of the polyA tail and thereby suppressing the amplification of by-products. Figure 3 shows the results of analyzing the size distribution of amplification products obtained after performing the steps from reverse transcription to amplification on total RNA. Figure 3A shows the results under control conditions. Figure 3B shows the results when some of the dATP in the TdT reaction solution used for poly(A) addition was replaced with 2'-fluoro-modified nucleotides (2'-fluoro-modified ATP). Figure 4 shows the results of analyzing the size distribution of amplification products obtained after reverse transcription and amplification of total RNA. Figure 4A shows the results under control conditions. Figure 4B shows the results when poly(A) addition was performed in the presence of tetramethylammonium chloride (TMAC). Figure 5 shows the results of evaluating the size distribution of amplification products when poly(A) addition was performed under various conditions during reverse transcription and amplification of total RNA. The vertical axis of the figure shows the ratio of by-products between 50 and 300 bp (noise, N) to cDNA between 300 and 9000 bp (signal, S) (N / S ratio) calculated based on the size distribution. The Z-score was calculated from the N / S ratio. A lower Z-score indicates a lower amount of by-products relative to the amount of cDNA. Figure 5A shows the Z-scores for the proportion of by-products (N / S ratio) when polyadenylation was performed in the presence of various modified nucleotides, while Figure 5B shows the results when polyadenylation was performed under low temperature conditions (30°C and 32.3°C) and control conditions (37.4°C).The bottom, inner line, and upper end of each box in each figure represent the first, median, and third quartile, respectively. Figure 6 shows the results of evaluating the size distribution of amplified products when poly(A) addition was performed in the presence of various concentrations of tetramethylammonium chloride (TMAC) during the process from reverse transcription to amplification of total RNA. The bottom, inner line, and upper end of each box in each figure represent the first, median, and third quartile, respectively. Figure 7 shows the results of evaluating the size distribution of amplified products when poly(A) addition was performed in the presence of various ammonium cations (TMAC, tetramethylammonium chloride; TEAC, tetraethylammonium chloride; TPrAC, tetrapropylammonium chloride; TBAC, tetrabutylammonium chloride) during the process from reverse transcription to amplification of total RNA. The bottom, inner line, and upper end of each box in each figure represent the first, median, and third quartile, respectively. Figure 8 shows the results of measuring the chain length of oligo-dT primers after poly(A) addition to the 3' end. Figure 8A shows the results without adding terminal transferase (TdT). Figure 8B shows the results with TdT added but without adding 2'-fluoro-modified nucleotides (2'-Fluoro-dATP) and tetramethylammonium chloride (TMAC). Figure 8C shows the results with adding TdT and 2'-fluoro-modified nucleotides. Figure 8D shows the results with adding TdT and TMAC. Figure 9 shows the results of evaluating the size distribution of the amplified products when reverse transcription was performed on total RNA using natural oligo-dT primer 1 (Control in the figure) or modified oligo-dT primer 1 (SuperT in the figure) during the process from reverse transcription to amplification. Figure 10 shows the results of measuring the chain length of natural oligo-dT primer 2 (Control in the figure) or modified oligo-dT primer 2 (SuperT in the figure) after adding poly(A). Figure 11 shows the results of verifying the effectiveness of the suppression tagging method when applied to various single-cell RNA sequencing technologies. Figure 11A shows the results using the 10x genomics single-cell RNA sequencing platform.Figure 11B shows the results of Split-seq.

[0021] 1. PolyA Sequence Addition Method 1-1. Overview A first aspect of the present invention is a method for adding a polyA sequence to the 3'-end of a DNA strand (hereinafter, may be referred to as the "polyA sequence addition method" or the like). The polyA sequence addition method of the present invention is characterized in that, when adding a polyA sequence to the 3'-end of a DNA strand obtained by reverse transcription, conditions are used that stabilize intramolecular base pairing between A bases and T bases. The polyA sequence addition method of the present invention can reduce the amount of by-products derived from unreacted oligo-dT primers remaining after the reverse transcription reaction in cDNA amplification.

[0022] 1-2. Definition of Terms In this specification, the term "nucleic acid chain" refers, in principle, to a polymer composed of nucleosides as structural units, which are linked by internucleoside bonds. Examples of nucleic acid chains include DNA chains composed of DNA nucleosides and RNA chains composed of RNA nucleosides.

[0023] As used herein, "polyA" or "polyA sequence" refers to a base sequence consisting of two or more consecutive A (adenine) bases. Examples include base sequences consisting of 3 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more consecutive A bases. There is no particular upper limit to the number of consecutive A bases constituting the polyA sequence, but it may be, for example, 5,000 or less, 4,000 or less, 3,000 or less, 2,000 or less, 1,000 or less, 500 or less, 200 or less, or 100 or less. Exemplary ranges for the length of consecutive A bases constituting the polyA sequence include lengths of 3 to 5,000 bases, 10 to 1,000 bases, 50 to 500 bases, and 100 to 300 bases.

[0024] As used herein, a "nucleic acid primer" or "primer" refers to a nucleic acid molecule that specifically binds to a portion of a nucleic acid strand and provides a starting point for a nucleic acid synthesis reaction catalyzed by a polymerase enzyme such as DNA polymerase or reverse transcriptase. A nucleic acid primer is usually single-stranded.

[0025] As used herein, an "oligo dT primer" refers to a primer containing two or more consecutive thymine (T) bases (referred to herein as an "oligo dT sequence") that can bind to a poly(A) sequence. Nucleosides containing two or more consecutive T bases in an oligo dT primer are, in principle, DNA nucleosides. The length of the oligo dT sequence is not particularly limited, as long as it can bind to an RNA strand having a poly(A) sequence at its 3'-end to provide a starting point for a reverse transcription reaction using the RNA strand as a template, and / or can bind to a DNA strand having a poly(A) sequence at its 3'-end to provide a starting point for a DNA strand synthesis reaction using the DNA strand as a template. Examples of the length include at least 3 bases, at least 5 bases, at least 10 bases, at least 12 bases, at least 15 bases, or at least 18 bases, and / or at most 50 bases, at most 40 bases, at most 30 bases, or at most 25 bases. Exemplary length ranges include 3 to 50 bases, 8 to 30 bases, 12 to 30 bases, and 15 to 20 bases. Typically, the oligo-dT sequence in an oligo-dT primer is located at its 3' end. An oligo-dT primer can consist solely of an oligo-dT sequence, but can also contain an adapter sequence and / or a barcode sequence, as described below, and can further contain a unique molecular identifier (UMI) sequence, which contains random bases or a random sequence in at least a portion and indicates that the primers have been amplified from the same or different molecules. In this specification, when distinguishing between the oligo-dT primer used in the reverse transcription step and the oligo-dT primer used in the complementary strand synthesis step, they may be referred to as the "first oligo-dT primer" and the "second oligo-dT primer," respectively.

[0026] As used herein, the term "adapter sequence" refers to a sequence that contains at least a sequence homologous to or identical to the nucleic acid primer used for nucleic acid amplification in the amplification step described below. Typically, the adapter sequence is configured to contain a base sequence identical to at least several bases (e.g., 8 to 30 bases, 12 to 20 bases, or 15 to 18 bases) located at the 3' end of the nucleic acid primer. The specific base sequence of the adapter sequence is not limited, and those skilled in the art can design an appropriate sequence taking into account nucleic acid amplification conditions such as annealing temperature. The base length of the adapter sequence is not limited as long as the nucleic acid primer can bind to its complementary sequence. An appropriate length may be selected depending on the length of the nucleic acid primer; however, the adapter sequence is typically 8 bases or more, 10 bases or more, 12 bases or more, 15 bases or more, or 18 bases or more, and / or 50 bases or less, 40 bases or less, 30 bases or less, or 25 bases or less. Exemplary ranges include 8 to 50 bases, 12 to 30 bases, and 18 to 25 bases.

[0027] As used herein, the term "barcode sequence" refers to a base sequence contained in a primer sequence for distinguishing between two or more samples from which an RNA strand is derived. For example, when performing the reverse transcription step described below on RNA strands derived from multiple single cells or multiple tissues using oligo-dT primers containing different barcode sequences for each sample, DNA strands derived from the same single cell or tissue will contain a common barcode sequence. As a result, even if nucleic acids from different samples are pooled and processed in subsequent steps, their origins can be identified based on the sequence information obtained by sequencing. The specific base sequences of barcode sequences are not limited as long as they are different from each other, and may be 1 to 3 bases long, although sequences of 4 bases or longer are typically used. For example, the barcode sequences may be 3 bases or longer, 5 bases or longer, 10 bases or longer, 12 bases or longer, 15 bases or longer, or 18 bases or longer, and / or 500 bases or shorter, 200 bases or shorter, 100 bases or shorter, 50 bases or shorter, or 25 bases or shorter. Exemplary ranges include 2-200 bases in length, 8-100 bases in length, 12-50 bases in length, and 20-30 bases in length.

[0028] As used herein, "terminal deoxynucleotidyl transferase" (also referred to as "terminal transferase," and often abbreviated as "TdT") refers to an enzyme that adds deoxynucleotides to the 3'-end of a nucleic acid chain. TdT can add deoxynucleotides to the 3'-end of both single-stranded and double-stranded DNA, but its activity toward double-stranded DNA is known to be significantly affected by the structure of the 3'-end. TdT exhibits high activity in adding poly(A) to protruding 3'-ends, but significantly lower activity in adding poly(A) to recessed 3'-ends (Thomas, KR and Olivera BM, J. Biol. Chem. (1978), 253(2):424-9; Deng G. and Wu R., Nucleic Acids Res. (1981), 9(16):4173-88). Furthermore, TdT can be added to the 3'-end of a nucleic acid chain regardless of the deoxynucleotide having any base (e.g., dATP, dTTP, dCTP, dGTP), but it is known to have particularly high activity toward deoxynucleotides having the A base (adenosine nucleotides). While the source of TdT is not particularly limited herein, bovine TdT (e.g., recombinant TdT derived from calf thymus) is often used in the art.

[0029] As used herein, the term "active fragment" of TdT refers to any fragment of TdT that has TdT activity, for example, a fragment that has 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of wild-type TdT, or an activity equivalent to or greater than these.

[0030] As used herein, the term "extension," when used with respect to a primer, refers to the addition of one or more nucleotides to the 3' end of the primer. Furthermore, as used herein with respect to a primer such as an oligo-dT primer, the term "unreacted" refers to the absence of any nucleotides added to the 3' end of the primer used in a polymerase reaction such as a reverse transcription reaction.

[0031] As used herein, the term "sample" refers to any sample containing an RNA strand that can be subjected to the poly(A) sequence addition method of the present invention or the nucleotide sequencing method described below. The sample can be derived from any subject, including any eukaryote, such as an animal, a plant, or yeast, or a prokaryote, such as bacteria (e.g., Escherichia coli) or archaea. The animal may be a mammal, such as a human, a primate, including a chimpanzee, a pet animal, such as a dog or cat, a livestock animal, such as a cow, a horse, a sheep, or a goat, a rodent, such as a mouse or a rat, or an animal kept in a zoo. Specific examples of samples include cells, tissues, cell extracts, tissue extracts, tissue fluids, body fluids, feces, and hair. The type of cell or tissue is not particularly limited. The cell may be a single cell or a cell population containing two or more cells. In addition to the above samples, samples may also be extracted or purified from these samples (extracts or purified products).

[0032] The polyA sequence addition method of the present invention includes a reverse transcription step and an addition step as essential steps, and includes an RNA extraction step, an RNA strand degradation step, a complementary strand synthesis step, and / or an amplification step as optional steps. Each step in the polyA sequence addition method of the present invention will be specifically described below.

[0033] (RNA extraction step) The "RNA extraction step" is a step of extracting an RNA strand from an arbitrary sample. This step is a selective step.

[0034] As used herein, "RNA" or "RNA strand" includes total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA, and synthetic RNA.

[0035] The method for extracting RNA from a sample in this step is not particularly limited. When extracting RNA from cells, RNA can be liberated and extracted by lysing the cells using a cell lysis reagent containing a surfactant. Alternatively, RNA can be extracted using the standard acid phenol method, or an RNA extraction reagent containing acid phenol can be used. RNA extraction reagents and RNA extraction kits are commercially available from various life science manufacturers, such as Qiagen, Takara Bio, Toyobo, Thermo Fisher Scientific, and Promega, and these can also be used. Furthermore, the RNA extracted can be purified as needed.

[0036] (Reverse transcription step) The "reverse transcription step" is a step in which an RNA strand having a polyA sequence at its 3' end is used as a template and reverse transcribed using a first oligo-dT primer to obtain a DNA strand extended toward the 3' end. This step produces a DNA strand in which a base sequence complementary to at least a portion of the RNA strand is linked to the 3' end of the first oligo-dT primer. For example, when mRNA is used as a template in this step, a cDNA strand containing a base sequence complementary to the base sequence other than the polyA of the mRNA is obtained. Furthermore, when non-coding RNA such as miRNA to which a polyA sequence has been added is used as a template in this step, a DNA strand containing a base sequence complementary to the non-coding RNA can be obtained.

[0037] The "RNA strand having a polyA sequence at its 3' end" used as a template for reverse transcription in this step may be any of the RNA strands described above. Examples include eukaryotic mRNA having a polyA sequence at its 3' end. RNA strands other than eukaryotic mRNA usually do not have a polyA sequence at their 3' end, but they can be subjected to this step by adding a polyA sequence to the 3' end of the RNA strand using, for example, a polyA polymerase derived from Escherichia coli or yeast. For example, non-coding RNA with a polyA sequence added to its 3' end can be subjected to this step.

[0038] The reverse transcription in this step can be carried out using any reverse transcriptase, typically in the presence of a combination of deoxyribonucleotides containing adenine (A), cytosine (C), guanine (G), and thymine (T) (sometimes referred to herein as "dNTPs"). The conditions for the reverse transcription reaction are not particularly limited. For example, reverse transcription can be carried out according to the reverse transcription method described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press. Reverse transcription can typically be carried out at a temperature of 35°C to 60°C for 5 minutes to 6 hours.

[0039] The amount of the first oligo-dT primer used in this step is not particularly limited, as long as it is greater than the amount of the RNA strand used as a template. The concentration of the first oligo-dT primer may be, for example, 0.1 nM or more, 1 nM or more, 10 nM or more, or 100 nM or more, and / or 100 μM or less, 10 μM or less, or 1 μM or less.

[0040] The amount of RNA strand used as a template in this step is also not limited, and may be, for example, 0.001 ng or more, 0.01 ng or more, or 0.1 ng or more, and / or 100 ng or less, 1 ng or less, or 0.1 ng or less.

[0041] (Addition Step) The "addition step" is a step in which a polyA sequence is added to the 3'-end of a DNA strand using TdT or an active fragment thereof. This step is characterized by being carried out under conditions that stabilize intramolecular base pairing between the A base and the T base. In this step, the A base added to the 3'-end of the first oligo-dT primer that was not extended to the 3'-end in the reverse transcription step described above forms a base pair with the T base constituting the first oligo-dT primer, and as a result, this extended strand forms a loop structure and self-associates, forming an intramolecular double-stranded structure, which can suppress further addition of polyA to the 3'-end by TdT.

[0042] As used herein, "intramolecular base pairing" refers to base pairing formed between two or more bases in the same nucleic acid strand. Examples include base pairing formed between an A base and a T base in the same nucleic acid strand, and base pairing formed between a G base and a C base in the same nucleic acid strand. In this step, intramolecular base pairing refers to base pairing between the T base constituting the first oligo-dT primer and the A base added to its 3'-end.

[0043] As used herein, "conditions under which intramolecular base pairing between an adenine (A) base and a thymine (T) base is stabilized" refer to conditions under which base pairing between an A base and a T base is stabilized and / or enhanced compared to conditions typically used for TdT reaction conditions. More specifically, the conditions may be such that base pairing between an A base and a T base is stabilized compared to base pairing between a G base and a C base. Preferably, the conditions are such that only base pairing between an A base and a T base is stabilized, and base pairing between a G base and a C base is not substantially stabilized.

[0044] In the art, the stability of base pairing between bases can be expressed as the melting temperature (Tm). The "melting temperature (Tm)" refers to the temperature at which 50% of a double-stranded nucleic acid population dissociates into single strands. Therefore, conditions under which base pairing between the aforementioned A base and T base is stabilized are, for example, conditions under which the melting temperature between a polynucleotide chain composed only of A bases and a polynucleotide chain composed only of T bases increases, including conditions under which the Tm value increases by 1°C or more (e.g., conditions under which the Tm value increases by 1°C to 10°C, 2°C to 9°C, 3°C to 8°C, 4°C to 7°C, or 5°C to 6°C). It is preferable that, under these conditions, the melting temperature between a polynucleotide chain composed only of G bases and a polynucleotide chain composed only of C bases does not substantially increase, or that the degree of increase is smaller than the degree of increase in the melting temperature between a polynucleotide chain composed only of A bases and a polynucleotide chain composed only of T bases.

[0045] In this step, specific examples of "conditions under which intramolecular base pairing between an adenine (A) base and a thymine (T) base is stabilized" include (i) a condition in which an ammonium cation is present, (ii) a condition in which a modified adenine nucleotide is present, (iii) a temperature condition of 34°C or less, and (iv) a condition in which the first oligo-dT primer contains a modified thymine nucleoside, as well as any combination of two or more of (i) to (iv).

[0046] As used herein, "ammonium cation" refers to an ammonium ion (NH4 + The ammonium cation may be any of a primary ammonium cation, a secondary ammonium cation, a tertiary ammonium cation, or a quaternary ammonium cation. The ammonium cation is preferably a quaternary ammonium cation, such as a tetraalkylammonium cation. Specific examples of tetraalkylammonium cations include tetramethylammonium cation, tetraethylammonium cation, tetrapropylammonium cation, tetrabutylammonium cation, tetrapentylammonium cation, and tetrahexylammonium cation. The ammonium cation can be used as an ammonium salt in combination with any anion such as chloride ion, fluoride ion, bromide ion, or iodide ion.

[0047] In the above condition (i), the concentration of ammonium cation is not limited as long as it stabilizes or enhances base pairing between A and T bases, but is preferably a concentration that does not suppress or inhibit polyA addition back to TdT, such as 1 nM or more, 10 nM or more, 100 nM or more, 1 μM or more, 0.01 mM or more, 0.1 mM or more, 0.2 mM or more, 0.5 mM or more, 1 mM or more, 2 mM or more, 5 mM or more, or 10 mM or more, and / or 200 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less, 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, or 20 mM or less. Exemplary concentration ranges for ammonium cations include 0.5 mM to 100 mM, 1 mM to 50 mM, or 1 mM to 100 mM, preferably 2 mM to 30 mM or 5 mM to 20 mM. Exemplary concentrations include 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, and 50 mM.

[0048] The condition (ii) above, in which modified adenine nucleotides are present, refers to the presence of modified adenine nucleotides in the TdT reaction solution used in this step, specifically modified adenosine triphosphate (modified dATP), which can serve as a TdT substrate. The concentration of modified adenine nucleotides is not limited and may be, for example, 1 nM or more, 10 nM or more, 100 nM or more, 1 μM or more, 0.01 mM or more, 0.1 mM or more, 0.2 mM or more, or 0.5 mM or more, and / or 100 mM or less, 10 mM or less, 5 mM or less, 2 mM or less, or 1 mM or less. Examples of the concentration range of modified adenine nucleotides include 10 nM to 50 mM, 100 nM to 10 mM, and 1 mM to 5 mM. When the TdT reaction solution used in this step contains modified adenine nucleotides, the reaction solution may or may not also contain unmodified dATP. For example, the ratio of modified adenine nucleotides to unmodified dATP may be 1% to 50%, 5% to 40%, or 10% to 30%. The type of modified adenine nucleotides may be a single type or a combination of multiple types.

[0049] As used herein, the term "modified adenine nucleotide" or "modified adenosine nucleotide" refers to a nucleotide having an adenine base as a base, and including a modified adenine base and / or a modified sugar moiety. The modified sugar moiety may be, for example, a bicyclic sugar moiety or a 2'-modified sugar moiety. Examples of modified adenine nucleotides include 2'-O-methyl-modified nucleotides having an adenine base, 2'-fluoro-modified nucleotides having an adenine base, 2'-O-methoxyethyl-modified nucleotides having an adenine base, and LNA nucleotides having an adenine base; as well as 2'NH2-dATP and dZTP.

[0050] As used herein, the term "modified adenine base" refers to an adenine base containing any modification group, preferably one that can improve the stability of base pairing with thymine base compared to natural adenine base. Specific examples of modified adenine bases include 2'-fluoro-modified adenine base and 2'-aminoadenine (2-Amino-dA) base.

[0051] As used herein, "modified sugar" refers to a sugar having a substitution and / or any change from a natural sugar moiety (i.e., a sugar moiety found in DNA (2'-H) or RNA (2'-OH)), and "sugar modification" refers to a substitution and / or any change from a natural sugar moiety.

[0052] As used herein, a "bicyclic sugar moiety" refers to a sugar in which the 2'- and 4'-carbon atoms are bridged by two or more atoms. Examples of bicyclic sugar moieties are known to those skilled in the art and include methyleneoxy (4'-CH2-O-2') BNA (also known as "LNA"), ethyleneoxy (4'-(CH2)2-O-2') BNA (also known as "ENA"), cEt BNA, cMOE BNA, AmNA, GuNA, etc. Modified nucleosides containing a bicyclic sugar moiety are sometimes referred to as bicyclic nucleosides or bridged nucleic acids (BNA).

[0053] As used herein, the term "2'-modified sugar moiety" refers to a furanosyl sugar modified at the 2'-position. Specific examples of the 2'-modified sugar moiety include a 2'-O-methyl-modified sugar moiety, a 2'-fluoro-modified sugar moiety, a 2'-amine-modified sugar moiety, a 2'-azide-modified sugar moiety, and a 2'-O-methoxyethyl-modified sugar moiety.

[0054] In the above condition (iii), the temperature condition of 34°C or less has no lower limit as long as it is a temperature at which TdT activity can be obtained, for example, 25°C or higher, 26°C or higher, 27°C or higher, 28°C or higher, 29°C or higher, 30°C or higher, or 31°C or higher, and / or 34°C or lower, 33°C or lower, or 32°C or lower, and exemplary temperature ranges include 29°C to 34°C, 30°C to 33°C, or 31°C to 32°C.

[0055] In the above condition (iv), the phrase "the first oligo-dT primer contains a modified thymine nucleoside" means that the first oligo-dT primer contains at least one modified thymine nucleoside. The number of modified thymine nucleosides contained in the first oligo-dT primer is not limited, and may be, for example, 1 or more, 2 or more, 3 or more, or 4 or more, and / or 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, or 5 or less. The position at which the modified thymine nucleoside is located in the first oligo-dT primer is not limited, and may be, for example, the 5' end and / or 3' end, a position other than the terminal (non-terminal position), or any combination thereof. Furthermore, the type of modified thymine nucleoside may be a single type, or multiple types may be combined.

[0056] Furthermore, as used herein, the term "modified thymine nucleotide" or "modified thymidine nucleotide" refers to a nucleotide having a thymine base as a base, which contains a modified thymine base and / or a modified sugar moiety. The modified sugar moiety may be, for example, a bicyclic sugar moiety or a 2'-modified sugar moiety. Examples of modified thymine nucleotides include 2'-O-methyl-modified nucleotides having a thymine base, 2'-fluoro-modified nucleotides having a thymine base, 2'-O-methoxyethyl-modified nucleotides having a thymine base, and LNA nucleotides having a thymine base.

[0057] As used herein, the term "modified thymine base" refers to a thymine base containing any modification group, preferably one that can stabilize base pairing with the A base compared to natural thymine bases. An example of a modified thymine base is 5-hydroxybutynl-2'-deoxyuridine (sometimes referred to as "Super T").

[0058] Conditions for this step other than those described above are not particularly limited, and any TdT reaction conditions known in the art can be used. Reaction conditions can be appropriately determined by referring to, for example, Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press. For example, in the presence of 0.5 mM to 10 mM unmodified dATP, a required amount of TdT or an active fragment thereof is added to the reaction solution, and the reaction can be carried out at a temperature of 34°C to 42°C for a period of several seconds to several hours, such as 10 seconds to 2 hours, 20 seconds to 1 hour, 30 seconds to 10 minutes, 40 seconds to 5 minutes, or 1 minute to 2 minutes, except for the conditions described in (iii) above.

[0059] (RNA strand degradation step) The "RNA strand degradation step" is a step in which the RNA strand after the reverse transcription step is degraded using RNase H enzyme. This step is a selective step. In this step, a heteroduplex formed by hybridization of a DNA strand extended on the 3' end side with a template RNA strand is recognized by RNase H enzyme, and the RNA strand is cleaved to obtain a single-stranded DNA strand.

[0060] This step can be carried out before, simultaneously with, or after the above-mentioned addition step. For example, when this step is carried out simultaneously with the addition step, RNase H enzyme can be added to the TdT reaction solution in the addition step to carry out the poly(A) sequence addition reaction and the RNA strand degradation reaction simultaneously.

[0061] (Complementary strand synthesis step) The "complementary strand synthesis step" is a step in which a complementary strand is synthesized using a second oligo-dT primer, with the DNA strand to which the poly-A sequence has been added in the addition step as a template. This step is selective. In this step, the second oligo-dT primer binds to the poly-A sequence added to the DNA strand, providing a starting point for complementary strand synthesis.

[0062] The complementary strand synthesis in this step can be carried out using any DNA polymerase in the presence of dNTPs. The reaction conditions are not particularly limited. For example, the reaction can be carried out according to the DNA polymerase reaction conditions described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press.

[0063] In one embodiment, the first and second oligo-dT primers contain the same or different adapter sequences, for example, at the 5' end of the oligo-dT sequence.

[0064] In another embodiment, the first oligo-dT primer does not contain an adapter sequence and the second oligo-dT primer does contain an adapter sequence. In yet another embodiment, the first oligo-dT primer contains an adapter sequence and the second oligo-dT primer does not contain an adapter sequence. In yet a further embodiment, the first oligo-dT primer and the second oligo-dT primer do not contain an adapter sequence.

[0065] In further embodiments, the first oligo-dT primer and / or the second oligo-dT primer contain a barcode sequence in addition to the same or different adapter sequence. For example, the first oligo-dT primer contains a barcode sequence and the second oligo-dT primer does not contain a barcode sequence, or the second oligo-dT primer contains a barcode sequence and the first oligo-dT primer does not contain a barcode sequence, or both the first oligo-dT primer and the second oligo-dT primer contain a barcode sequence. The position of the barcode sequence in the first oligo-dT primer and / or the second oligo-dT primer is not limited; for example, the barcode sequence may be located between the adapter sequence and the oligo-dT sequence. In certain embodiments, the first oligo-dT primer and / or the second oligo-dT primer contain, in order from the 5' end, an adapter sequence, a barcode sequence, and an oligo-dT sequence.

[0066] In yet another embodiment, the first oligo-dT primer and the second oligo-dT primer both contain an adapter sequence and do not contain a barcode sequence.

[0067] (Amplification step) The "amplification step" is a step of amplifying double-stranded DNA composed of the DNA strand and the complementary strand after the complementary strand synthesis step. This step is a selective step. In this step, amplification is performed using a pair of nucleic acid primers, each of which contains a base sequence homologous to or identical to the above-mentioned adapter sequence at its 3' end. The length of the nucleic acid primers used for amplification is usually 8 bases or more, 10 bases or more, 12 bases or more, 15 bases or more, or 18 bases or more, and / or 50 bases or less, 40 bases or less, 30 bases or less, or 25 bases or less. Exemplary ranges include 8 to 50 bases, 12 to 30 bases, and 18 to 25 bases.

[0068] Any known nucleic acid amplification method can be used in this step. As used herein, "nucleic acid amplification method" refers to a method in which nucleic acids are amplified by a nucleic acid polymerase, optionally using primers. Examples include PCR, NASBA, ICAN (isothermal gene amplification), LAMP (registered trademark), and RCA. While the nucleic acid amplification method used in the present invention is not limited, PCR is preferred, typically using a thermostable DNA polymerase. Each nucleic acid amplification method is known in the art, and can be performed using the conditions described in various protocols. Nucleic acid amplification kits are also commercially available from life science manufacturers, and these kits can also be used. In the amplification step, for example, PCR can be used to perform several amplification cycles (e.g., 1 to 40 cycles, 2 to 30 cycles, 3 to 20 cycles, 4 to 15 cycles, or 5 to 10 cycles).

[0069] 1-4. Effects: According to the poly(A) sequence addition method of the present invention, in unreacted oligo-dT primers that were not extended to the 3' end in the reverse transcription step, the A base added to the 3' end in the addition step can form a base pair with the T base constituting the oligo-dT sequence within the molecule. This is because the addition step is performed under conditions that stabilize intramolecular base pairing between the A base and the T base. Because terminal transferase activity is low at the recessed 3' end, the addition of additional A bases to the 3' end of the oligo-dT primer is suppressed by the intramolecular base pairing, and subsequent complementary strand synthesis is also suppressed. Therefore, the generation of by-products (double-stranded DNA derived from unreacted oligo-dT primers that were not extended to the 3' end in the reverse transcription step and amplification products amplified using the unreacted oligo-dT primers as templates) can be suppressed.

[0070] 2. Nucleotide Sequencing Method The second aspect of the present invention is a method for determining the nucleotide sequence of an RNA strand having a poly(A) sequence at the 3' end (hereinafter, sometimes referred to as the "nucleotide sequencing method" or the "sequencing method of the present invention").

[0071] The determination method of the present invention includes a nucleotide sequencing step of determining the nucleotide sequence of the amplification product obtained by the polyA sequence addition method of the first embodiment. In other words, the determination method of the present invention includes, as essential steps, the reverse transcription step, addition step, complementary strand synthesis step, and amplification step described in the first embodiment, as well as a nucleotide sequencing step. The determination method of the present invention also includes, as optional steps, an RNA extraction step and / or an RNA strand degradation step. In this embodiment, the reverse transcription step, addition step, complementary strand synthesis step, and amplification step are configured in accordance with the description of the first embodiment, and detailed description thereof will be omitted here.

[0072] The base sequencing method used in the base sequencing step may be any base sequencing method known in the art. Examples of base sequencing methods include the Sanger method (dideoxy method) and next-generation sequencing. Next-generation sequencing methods may be second-generation or third-generation sequencing methods. Examples of second-generation sequencing methods include pyrosequencing, synthetic sequencing, ligation sequencing, DNBSEQ, a DNA nanoball-based sequencing technology, and ion semiconductor sequencing. Examples of third-generation sequencing methods include sequencing methods using single-molecule sequencers or nanopore sequencers. These methods are known in the art and can be performed by referring to various protocol collections and methods disclosed on various company websites. Sequencers sold by various life science manufacturers, such as Illumina and Pacific Biosciences, can also be used. In determining the base sequences in this step, it is preferable to use a next-generation sequencing method that can simultaneously determine the base sequences of multiple nucleic acid molecules.

[0073] In this embodiment, the RNA strand subjected to the reverse transcription step is an RNA strand having a poly(A) sequence at the 3' end, but the origin of the RNA strand is not particularly limited, and it can be derived from, for example, a cell (e.g., a single cell), a cell population composed of multiple cells, a tissue, or an organ. The cell, cell population, tissue, or organ may be isolated or cultured. The cultured cell population, tissue, or organ may be an organoid.

[0074] In one embodiment, the determination method of the present invention is the Quartz-Seq method. As used herein, the "Quartz-Seq method" refers to a method for determining the base sequence of RNA in any sample, such as a single cell, a small number of cells, or a tissue, based on the method reported in Non-Patent Document 3, and is characterized herein by any one of the following (I) to (III), or any combination thereof, preferably all of them:

[0075] (I) The determination method of the present invention includes a primer degradation step, which is performed after the reverse transcription step and before the addition step, in which the first oligo-dT primer is degraded by an exonuclease such as exonuclease I. In this case, the concentration of the first oligo-dT primer used in the reverse transcription step is preferably low, for example, 10 μM or less, 1 μM or less, 0.1 μM or less, 10 nM or less, or 1 nM or less.

[0076] (II) The amplification step uses suppression PCR. Specifically, the first oligo-dT primer used in the reverse transcription step and the second oligo-dT primer used in the complementary strand synthesis step contain the same adapter sequence at the 5' end of the oligo-dT sequence, and amplification is performed using a pair of nucleic acid primers, each containing a base sequence complementary to this adapter sequence. When the amplification step is performed under these conditions, each strand constituting the double-stranded DNA derived from the unreacted oligo-dT primer that was not extended to the 3' end in the reverse transcription step contains the adapter sequence and its complementary sequence at both ends, and therefore, due to the short length of the double-stranded DNA, it is likely to bind to each other intramolecularly. Therefore, in the amplification step, the binding of the nucleic acid primer to the by-product is blocked, thereby suppressing the amplification of the by-product.

[0077] (III) The reaction time for adding the polyA sequence in the addition step is limited to a short time. For example, the reaction time is set to 10 minutes or less, 5 minutes or less, or 4 minutes or less, preferably 3 minutes or less, 2 minutes or less, or 1 minute or less. Exemplary reaction times include 5 seconds to 5 minutes, 10 seconds to 3 minutes, 30 seconds to 2 minutes, or 60 seconds to 90 seconds.

[0078] In an embodiment in which the determination method of the present invention is the Quartz-Seq method, in addition to the above (I) to (III), the amount of by-products produced can be further reduced by performing the additional step in the presence of topoisomerase V and / or using MightyAmp DNA polymerase (Takara Bio, Inc., Tokyo, Japan) or Terra PCR Direct Polymerase (Clontech, Mountain View, CA, USA) in the amplification step.

[0079] In one embodiment, the determination method of the present invention is the Quartz-Seq2 method. As used herein, the term "Quartz-Seq2 method" refers to a method for determining the base sequence of RNA in any sample, such as a single cell, a small number of cells, or a tissue, based on the method reported in Non-Patent Document 4. In this specification, when the above-mentioned Quartz-Seq method is performed on RNA derived from multiple samples (e.g., single cells), the first oligo-dT primer used in the reverse transcription step and / or the second oligo-dT primer used in the complementary strand synthesis step contain, in addition to the oligo-dT sequence and adapter sequence, a barcode sequence unique to each sample (e.g., each single cell) (a barcode sequence that differs for each sample or single cell). In this embodiment, the first oligo-dT primer and / or the second oligo-dT primer may further contain a UMI sequence that includes, at least in part, random bases or a random sequence.

[0080] In an embodiment in which the determination method of the present invention is the Quartz-Seq2 method, the determination method of the present invention can efficiently determine the base sequences of RNA strands derived from two or more single cells (e.g., 4 or more, 8 or more, 16 or more, 32 or more, 48 or more, 96 or more, or 384 or more). For example, single cells can be sorted one cell at a time into each well of a multi-well plate, such as a 96-well or 384-well plate, and an RNA extraction step can be performed independently in each well. Then, a reverse transcription step can be performed using a first oligo-dT primer containing a different barcode sequence for each well, and / or a complementary strand synthesis step can be performed using a second oligo-dT primer containing a different barcode sequence for each well. This allows for the assignment of a common barcode sequence among sequences derived from the same cell. DNA strands thus assigned with a barcode sequence can be pooled with DNA strands from different wells after the reverse transcription step or complementary strand synthesis step for subsequent steps, such as an amplification step, without losing information about the cell of origin, which is advantageous in terms of high throughput. Furthermore, in this embodiment, the amount of double-stranded DNA obtained after the amplification step can be increased by using a specific buffer (e.g., a buffer prepared by diluting ThermoPol (registered trademark) Reaction Buffer or RNase H Reaction Buffer manufactured by New England Biolabs by 0.55 times) for the polyA sequence addition reaction in the addition step.

[0081] The sequencing method of the present invention enables efficient base sequencing by suppressing the generation of by-products (double-stranded DNA derived from unreacted oligo-dT primers that were not extended to the 3' end in the reverse transcription step). For example, by applying the sequencing method of the present invention to the Quartz-Seq or Quartz-Seq2 method, single-cell RNA sequencing can be performed with higher efficiency than the methods reported in Non-Patent Documents 3 and 4.

[0082] 3. Poly-T Sequence Addition Method 3-1. Overview A third aspect of the present invention is a method for adding a poly-T sequence to the 3'-end of a DNA strand (hereinafter, may be referred to as the "poly-T sequence addition method" or the like). The poly-T sequence addition method of the present invention is characterized in that, when adding a poly-T sequence to the 3'-end of a DNA strand obtained by reverse transcription, conditions are used that stabilize intramolecular base pairing between T bases and A bases. The poly-T sequence addition method of the present invention can reduce the amount of by-products derived from unreacted oligo dA primers remaining after the reverse transcription reaction in cDNA amplification.

[0083] 3-2. Definition of Terms As used herein, "poly-U" or "poly-U sequence" refers to a base sequence consisting of two or more consecutive U (uracil) bases. Examples include base sequences consisting of 3 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, or 40 or more consecutive U bases. The number of U bases constituting the poly-U sequence is not particularly limited, but may be, for example, 5,000 or less, 4,000 or less, 3,000 or less, 2,000 or less, 1,000 or less, 500 or less, 200 or less, or 100 or less. Exemplary ranges for the length of consecutive U bases constituting the poly-U sequence include lengths of 3 to 5,000 bases, 10 to 1,000 bases, 50 to 500 bases, and 100 to 300 bases.

[0084] Furthermore, the term "poly T" or "poly T sequence" refers to a base sequence consisting of two or more consecutive T (thymine) bases. The number of T bases in a poly T sequence conforms to the above-mentioned range for the number of consecutive U bases in a poly U sequence, and exemplary ranges for the number of consecutive T bases constituting a poly T sequence include a length of 3 to 5,000 bases, a length of 10 to 1,000 bases, a length of 50 to 500 bases, and a length of 100 to 300 bases.

[0085] As used herein, an "oligo dA primer" refers to a primer containing two or more consecutive A bases (referred to herein as an "oligo dA sequence") that can bind to a poly-U sequence located at the 3'-end of an RNA strand and / or a poly-T sequence located at the 3'-end of a DNA strand. The base length of the oligo dA sequence is not particularly limited as long as it can bind to an RNA strand having a poly-U sequence at its 3'-end to provide a starting point for a reverse transcription reaction using the RNA strand as a template and / or can bind to a DNA strand having a poly-T sequence at its 3'-end to provide a starting point for a complementary strand synthesis reaction using the DNA strand as a template. For example, the oligo dA sequence may be 3 or more bases long, 5 or more bases long, 10 or more bases long, 12 or more bases long, 15 or more bases long, or 18 or more bases long, and / or 50 or less bases long, 40 or less bases long, 30 or less bases long, or 25 or less bases long. Exemplary length ranges include 3 to 50 bases, 8 to 30 bases, 12 to 30 bases, and 15 to 20 bases. Typically, the oligo dA sequence is located at the 3' end of an oligo dA primer. An oligo dA primer may consist of only an oligo dA sequence, but may also contain the adapter sequence and / or barcode sequence described above. In this specification, the oligo dA primer used in the reverse transcription step and the oligo dA primer used in the complementary strand synthesis step may be referred to as the "first oligo dA primer" and the "second oligo dA primer," respectively, to distinguish them from each other.

[0086] The poly-T sequence addition method of the present invention includes a reverse transcription step and an addition step as essential steps, and includes an RNA extraction step, an RNA strand degradation step, a complementary strand synthesis step, and / or an amplification step as optional steps. Each step in the poly-T sequence addition method of the present invention will be described below.

[0087] (RNA Extraction Step) In this embodiment, the RNA extraction step conforms to the configuration of the RNA extraction step in the first embodiment, and therefore a detailed description thereof will be omitted here.

[0088] (Reverse Transcription Step) In this embodiment, the reverse transcription step is a step of obtaining a DNA strand extended toward the 3' end by performing reverse transcription using a first oligo dA primer with an RNA strand having a poly U sequence at its 3' end as a template. This step obtains a DNA strand in which a base sequence complementary to at least a portion of the RNA strand is linked to the 3' end of the first oligo dA primer. An example of the "RNA strand having a poly U sequence at its 3' end" used as a template for reverse transcription in this step is one obtained by adding a poly U sequence to any RNA strand. Note that the poly U sequence can be obtained by adding a poly A sequence to the 3' end of an RNA strand using, for example, poly U polymerase. The rest of the configuration of this step is similar to the description of the reverse transcription step in the first embodiment, and therefore will not be described here.

[0089] (Addition Step) In this embodiment, the addition step is a step of adding a poly-T sequence to the 3'-end of a DNA strand using TdT or an active fragment thereof. This step is characterized by being carried out under conditions that stabilize intramolecular base pairing between the T base and the A base. In this step, the T base added to the 3'-end of the oligo dA primer that was not extended to the 3'-end in the above-mentioned reverse transcription step forms a base pair with the A base constituting the oligo dA primer, forming a loop structure and self-associating to form an intramolecular double-stranded structure, thereby suppressing the addition of additional T bases to the 3'-end by TdT.

[0090] In this step, specific examples of "conditions under which intramolecular base pairing between thymine (T) and adenine (A) bases is stabilized" include (i) the presence of ammonium cations, (ii) the presence of modified thymine nucleotides, (iii) a temperature of 34°C or less, and (iv) the first oligo dA primer containing a modified adenine nucleoside, as well as any combination of two or more of (i) to (iv). Conditions (i) and (iii) are similar to those described in the first embodiment, and therefore will not be described here.

[0091] In this embodiment, the condition (ii) above, in which modified thymine nucleotides are present, refers to the presence of modified thymine nucleotides in the TdT reaction solution used in this step, specifically modified thymidine triphosphate (modified dTTP), which can serve as a TdT substrate. The concentration of the modified thymine nucleotides is not limited and may be, for example, 1 nM or more, 10 nM or more, 100 nM or more, 1 μM or more, 0.01 mM or more, 0.1 mM or more, 0.2 mM or more, or 0.5 mM or more, and / or 100 mM or less, 10 mM or less, 5 mM or less, 2 mM or less, or 1 mM or less. Examples of the concentration range for the modified thymine nucleotides include 10 nM to 50 mM, 100 nM to 10 mM, and 1 mM to 5 mM. When the TdT reaction solution used in this step contains modified thymine nucleotides, the reaction solution may or may not also contain unmodified dTTP. For example, the ratio of modified thymine nucleotides to unmodified dTTP may be 1% to 50%, 5% to 40%, or 10% to 30%. The type of modified thymine nucleotide may be a single type or a combination of multiple types.

[0092] In the above condition (iv) of this embodiment, the first oligo dA primer "contains a modified adenine nucleoside" means that the first oligo dA primer contains at least one modified adenine nucleoside. The number of modified adenine nucleosides contained in the first oligo dA primer is not limited, and may be, for example, 1 or more, 2 or more, 3 or more, or 4 or more, and / or 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, or 5 or less. The position at which the modified adenine nucleoside is located in the first oligo dA primer is not limited, and may be, for example, the 5' end and / or 3' end, a position other than the terminal (non-terminal position), or any combination thereof. Furthermore, the type of modified adenine nucleoside may be a single type, or multiple types may be combined.

[0093] The conditions for this step other than those described above are not particularly limited, and any TdT reaction conditions known in the art can be used. For example, reaction conditions can be appropriately determined by referring to Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press. For example, the required amount of TdT or an active fragment thereof can be added to the reaction solution in the presence of 0.5 mM to 10 mM unmodified dTTP, and the reaction can be carried out for several tens of seconds to several hours at a temperature of 34°C to 42°C, other than the condition (iii) above.

[0094] (RNA strand degradation step) In this embodiment, the RNA strand degradation step conforms to the configuration of the RNA strand degradation step in the first embodiment, and therefore a detailed description thereof will be omitted here.

[0095] (Complementary strand synthesis step) In this embodiment, the complementary strand synthesis step is a step of synthesizing a complementary strand using the DNA strand to which the poly-T sequence has been added in the addition step as a template, and a second oligo dA primer. In this step, the second oligo dA primer binds to the poly-T sequence added to the DNA strand, providing a starting point for complementary strand synthesis.

[0096] The complementary strand synthesis in this step can be carried out using any DNA polymerase in the presence of dNTPs. The reaction conditions are not particularly limited. For example, the reaction can be carried out according to the DNA polymerase reaction conditions described in Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press.

[0097] In one embodiment, the first oligo dA primer and the second oligo dA primer contain the same adapter sequence at the 5' end of the oligo dT sequence.

[0098] In another embodiment, the first oligo dA primer and the second oligo dA primer contain different adapter sequences at the 5' end of the oligo dA sequence.

[0099] In a further embodiment, the first oligo dA primer and / or the second oligo dA primer comprises a barcode sequence of 8 to 200 bases in length between the adapter sequence and the oligo dT sequence.

[0100] In another further embodiment, the first oligo dA primer and / or the second oligo dA primer does not comprise a barcode sequence.

[0101] (Amplification step) In this embodiment, the amplification step is a step of amplifying double-stranded DNA composed of the DNA strand and the complementary strand after the complementary strand synthesis step. In this step, amplification is carried out using a pair of nucleic acid primers, each of which contains a base sequence identical to the above-mentioned adapter sequence at its 3' end. The specific configuration of this step is similar to the amplification step in the first embodiment, so a detailed description will be omitted here.

[0102] 3-4. Effect The poly T sequence addition method of this embodiment, like the poly A sequence addition method of the first embodiment, can suppress the generation of by-products (double-stranded DNA derived from unreacted oligo dA primer that was not extended to the 3' end in the reverse transcription step, and the amplification products obtained by amplifying the double-stranded DNA).

[0103] The present invention also provides a method for determining the nucleotide sequence of an RNA strand having a poly-U sequence at its 3'-end. This method includes a nucleotide sequencing step of determining the nucleotide sequence of an amplification product obtained by any of the above-mentioned poly-T sequence addition methods, and can be configured in the same manner as the method of the second aspect. This method allows the nucleotide sequence of an RNA strand having a poly-U sequence at its 3'-end to be determined.

[0104] 4. Composition or Kit 4-1. Overview A fourth aspect of the present invention is a composition or kit for inhibiting the production of by-products caused by terminal deoxynucleotidyl transferase (hereinafter often referred to as the "composition or kit of the present invention").

[0105] In this embodiment, the "by-product of terminal deoxynucleotidyl transferase" refers to (A) an oligo-dT primer that was not extended to its 3' end in the reverse transcription reaction, to which a poly-A sequence has been added, and / or (B) an oligo-dT primer that was not extended to its 3' end in the reverse transcription reaction, to which a poly-A sequence has been added.

[0106] 4-2. Configuration In one embodiment, the composition or kit of the present invention is intended to suppress the production of the by-product (A) above. In this embodiment, the composition or kit of the present invention comprises, as essential components, a modified oligo-dT primer containing an ammonium cation, a modified adenine nucleotide (e.g., modified dATP), and / or a modified thymine nucleoside.

[0107] In another embodiment, the composition or kit of the present invention is for suppressing the production of the by-product (B) above. In this embodiment, the composition or kit of the present invention comprises, as essential components, a modified oligo dA primer containing an ammonium cation, a modified thymine nucleotide (e.g., modified dTTP), and / or a modified adenine nucleoside.

[0108] One embodiment of the composition or kit of the present invention is a composition. In addition to the essential components described above, the composition may optionally contain a solvent and / or additives. The solvent may be, for example, water, an aqueous solution, or an organic solvent. The aqueous solution may be, for example, physiological saline, phosphate buffer, sodium acetate buffer, Tris buffer, etc. Examples of additives include chelating agents, pH adjusters, suspending agents, surfactants, stabilizers, excipients, preservatives, diluents, isotonicity agents, buffers, and solubilizing agents. In particular, chelating agents such as EDTA and EGTA are preferred for stabilizing nucleic acid primers because they can inactivate trace amounts of nucleases when they are present.

[0109] One embodiment of the composition or kit of the present invention is a kit, which may contain, in addition to the above-mentioned essential components, other reagents necessary for RNA extraction, reverse transcription, TdT reaction, complementary strand synthesis, nucleic acid amplification, and / or base sequencing, such as reverse transcriptase, TdT or an active fragment thereof, DNA polymerase (e.g., thermostable DNA polymerase), buffer (e.g., the above-mentioned T55 buffer), dNTP, ATP, DTT, additives, and / or instructions describing how to use the kit.

[0110] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0111] Example 1: Development of Suppression Tagging Method (Objective) As mentioned above, an unresolved problem with the Poly-A tagging method is that the oligo-dT primer used in the reverse transcription reaction remains unreacted, resulting in terminal transferase adding a poly-A sequence to the 3' end of the unreacted oligo-dT primer. Furthermore, complementary strand synthesis using this as a template results in the generation of amplifiable double-stranded DNA that does not contain mRNA-derived sequences as a by-product (Figure 2A). In this example, based on the concept described above in this specification, we performed poly-A addition by terminal transferase under various conditions that stabilize intramolecular base pairing between A and T bases, and verified that the proportion of by-products can be effectively reduced.

[0112] (Method) As a control condition in this example, the steps from reverse transcription to amplification were carried out according to the methods described in (1) to (5) below.

[0113] (1) Reverse transcription: Reverse transcription was performed using total RNA prepared from human iPS cells and Universal Mouse Reference RNA (UMRR) as templates with an oligo-dT primer. Specifically, 10 μL of RT premix containing 2× ThermoPol buffer, SuperScript III (1.25 U / μL), and RNasin Plus (0.137 U / μL) was added to 10 μL of lysis solution containing total RNA (1 ng), NP-40 (0.3%), dNTPs (0.12 mM), oligo-dT primer for reverse transcription (0.111 μM), and RNasin Plus (1 U / μL). The sequence of the oligo-dT primer for reverse transcription was 5'-TATAGAATTCGCGGCCGCTCGCGATACnnnnnnnnnnnnnnnnNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 1; "n" and "N" each represent a random base assigned with a 25% probability).

[0114] The resulting mixture was incubated at 35° C. for 5 minutes, and then maintained at 50° C. for 50 minutes to carry out reverse transcription. After the reaction, the reaction solution was purified using Zymo DNA Clean & Concentrator-5.

[0115] (2) Poly-A tailing: 20 μL of the purified cDNA solution was mixed with 25 μL of TdT premix containing 1× ThermoPol buffer, dATP (2.4 mM), RNase H (0.0384 U / μL), and terminal transferase (TdT) (Calf Thymus, recombinant, terminal transferase; Roche) (26.88 units / μL). The mixture was then incubated at 37°C or 37.4°C for 75 seconds to allow for RNA degradation by RNase H and poly-A tailing. The reaction mixture was then inactivated by incubating at 65°C for 10 minutes.

[0116] (3) Synthesis of complementary strand: 46 μL of tagging premix containing 1.084× Mighty amp buffer version 2, tagging oligo-dT primer (0.0693 μM), and Mighty amp (0.0541 U / μL) was added to 11 μL of the inactivated reaction mixture, mixed, and denatured at 98°C. The mixture was then cooled to 40°C, heated from 40°C to 68°C at a rate of 0.2°C / sec, and incubated at 68°C for 5 minutes to synthesize complementary strand. The sequence of the tagging oligo-dT primer was 5'-TATAGAATTCGCGGCCGCTCGCGATTTTTTTTTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 2).

[0117] (4) PCR amplification: 50 μL of PCR premix containing 1× mighty amp buffer version 2 and an amplification primer (1.895 μM) was added to the reaction mixture after complementary strand synthesis, and a PCR reaction was performed. The amplification primer was 5'-GTATAGAATTCGCGGCCGCTCGCGAT-3' (SEQ ID NO: 3), and the number of amplification cycles was 12. The amplification product after the amplification reaction was purified using Qiagen MinElute and Ampure XP.

[0118] (5) Size Distribution Analysis: The size distribution of the purified amplified products was analyzed using the Bioanalyzer 2100 High-sensitivity kit. Based on the size distribution obtained, nucleic acids between 50 bp and 300 bp were considered as by-products (noise, N) and nucleic acids between 300 bp and 9000 bp were considered as cDNA (signal, S). The signal-to-noise ratio (N / S ratio) was calculated based on the sum of the fluorescence intensity values ​​in each range. Furthermore, a Z-score was calculated from the N / S ratio based on the results of simultaneous experiments under each condition.

[0119] (6) Poly(A) Addition Conditions in the Suppression Tagging Method: When the poly(A) addition described in (2) above was performed in the presence of modified nucleotides, a TdT premix in which 25% or 10% of the dATP was substituted with various modified nucleotides containing adenine bases was used in the reaction described in (2). When the poly(A) addition described in (2) above was performed in the presence of ammonium cations, a TdT premix supplemented with 10 mM to 100 mM ammonium cations was used in the reaction described in (2). Furthermore, when the poly(A) addition described in (2) above was performed under low-temperature conditions, the 75-second incubation in the reaction described in (2) above was carried out at 30°C or 32.3°C. Other conditions were the same as those in the methods described in (1) to (5) above.

[0120] (Results) Figure 3B shows the results when 25% of the dATP in the TdT premix used for poly(A) addition was replaced with 2'-fluoro-modified nucleotides (2'-fluoro-modified ATP). Figure 4B shows the results when 10 mM tetramethylammonium chloride (TMAC) was added to the TdT premix used for poly(A) addition. In both cases, the peaks corresponding to by-products in the 50-300 bp range observed under control conditions (Figures 3A and 4A) were significantly reduced when poly(A) addition was performed in the presence of modified nucleotides or TMAC, but the amount of cDNA amplified in the 300-9000 bp range was equal to or greater than that under the control conditions.

[0121] Figure 5A shows the Z-scores of the ratio of by-products (N / S ratios) generated when polyadenosyltransferase (PA) was added in the presence of various modified nucleotides. A lower Z-score indicates a lower ratio of by-products (N) to cDNA (S). Figure 5A shows that polyadenosyltransferase (PA) added in the presence of Z-modified ATP (dZTP; 2-amino-2′-deoxyadenosine-5′-triphosphate), 2′-azido-ATP (2′-azido-ATP), 2′-amine-modified ATP (2′-NH2-dATP), 2′-methyl-modified ATP (2′-OMe-ATP), 2′-fluoro-modified ATP (2′-Fluoro-dATP), and cross-linked ATP (adenine-containing LNA nucleotide; LNA-ATP) resulted in lower Z-scores, indicating reduced amplification of by-products, compared with polyadenosyltransferase (PA) added in the absence of modified ATP.

[0122] Figure 5B shows the results of polyA addition under low-temperature conditions. Compared with the control condition of polyA addition at 37.4°C, the Z-scores were lower when polyA addition was performed at low temperatures of 30°C and 32.3°C, indicating reduced amplification of by-products.

[0123] Figure 6 shows the results of the Z-score evaluation of the proportion of by-products generated when poly(A) addition was performed in the presence of various concentrations of TMAC. The results in Figure 6 show that when poly(A) addition was performed in solutions containing TMAC at concentrations of 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, and 100 mM, the Z-scores were lower than when poly(A) addition was performed in the absence of TMAC, indicating that the amount of by-product amplification was reduced. In this experiment, TMAC remained in the reaction solution after poly(A) addition, but did not inhibit PCR amplification in subsequent steps, demonstrating that it is not necessary to remove TMAC after poly(A) addition.

[0124] Figure 7 shows the results of evaluating the proportion of by-products when poly(A) addition was performed in the presence of various ammonium cations, based on Z scores. The results in Figure 7 show that poly(A) addition performed in the presence of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride (TPrAC), or tetrabutylammonium chloride (TBAC) resulted in lower Z scores, indicating a reduced amount of by-product amplification, compared to poly(A) addition performed in the absence of ammonium cations.

[0125] Furthermore, the above results also demonstrate that the generation of by-products can be significantly suppressed without removing the oligo-dT primer from the reaction solution before PCR amplification, demonstrating that this method is highly convenient.

[0126] Example 2: Evaluation of poly(A) chain length (Objective) To evaluate the chain length after poly(A) addition to an oligo-dT primer in the presence of 2'-fluoro-modified ATP (2'-Fluoro-dATP) or tetramethylammonium chloride (TMAC).

[0127] (Method) Poly(A) was added to a 24-mer oligo(dT) primer (5'-[FAM]TTTTTTTTTTTTTTTTTTTTTTTTTT-3', SEQ ID NO: 4) fluorescently labeled at the 5' end by the following method: A poly(A) tailing solution containing potassium acetate (20 mM), Tris-acetate (8 mM, pH 7.9), magnesium acetate (4 mM), DTT (0.4 mM), oligo(dT) primer (0.3 μM), terminal transferase (TdT) (14.93 U / μL), and dATP (1.33 mM) was prepared. The reaction was carried out at 37°C for 7 minutes, followed by incubation at 65°C for 10 minutes to inactivate the enzyme.

[0128] When poly(A) addition was performed in the presence of 2'-fluoro-dATP, dATP in the Poly-A tailing solution was replaced with 1.33 mM 2'-fluoro-dATP, and when poly(A) addition was performed in the presence of tetramethylammonium chloride (TMAC), 10 mM TMAC was added to the Poly-A tailing solution.

[0129] The reaction mixture after inactivation was diluted with nuclease-free water, and a size standard (120 LIZ) was added. The chain length of the nucleic acid in the reaction mixture was measured using an ABI 3130xl (ThermoFisher Scientific). The measurement results showed that 24-base lengths correspond to unreacted oligo-dT primers, while nucleic acid chains greater than 24 bases but less than 48 bases in length correspond to those that can self-associate to form recessed ends. Nucleic acid chains greater than 48 bases in length correspond to those that can form protruding ends.

[0130] (Results) The results of poly(A) addition in the absence of 2'-fluoro-modified ATP and TMAC are shown in Figure 8B. It was revealed that most of the poly(A)-added nucleic acid strands were significantly longer than 48 bases, and were elongated to 100-200 bases or more.

[0131] Next, the results in the presence of 2'-fluoro-modified ATP or TMAC are shown in Figures 8C and 8D, respectively. Under both conditions, most of the nucleic acid strands were shorter than 48 bases, and elongated oligo-dT primers were hardly observed. These results demonstrate that poly(A) addition to oligo-dT primers was significantly suppressed in the presence of 2'-fluoro-modified ATP or TMAC.

[0132] Example 3: Suppression tagging method based on modified oligo-dT primers (Objective) Base pairing between A and T bases can also be stabilized by modified nucleosides containing T bases. Therefore, in this example, poly(A) addition is performed on oligo-dT primers consisting of natural nucleosides and oligo-dT primers containing modified nucleosides, and the chain lengths of the nucleic acid chains obtained in each case are compared.

[0133] (Methods and Results) (1) Preparation of Oligo dT Primer In this example, 5-hydroxybutynl-2'-deoxyuridine (hereinafter referred to as "Super T") was used as the modified nucleoside introduced into the oligo dT primer. Super T is the base moiety in the nucleoside shown in Formula I below, and base pairs with the A base. It is known that the Tm increases by approximately 2°C for each replacement of one natural T base in an oligonucleotide with Super T.

[0134]

[0135] The sequences of the oligo dT primers compared in this example are shown in Table 1 below.

[0136]

[0137] Natural oligo-dT primer 1 is composed of an adapter sequence, a barcode sequence, and a 24-base-long natural T base. Modified oligo-dT primer 1 is composed of an adapter sequence, a barcode sequence, and a 24-base-long T base, and in the oligo-dT region composed of 24 T bases, the 2nd, 8th, 14th, and 20th bases are Super T bases, and all other bases are composed of natural T bases.

[0138] The natural oligo dT primer 2 is 24 bases long and consists of natural T bases throughout, with the 5' end fluorescently labeled. The modified oligo dT primer 2 is 24 bases long, with the 2nd, 8th, 14th, and 20th bases from the 5' end being Super T bases, and all other bases are natural T bases, with the 5' end fluorescently labeled.

[0139] (2) By-product suppression effect in whole transcript amplification 1 ng of total RNA was subjected to the steps from reverse transcription to amplification by the method described in (1) to (5) of Example 1. However, the oligo dT primer used in (1) of Example 1 was the natural oligo dT primer 1 or modified oligo dT primer 1 described above.

[0140] The size distribution of the amplified nucleic acids was analyzed using the Bioanalyzer 2100 High-sensitivity kit, and the results are shown in Figure 11. When reverse transcription was performed using modified oligo dT primer 1 (Figure 9, "Super T"), the amount of by-products with lengths of 100 to 500 bases was significantly reduced, while the amount of cDNA with lengths of 500 to 7000 bases was increased, compared to when reverse transcription was performed using natural oligo dT primer 1 (Figure 9, "Control").

[0141] (3) Evaluation of chain length after poly(A) addition. Poly(A) was added to either the native oligo(dT) primer 2 or the modified oligo(dT) primer 2 as follows. A poly(A) tailing solution containing potassium acetate (20 mM), Tris-acetate (8 mM, pH 7.9), magnesium acetate (4 mM), DTT (0.4 mM), oligo(dT) primer (0.3 μM), terminal transferase (TdT) (14.93 U / μL), and dATP (1.33 mM) was prepared and reacted at 37°C for 7 minutes. The enzyme was then inactivated by incubation at 65°C for 10 minutes. The reaction mixture was diluted with nuclease-free water, a size standard (120 L / L) was added, and the chain length of the nucleic acid in the reaction mixture was measured using an ABI 3130xL (ThermoFisher Scientific).

[0142] The measurement results are shown in Figure 10. With natural oligo dT primer 2, many of the poly(A)-added nucleic acid chains were significantly longer than 48 bases, and a large amount of elongated nucleic acids was produced. On the other hand, with modified oligo dT primer 2, poly(A) addition was significantly suppressed, and the production of elongated nucleic acids was significantly reduced.

[0143] Example 4: Application to single-cell RNA sequencing (Objective) The suppression tagging method of the present invention will be applied to the 10x genomics single-cell RNA sequencing platform and single-cell RNA sequencing using Split-seq, and the effect on the size distribution of amplified cDNA will be examined.

[0144] (Methods and Results) (1) 10x genomics' single-cell RNA sequencing platform: 10x genomics' Chromium Single Cell 3' Reagent Kits (v3.1) were used. Single-stranded cDNA was synthesized from mouse ES cells (5G6GR) according to the product manual and then purified. The purified single-stranded cDNA solution was divided into equal volumes to prepare cDNA solutions. To 20 μL of the cDNA solution, 25 μL of TdT premix A (1× ThermoPol buffer, 2.4 mM dATP, 0.0384 U / μL RNase H, 26.88 units / μL of terminal transferase (TdT)) without 2'-fluoro-ATP and tetraammonium chloride (TMAC) or 25 μL of TdT premix B (1× ThermoPol buffer, 1.8 mM dATP, 0.6 mM Fluoro-ATP, 10 mM tetraammonium chloride, 0.0384 U / μL RNase H, 26.88 units / μL of terminal transferase (TdT)) containing 2'-fluoro-ATP and TMAC was added and mixed thoroughly. The mixture was then heated at 37°C for 75 seconds to carry out the polyadenylation reaction. To 11 μL of the reaction mixture, 46 μL of Tagging premix (1.084× Mighty amp buffer version 2, 0.0693 μM Tagging_10xTSO primer, Mighty amp 0.0541 U / μL) was added for tagging reaction. The sequence of the Tagging_10xTSO primer was 5'-AAGCAGTGGTATCAACGCAGAGTACATtttttttttttttttttttttttt-3' (SEQ ID NO: 7). Subsequently, 50 μL of PCR premix (1× mighty amp buffer version 2, 1.895 μM FullRead1 primer, 1.895 μM 10xpTSO primer) was added and PCR reaction was performed.The sequence of the FullRead1 primer was 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' (SEQ ID NO: 8), and the sequence of the 10xpTSOprimer was 5'-AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO: 9). The PCR reaction mixture was purified using Qiagen MinElute and Ampure XP. The size distribution of the amplified DNA was measured using a Bioanalyzer 2100 High-sensitivity kit.

[0145] The size distribution of the amplified products is shown in Figure 11A. When polyadenylation was performed under conditions containing 2'-fluoro-modified ATP and TMAC (Figure 11A, bottom), the generation of by-products of 400 bp or less was significantly suppressed compared to when polyadenylation was performed under conditions lacking 2'-fluoro-modified ATP and TMAC (Figure 11A, top). However, the amount of amplified cDNA of 400 bp or more was equal to or greater than that under the control condition.

[0146] (2) Split-Seq: 10 μL of RT premix (composition: 2× ThermoPol buffer, SuperScript III 1.25 U / μL, RNasin plus 0.137 U / μL) was added to 10 μL of lysis solution (composition: 1 ng total RNA, 0.3% NP-40, 0.12 mM dNTPs, 0.111 μM Split-seq RT primer, RNasin plus 1 U / μL), mixed well, and then reverse transcription was performed. The sequence of the Split-seqRT primer was 5'-CAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNGTGGCCGATGTTTCGCATCGGCGTACGACTNNNNNNNNATCCACGTGCTTGAGAGGCCAGAGCATTCGNNNNNNNNTTTTTTTTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 10; "N" represents a random base, with each base assigned with a 25% probability). The reverse transcription reaction was carried out at 35°C for 5 minutes and at 50°C for 50 minutes. The reaction solution after the reverse transcription reaction was purified using Zymo DNA Clean & Concentrator-5. To 20 μL of the purified cDNA solution, 25 μL of TdT premix A (1× ThermoPol buffer, 2.4 mM dATP, 0.0384 U / μL RNase H, 26.88 units / μL terminal transferase (TdT)) without 2'-fluoro-ATP and tetraammonium chloride (TMAC) or TdT premix B (1× ThermoPol buffer, 1.8 mM dATP, 0.6 mM Fluoro-ATP, 10 mM tetraammonium chloride, 0.0384 U / μL RNase H, 26.88 units / μL terminal transferase (TdT)) containing 2'-fluoro-ATP and TMAC was added and mixed thoroughly. The mixture was then heated at 37°C for 75 seconds for polyadenylation.To 11 μL of the reaction mixture after poly(A) addition, 46 μL of tagging premix (composition: 1.084× Mighty amp buffer version 2, 0.0693 μM Split-seq Tagging primer, Mighty amp 0.0541 U / μL) was added, and the tagging reaction was performed. The sequence of the Split-seq Tagging primer was 5'-AAGCAGTGGTATCAACGCAGAGTGAATTTTTTTTTTTTTTTTTTTTTTTTTT-3' (SEQ ID NO: 11). Subsequently, 50 μL of PCR premix (composition: 1× Mighty amp buffer version 2, 0.947 μM Split-seq PCR1 primer, 0.947 μM Split-seq PCR2 primer) was added, and the PCR reaction was performed. The sequence of Split-seqPCR1 primer was 5'-AAGCAGTGGTATCAACGCAGAGT-3' (SEQ ID NO: 12), and the sequence of Split-seqPCR2 primer was 5'-CAGACGTGTGCTCTTCCGATCT-3' (SEQ ID NO: 13). The PCR reaction mixture was purified using Qiagen MinElute and Ampure XP. The size distribution of the amplified DNA was detected using a Bioanalyzer 2100 High-sensitivity kit.

[0147] The size distribution of the amplified products is shown in Figure 11B. When polyadenylation was performed with 2'-fluoro-modified ATP and TMAC (Figure 11B, bottom), the generation of by-products of 400 bp or less was significantly suppressed compared with when polyadenylation was performed without 2'-fluoro-modified ATP and TMAC (Figure 11B, top). However, the amount of amplified cDNA of 400 bp or more was comparable to that of the control condition.

[0148] These results demonstrate that the suppression tagging method of the present invention can improve the performance of various techniques and platforms used for trace RNA sequencing analysis, such as single-cell RNA sequencing. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. A method for adding a polyA sequence to the 3' end of a DNA strand, comprising: a reverse transcription step in which an RNA strand having a polyA sequence at its 3' end as a template is reverse transcribed using a first oligo-dT primer to obtain a DNA strand extended to the 3' end; and an addition step in which the polyA sequence is added to the 3' end of the DNA strand using terminal deoxynucleotidyl transferase or an active fragment thereof under conditions that stabilize intramolecular base pairing between an adenine base added to the 3' end of the first oligo-dT primer that was not extended to the 3' end in the reverse transcription step and a thymine base, wherein the addition step adds the polyA sequence in the presence of ammonium cations and / or modified adenine nucleotides and / or at a temperature of 34°C or lower, and / or the first oligo-dT primer used in the reverse transcription step contains a modified thymine nucleoside.

2. The method of claim 1, wherein the RNA strand is mRNA and / or non-coding RNA with a polyA sequence added to the 3' end.

3. The method of claim 1, wherein the ammonium cation is a tetraalkylammonium cation.

4. The method of claim 3, wherein the tetraalkylammonium cation is selected from the group consisting of tetramethylammonium cation, tetraethylammonium cation, tetrapropylammonium cation, tetrabutylammonium cation, tetrapentylammonium cation, and tetrahexylammonium cation.

5. The method of claim 1, wherein the ammonium cation is 1 mM to 100 mM.

6. The method of claim 1, wherein the modified adenine nucleotide and / or the modified thymine nucleoside comprises a bicyclic sugar moiety, a 2'-modified sugar moiety, and / or a modified nucleobase.

7. The method of claim 6, wherein the bicyclic sugar moiety comprises a methyleneoxy group bridging the 4' and 2' positions, and / or the 2'-modified sugar moiety comprises a 2'-O-methyl-modified sugar moiety, a 2'-fluoro-modified sugar moiety, a 2'-amine-modified sugar moiety, a 2'-azido-modified sugar moiety, and / or a 2'-O-methoxyethyl-modified sugar moiety.

8. The method of claim 1, further comprising an RNA strand degradation step in which the RNA strand after the reverse transcription step is degraded using an RNase H enzyme.

9. The method of claim 1, further comprising a complementary strand synthesis step in which a complementary strand is synthesized using a second oligo dT primer, with the DNA strand to which the poly A sequence has been added in the addition step as a template.

10. The method of claim 9, wherein the first oligo-dT primer and the second oligo-dT primer contain the same or different adapter sequences at the 5' end of the oligo-dT sequence.

11. The method of claim 10, wherein the first oligodT primer and / or the second oligodT primer comprises a barcode sequence of 8 to 200 bases in length between the adapter sequence and the oligodT sequence.

12. The method of claim 10, wherein the first oligo-dT primer and / or the second oligo-dT primer does not contain a barcode sequence.

13. The method according to claim 10, further comprising an amplification step of amplifying double-stranded DNA composed of the DNA strand and the complementary strand after the complementary strand synthesis step, using a pair of nucleic acid primers each containing, at their 3' ends, a base sequence identical to the same or different adapter sequence, to obtain an amplification product.

14. A method for determining the base sequence of an RNA strand having a polyA sequence at the 3' end, comprising a base sequencing step of determining the base sequence of the amplification product obtained according to the method of claim 13.

15. The method of claim 14, wherein the RNA strand is derived from an isolated or cultured cell, a cell population consisting of multiple cells, a tissue, or an organ.

16. The method according to claim 15, which is the Quartz-Seq method or the Quartz-Seq2 method.

17. A method for adding a poly-T sequence to the 3'-end of a DNA strand, comprising: a reverse transcription step of performing reverse transcription using a first oligo dA primer and an RNA strand having a poly-U sequence at its 3'-end as a template to obtain a DNA strand extended at its 3'-end; and an addition step of adding a poly-T sequence to the 3'-end of the DNA strand using terminal deoxynucleotidyl transferase or an active fragment thereof under conditions that stabilize intramolecular base pairing between a thymine base added to the 3'-end of the first oligo dA primer that was not extended at its 3'-end in the reverse transcription step and an adenine base, wherein the addition step adds the poly-T sequence in the presence of ammonium cations and / or modified thymine nucleotides and / or at a temperature of 34°C or lower, and / or the first oligo dA primer used in the reverse transcription step contains a modified adenine nucleoside.

18. A composition or kit for suppressing the production of by-products by terminal deoxynucleotidyl transferase, wherein: (i) the by-products are oligo-dT primers that are not extended to the 3' end in a reverse transcription reaction, and a poly-A sequence is added to the 3' end of the oligo-dT primer, the composition or kit comprising a modified oligo-dT primer comprising an ammonium cation, a modified adenine nucleotide, and / or a modified thymine nucleoside; and / or (ii) the by-products are oligo-dA primers that are not extended to the 3' end in a reverse transcription reaction, and a poly-T sequence is added to the 3' end of the oligo-dA primer, the composition or kit comprising an ammonium cation, a modified thymine nucleotide, and / or a modified adenine nucleoside.

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