A library construction method for direct sequencing of single-stranded nucleic acids

Abstract

The application provides a library construction method for direct sequencing of single-stranded nucleic acid, which comprises providing a sequence fragment to be sequenced, and the preparation method of the sequence fragment to be sequenced comprises cutting a double-stranded molecule sequence formed by internal base complementary pairing of the single-stranded nucleic acid by using a transposase, so as to form the sequence fragment to be sequenced. Compared with the existing library construction method for direct sequencing of RNA, the application utilizes the complementary pairing structure formed by the bases of RNA or single-stranded DNA itself, so that the transposase complex can cut and connect the same. This is a previous attempt, and this library construction method greatly improves the library construction efficiency.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to the earlier application filed on February 8, 2024, with patent application number 202410177526.0 and entitled "A Library Construction Method for Direct Sequencing of Single-Stranded Nucleic Acids". The entire contents of the earlier application are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of gene sequencing technology, and in particular to a library construction method for direct sequencing of single-stranded nucleic acids. Background Technology

[0004] Nanopore sequencing is a revolutionary next-generation sequencing technology. In nanopore sequencing, nanopore proteins are immobilized on an electrically insulating monolayer biomimetic membrane, separating the electrolyte solution into two parts. When a constant voltage is applied across the biomimetic membrane, electrolyte ions pass through the nanopore, generating an electric current. Since nucleic acids are negatively charged, they can pass through the nanopore under the influence of the electric field. The different volumes and charges of different nucleotides cause changes in the current across the membrane, allowing for the determination of nucleic acid sequences by recording these changes. Compared to other sequencing technologies, nanopore sequencing offers advantages such as portability, real-time processing, ultra-long read lengths, and the ability to detect epigenetic modifications in nucleic acids, making it a future direction for nucleic acid sequencing technology.

[0005] RNA plays a crucial role in gene expression within organisms, and RNA sequencing helps us understand important information such as gene expression levels, differential splicing, and transcriptional regulation. Compared to DNA, RNA typically exists in single-stranded form and exhibits a variety of base modifications, with over a hundred identified. RNA viruses account for more than 80% of all human pathogenic viruses, and most highly contagious viral diseases are caused by RNA viruses. Therefore, establishing a rapid, high-throughput RNA gene sequencing technology platform is of great significance. Traditional RNA sequencing methods, such as transcriptome sequencing (RNA-Seq), usually require reverse transcription and amplification steps, which can introduce bias and errors and are time-consuming. Furthermore, the base modification information of RNA is lost after reverse transcription and amplification. Nanopore sequencing is a direct sequencing technology that can not only sequence DNA but also directly determine RNA sequences. Oxford Nanopore Technologies has developed a direct RNA sequencing protocol, successfully achieving direct RNA sequencing. However, this method still requires a reverse transcription step during library construction, and the sequencing accuracy is not high. Therefore, developing simpler and more efficient direct RNA sequencing methods will greatly promote the development of RNA sequencing research.

[0006] Since its discovery in *E. coli*, Tn5 transposase has been widely used for fragmenting and labeling double-stranded DNA and RNA / DNA hybrids. Transposases can insert transposon DNA into the target sequence while fragmenting the target DNA, thus generating DNA fragments that can be directly amplified by PCR for library construction. Compared with traditional methods, this method combines DNA fragmentation and adapter ligation, significantly shortening library construction time and improving library construction efficiency. Therefore, Tn5 has been applied to various high-throughput sequencing methods. To investigate how Tn5 acts on different nucleic acid substrates, researchers conducted experiments using double-stranded DNA, single-stranded DNA, RNA strands, and DNA / RNA hybrids as substrates. They found that Tn5 can fragment single-stranded DNA but has almost no effect on single-stranded RNA (see: Zhang et al., *Genome Research*, 33:412–426, Tn5 tagments and transposes oligos to single-stranded DNA for strand-specific RNA sequencing). Summary of the Invention

[0007] This invention unexpectedly discovered that by utilizing the complementary bases of single-stranded RNA or single-stranded DNA to form pairing structures, transposases can randomly cleave single-stranded RNA or DNA into RNA or DNA fragments, which can then be sequenced. For RNA, by eliminating the reverse transcription step, the modification features present on the RNA are directly detected and recorded, greatly improving the accuracy and efficiency of sequencing. For some single-stranded DNA viruses in nature, whose structures are more complex, this invention provides a new method for single-stranded DNA sequencing with even higher detection accuracy and efficiency.

[0008] The first aspect of the present invention provides a method for labeling and fragmenting single-stranded nucleic acids, comprising using a transposase to cut the single-stranded nucleic acid into a double-stranded molecular sequence formed by internal base complementary pairing, thereby completing the fragmentation and labeling of the single-stranded nucleic acid.

[0009] In one embodiment of the present invention, the single-stranded nucleic acid includes single-stranded DNA (ssDNA) and / or single-stranded RNA (ssRNA).

[0010] In a preferred embodiment of the present invention, the single-stranded nucleic acid is single-stranded RNA (ssRNA).

[0011] In one embodiment of the present invention, the single-stranded RNA includes mRNA and repressive ssRNA (e.g., antisense ssRNA, siRNA or miRNA), tRNA and nucleic acid sequences from single-stranded RNA viruses.

[0012] In one embodiment of the present invention, the ssRNA (e.g., mRNA) includes any modified ssRNA.

[0013] In one embodiment of the present invention, the modification includes 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl) (2'-O-moe), 2'-fluorine (2'-F), thiophosphate (PS) bond substitution, m6A, etc.

[0014] ssDNA can be synthesized using in vitro methods. The in vitro methods covered for synthesizing ssDNA include methods that involve transcribing a DNA template encoding a promoter operatively linked to a nucleotide sequence to produce an RNA transcript; synthesizing an ssDNA / RNA duplex by reverse transcription of the RNA transcript; and degrading RNA from the ssDNA / RNA duplex using an RNA-degrading enzyme to produce ssDNA. Optionally, the method may also include purifying the ssDNA. Purification of ssDNA can be achieved by gel purification or other DNA purification protocols well known in the art. Promoters suitable for in vitro transcription of DNA templates are known in the art. Examples of suitable promoters include, but are not limited to, T7, T3, and SP6 promoters. Preferably, the promoter comprises a T7 promoter and transcription is performed using T7 RNA polymerase. The DNA can be derived from pathogen DNA, such as single-stranded DNA viruses.

[0015] The synthesis of ssDNA / RNA duplexes can be performed using RNA-dependent DNA polymerases such as reverse transcriptases. Suitable reverse transcriptase proteins for use in this invention can be obtained from various retroviruses, including but not limited to Moloney Murine Leukemia virus, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and reverse transcriptases isolated from various yeast and bacterial species. Preferably, the reverse transcriptase is Moloney Murine Leukemia virus.

[0016] Other in vitro methods can also be used to generate the ssDNA of the present invention. Suitable methods include: asymmetric PCR (see, for example, U.S. Patent 8,735,067), PCR using two oligonucleotide primers and one primer present at a limiting concentration (see, for example, U.S. Patent 5,066,584), and generating ssDNA from dsDNA molecules using a "nicking enzyme" and / or restriction endonuclease (see, for example, LsODN Preparation Kit - Biodynamics Laboratory Inc., Tokyo, Japan).

[0017] In one embodiment of the present invention, the single-stranded DNA and / or single-stranded RNA contains a self-complementary sequence, which allows a portion of the single-stranded DNA and / or single-stranded RNA to fold and pair with itself to form a double-stranded molecular sequence.

[0018] In one embodiment of the present invention, the complementary sequences of the single-stranded nucleic acid are linked together by hydrogen bonds to form a double-stranded molecular sequence. For example, in DNA, A and T, C and G are complementary. In RNA, A and U, C and G are complementary.

[0019] In one embodiment of the present invention, the double-stranded molecular sequence has local stability. These double-stranded molecular sequences are typically composed of specific nucleotide sequences, such as guanine-guanine (GG) and adenine-adenine (AA).

[0020] In one embodiment of the present invention, the double-stranded molecular sequence is located at any position of the single-stranded DNA and / or single-stranded RNA, and may be located at the 3' end, 5' end, or any intermediate position.

[0021] In one embodiment of the present invention, the double-stranded molecular sequence is located at multiple randomly distributed positions.

[0022] In one embodiment of the present invention, the internal bases may be adjacent bases or non-adjacent bases.

[0023] In one embodiment of the present invention, the transposase includes, but is not limited to, Tn transposases (e.g., Tn3, Tn5 or their functional derivatives (e.g., the mutant reported in CN106754811A), Tn7, Tn10, Tn552, Tn903), MuA transposases or their functional derivatives, or Vibhar transposases.

[0024] In some embodiments, the transposase is a Tn5 transposase, which contains a sequence having at least 80%, 90%, 95%, or 99% homology to SEQ ID NO:7; more preferably, it contains a mutation at at least at position 54 and position 372; for example, the mutant includes mutations at one or both positions of SEQ ID NO:7, such as an E54 single mutation, an L372 single mutation, or an E54+L372 mutation; preferably an E54K single mutation, an L372P single mutation, or an E54K+L372P mutation.

[0025] In some implementations, it further includes a mutation of at least one of R26, R62, E110, D156, P242, and E345;

[0026] The mutated amino acid is one or more of the neutral or basic amino acids, which affects its binding to nucleic acids, making the binding more robust.

[0027] Preferably, it is one or more of valine (V), arginine (R), tyrosine (Y), isoleucine (I), glutamic acid (E), tryptophan (W), proline (P), lysine (K), and alanine (A); preferably, it is one or more of tryptophan (W), proline (P), lysine (K), and alanine (A).

[0028] Preferably, it further includes at least one mutation of R26, R62, E110, D156, P242, and E345; more preferably, it further includes at least one mutation of R26W, R62P, E110K, D156K, P242A, and E345K.

[0029] Or sequences that have at least 80%, 90%, 95%, or 99% homology with the above-mentioned sequences.

[0030] More preferably, it is the transposase mutant shown in SEQ ID NO:13.

[0031] In some embodiments, the MuA transposase comprises a sequence having at least 80% or 90% or 95% or 99% homology with SEQ ID NO:14; more preferably, it comprises a mutation at at least one position of positions 179, 233, 254, 464, 487, 539, and 594; for example, it comprises mutations at one, two, three, four, five, six, or seven positions of positions 179, 233, 254, 464, 487, 539, and 594 of SEQ ID NO:14.

[0032] The mutated amino acid is one or more neutral or basic amino acids, which affects its binding to nucleic acids, making the binding more robust. Preferably, it is at least one of lysine (K), valine (V), arginine (R), tyrosine (Y), isoleucine (I), and glutamic acid (E); more preferably, it is at least one of valine (V), arginine (R), tyrosine (Y), and isoleucine (I).

[0033] Preferably, it is at least one of E233V, Q254R, F464Y, M487I, Q539R, Q594R, and E179V.

[0034] The mutated amino acid is one or more neutral or basic amino acids, which affects its binding to nucleic acids, making the binding more robust. Preferably, it is at least one of lysine (K), valine (V), arginine (R), tyrosine (Y), isoleucine (I), and glutamic acid (E); more preferably, it is at least one of lysine (K), arginine (R), and glutamic acid (E).

[0035] Preferably, it also includes a mutation of at least one of L106K, W110R, G136E, Q186E, I189K, L209R, A222K, Q229E, Q230R, N496R, V517R or A561E.

[0036] More preferably, it is the transposase mutant shown in SEQ ID NO:15.

[0037] In one embodiment of the present invention, the transposase exists in the form of a transposase complex, the transposase complex comprising the transposase and a transposase recognition sequence.

[0038] In one embodiment of the present invention, the transposase recognition sequence optionally includes a ligase-recognized linker, aptamer, antibody, biotin, digoxigenin or digoxigenin ligand, bioorthogonal reactive group, methylation fragment, fluorescently labeled fragment, capture fragment, adapter sequence, primer sequence, promoter sequence or tag sequence, thereby making the fragmented nucleic acid molecule suitable for a variety of detection or analysis occasions.

[0039] In one embodiment of the present invention, the transposase recognition sequence is a Tn5-Me or MuA-Me sequence.

[0040] In one embodiment of the present invention, the transposase complex randomly binds to any position of the double-stranded molecule sequence, thereby cleaving the single-stranded nucleic acid into multiple single-stranded fragments, and introducing the transposase recognition sequence into the single-stranded fragments.

[0041] In one embodiment of the present invention, the cleavage site is located at any position in the double-stranded molecule sequence.

[0042] In one embodiment of the present invention, the reaction conditions for cutting are: a reaction temperature of 15–55°C and a reaction time of 15–60 min. In a preferred embodiment of the present invention, the reaction temperature is 37°C.

[0043] In a second aspect, the present invention provides a library construction method for direct sequencing of single-stranded nucleic acids, comprising providing a sequence fragment to be tested; the method for preparing the sequence fragment to be tested is the method of the first aspect.

[0044] In one embodiment of the present invention, the database construction method includes:

[0045] 1) Using the method of the first aspect of the present invention, single-stranded nucleic acid is fragmented and introduced into the ligation ends;

[0046] 2) The fragmented single-stranded nucleic acids are ligated into sequencing adapter complexes to obtain sequencing libraries.

[0047] In one embodiment of the present invention, the sequencing adapter complex is an adapter complex for nanopore sequencing.

[0048] In one embodiment of the present invention, the sequencing adapter complex includes a region for ligation of the nucleic acid fragment to be analyzed; preferably, the region includes a complementary sequence for helicase binding and ligation of the double-stranded DNA fragment.

[0049] In one embodiment of the present invention, the transposase recognition sequence and the region for ligating the nucleic acid fragment to be analyzed have complementary sticky ends, and the ligation is achieved by a nucleic acid ligase; preferably, the ligation is achieved by a T4 DNA ligase.

[0050] This invention designs the sticky ends of the transposase recognition sequence so that the transfer strand in the transposase recognition sequence is linked to the sequencing adapter complex, thereby avoiding the gap repair step; preferably, the complementary sequences of the transfer strand in the transposase recognition sequence and the linking region of the nucleic acid fragment to be analyzed in the sequencing adapter complex have complementary overhangs (such as CAC and GTG).

[0051] In a third aspect, the present invention provides a sequencing library prepared by the method of the second aspect of the present invention.

[0052] In a fourth aspect, the present invention provides a sequencing method, the method comprising the steps of fragmenting and labeling single-stranded nucleic acids using the method of the first aspect of the present invention, or the steps of constructing a sequencing library using the method of the second aspect of the present invention, or the steps of sequencing the sequencing library of the third aspect of the present invention.

[0053] A fifth aspect of the present invention provides a composition for constructing single-stranded nucleic acid nanopore sequencing libraries, the composition comprising:

[0054] 1) Transposase and transposase recognition sequence;

[0055] 2) A sequencing adapter complex, the sequencing adapter complex including a region for ligation of the nucleic acid fragment to be analyzed;

[0056] 3) The transposase recognition sequence and the region used for linking the nucleic acid fragment to be analyzed have complementary sticky ends;

[0057] 4) Nucleotide ligase.

[0058] The sixth aspect of the present invention provides a transposase mutant, comprising:

[0059] A Tn5 transposase mutant comprising a sequence having at least 80%, 90%, 95%, or 99% homology to SEQ ID NO:7; and having one, two, or three of the E54K, L372P, and E345K mutation sites; more preferably, comprising the amino acid sequence shown in SEQ ID NO:13.

[0060] or

[0061] MuA transposase, the MuA transposase comprising a sequence having at least 80% or 90% or 95% or 99% homology with SEQ ID NO:14; and having one, two or more of the E179V, E233V, Q254R, F464Y, M487I, Q539R or Q594R mutation sites; more preferably, comprising the amino acid sequence shown in SEQ ID NO:15.

[0062] The present invention also provides a nucleic acid sequence, wherein the nucleic acid sequence is: 1) a sequence encoding the above-mentioned transposase mutant; or 2) a sequence complementary to 1).

[0063] The present invention also provides biological materials that express the above-mentioned transposase mutant or contain the above-mentioned nucleic acid sequence.

[0064] The present invention also provides the application of the above-mentioned transposase mutants, the above-mentioned nucleic acid sequences, or the above-mentioned biological materials in single-stranded nucleic acid molecular fragmentation or sequencing library construction.

[0065] Compared with the prior art, the present invention has the following beneficial effects:

[0066] (1) Compared with existing library construction methods based on direct RNA sequencing, this invention utilizes the complementary bases of RNA or single-stranded DNA to form a pairing structure, allowing the transposase complex to cleave and ligate it. This is an unprecedented approach, and this library construction method greatly improves efficiency.

[0067] (2) The helicase of the present invention moves directionally along the 5'- to 3'- ends of the RNA chain, and the pore-passing speed of the RNA chain is fast and uniform, reaching 300nt / s.

[0068] (3) From the perspective of the database construction process, the database construction of this invention is short and easy to operate.

[0069] (4) The library construction method for direct RNA sequencing of the present invention enables direct sequencing of RNA or single-stranded DNA, and can directly detect and record the modification features present on RNA. Attached Figure Description

[0070] Figure 1 is a schematic diagram of the present invention: Figure 1AA cartoon illustration of single-stranded nucleic acid labeling and fragmentation; Figure 1B A cartoon diagram illustrating the process of library construction for single-stranded nucleic acids; the transposase complex recognizes and cleaves local double-stranded regions of single-stranded nucleic acids and ligates short DNA fragments to the test fragments. Subsequently, the sequencing adapter complex ligates the DNA fragments via ligases, completing the library construction of the test sample.

[0071] Figure 2 A gel image showing the effect of the Tn5 transposase complex on RNA single-strand cleavage.

[0072] Figure 3 for Figure 2 Perforation signal diagram after single-stranded RNA library construction;

[0073] Figure 4 The image shows the perforation signal after the Tn5 transposase complex cleaves PhiX174 virion DNA single-stranded DNA for library construction.

[0074] Figure 5 A gel image showing the effect of the MuA transposase complex on RNA single-strand cleavage.

[0075] Figure 6 for Figure 5 Image of perforation signal after single-stranded RNA library construction. Detailed Implementation

[0076] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0077] Technical terms:

[0078] Tn5 transposase complex

[0079] The homodimer formed by the Tn5 enzyme and the transposase recognition sequence (including the ME sequence: CTGTCTCTTATACACATCT and other marker molecules or fragments) can insert the transposase recognition sequence into the target nucleic acid sequence while simultaneously cleaving the DNA double-stranded sequence, and introduce marker molecules or fragments at the same time. For the application of Tn5 transposase in genome research, please refer to: Li N, Jin K, Bai Y, et al. Tn5 Transposase Applied in Genomics Research[J]. International Journal of Molecular Sciences,2020,21(21):8329.

[0080] The terms "transpoase recognition sequence" or "transposon terminus" refer to a double-stranded nucleic acid molecule containing a nucleotide sequence necessary for the formation of a functional complex with a transposase in a transposition reaction. In this invention, "transposon terminus" and "transpoase recognition sequence" have the same meaning and are used interchangeably. The transposon terminus forms a "transposase complex" with a transposase that recognizes and binds to it, and this complex is capable of inserting or transposing the transposon terminus into a double-stranded nucleic acid molecule incubated with it in an in vitro transposition reaction, or into the double-stranded structure of a single-stranded nucleic acid molecule as described in this invention. The transposon terminus comprises two complementary sequences: a "transferred transposon terminus sequence" and a "non-transferred transposon terminus sequence." The nucleic acid strand containing the transferred transposon terminus sequence is called the "transferred strand." The nucleic acid strand containing the non-transferred transposon terminus sequence is called the "non-transferred strand." In an in vitro transposition reaction, the 3' end of the transferred strand binds to or transfers to a target nucleic acid molecule (e.g., a DNA molecule, an RNA molecule). In in vitro transposon reactions, the 5' end of the transposon terminal sequence complementary to the transposon terminal sequence (i.e., the non-transferred transposon terminal sequence) does not bind to or transfer to the target nucleic acid molecule, forming a 9 bp gap.

[0081] Preferably, the transposase of this invention comprises:

[0082] The amino acid sequence (M-Tn5) of the Tn5 transposase mutant (E54K, L372P, E345K) is SEQ ID NO:13.

[0083] MVTFMITSALHRAADWAKSVFSSAALGDPRRTARLVNVAAQLAKYSGKSITISSEGSKAMQEGAYRFIRNPNVSAEAIRKAGAMQTVKLAQEFPELLAIEDTTSLSYRHQVAE ELGKLGSIQDKSRGWWVHSVLLLEATTFRTVGLLHQEWWMRPDDPADADEKESGKWLAAAATSRLRMGSMMSNVIAVCDREADIHAYLQDKLAHNERFVVRSKHPRKDVESGLY LYDHLKNQPELGGYQISIPQKGVVDKRGKRKNRPARKASLSLRSGRITLKQGNITLNAVLAEEINPPKGETPLKWLLLTSEPVESLAQALRVIDIYTHRWRIEEFHKAWKTGA GAERQRMEKPDNLERMVSILSFVAVRLLQLRESFTPPQALRAQGLLKEAEHVESQSAETVLTPDECQLLGYLDKGKRKRKEKAGSLQWAYMAIARLGGFMDSKRTGIASWGALW

[0084] Tn5 transposase mutant expression and purification

[0085] The recombinant plasmid pET28a containing the Tn5 mutant was transformed into the BL21(DE3) *E. coli* expression host via heat shock. During the induction process, the host bacteria containing the expression plasmid were first cultured overnight at 37°C in LB medium supplemented with kanamycin. Then, the culture was scaled up at 37°C at a 1:100 ratio until the OD(600) value reached 0.4-0.6. The culture was then stopped and the bacteria were cooled to 4°C for 1 hour. Subsequently, isopropyl β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM was added, and expression was induced at 16°C for 12-16 hours. The bacteria were then collected by centrifugation at 15,000 rpm at 4°C. The cells were then autoclaved at 4°C, and the supernatant was collected by centrifugation at 4°C. The target protein was then purified step-by-step using nickel columns, heparin columns, and molecular sieves to obtain a large quantity of high-purity Tn5 transposase mutant protein.

[0086] The amino acid sequence (M-MuA) of the Escherichia phage MuA transposase mutant (1-76 truncated, E179V, E233V, Q254R, F464Y, M487I, Q539R, Q594R) is SEQ ID NO:15.

[0087] IARPTLEAHDYDREALWSKWDNASDSQRRLAEKWLPAVQAADEMLNQGISTKTAFATVAGHYQVSASTLRDKYYQVQKFAKPDWAAALVDGRGASRRNVHKSVFDEDAWQFLIADYLRPEKPAFRKCYERLELAAREHGWSIPSRA TAFRRIQQLDVAMVVACREGEHALMHLIPAQRRTVEHLDAMQWINGDGYLHNVFVRWFNGDVIRPKTWFWQDVKTRKILGWRCDVSENIDSIRLSFMDVVTRYGIPEDFHITIDNTRGAANKWLTGGAPNRYRFKVKEDDPKGLFLL MGAKMHWTSVVAGKGWGQAKPVERAFGVGGLEEYVDKHPALAGAYTGPNPQAKPDNYGDRAVDAELFLKTLAEGVAMFNARTGRETEMCGGKLSYDDVFEREYARTIVRKPTEEQKRILLLPAEAVNVSRKGEFTLKVGGSLKGAKN VYYNMALMNAGVKKVVVRFDPQRLHSTVYCYTLDGRFICEAECLAPVAFNDAAAGREYRRRQKQLKSATKAAIKAQKRMDALEVAELLPQIAEPAAPESRIVGIFRPSGNTERVKNQERDDEYETERDEYLNHSLDILEQNRRKKAI

[0088] MuA transposase mutant expression and purification

[0089] The pBAD recombinant plasmid containing the MuA mutant was transformed into the BL21(DE3) *E. coli* expression host via heat shock. During the induction of expression, the host bacteria containing the expression plasmid were first cultured overnight at 37°C in LB medium supplemented with Ampicillin resistance. Then, the culture was scaled up at 37°C at a 1:100 ratio until the OD(600) value reached 0.4-0.6. The culture was then stopped and the bacteria were cooled to 4°C for 1 hour. A final concentration of 0.006% arabinose was added, and expression was induced at 28°C for 4 hours. The bacteria were then collected by centrifugation at 15,000 rpm at 4°C. The cells were then autoclaved at 4°C, and the supernatant was collected by centrifugation at 4°C. The target protein was then purified using nickel column chromatography, heparin column chromatography, and molecular sieve chromatography to obtain a large quantity of high-purity MuA transposase mutant protein.

[0090] Tagging and fragmenting

[0091] In this invention, labeling and fragmentation has the common meaning in the art, that is, the introduction of labeling molecules such as antibodies, fluorescent fragments or tag fragments while fragmenting nucleic acid molecules; when a tag fragment is introduced, it has the same meaning as the common expression in the art, "tagmenting", that is, the introduction of a tag sequence for NGS sequencing, such as P5 or P7, or other complex adapters such as Y adapters, through transposase recognition sequences.

[0092] Single-stranded DNA and / or single-stranded RNA

[0093] In one embodiment of the invention, the length of the single-stranded DNA and / or single-stranded RNA typically varies between 6 and 30,000 bases, preferably 10 to 12,000, particularly 100 to 10,000, 150 to 8,000, 200 to 7,000, 250 to 6,000, or 300 to 5,000 bases. In one embodiment, the single-stranded DNA and / or single-stranded RNA has a length of at least 2,700 bases (e.g., at least 2,800, at least 2,900, at least 3,000, at least 3,100, at least 3,200, at least 3,300, at least 3,400, at least 3,500, at least 3,600, at least 3,700, at least 3,800, at least 3,900, at least 4,000, at least 4,100, at least 4,200, at least 4,300, at least 4,400, at least 4,500, at least 4,600, at least 4,700, at least 4,800, at least 4,900, at least 5,000 bases). The single-stranded DNA and / or single-stranded RNA used in this article have at least 3,500 bases (e.g., at least 3,600, at least 3,700, at least 3,800, at least 3,900, at least 4,000, at least 4,100, at least 4,200, at least 4,300, at least 4,400, at least 4,500, at least 4,600, at least 4,700, at least 4,800, at least 4,900). Single-stranded DNA and / or single-stranded RNA of at least 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, or 9,500 bases, preferably up to 15,000, such as up to 14,000, 13,000, or 12,000 bases.

[0094] In one embodiment of the present invention, the ssDNA can be bacteriophage virus DNA, including but not limited to Debruijin DNA and PhiX174 virion DNA.

[0095] In one embodiment of the present invention, the single-stranded RNA includes mRNA and repressive ssRNA (e.g., antisense ssRNA, siRNA, or miRNA), tRNA, and nucleic acid sequences from single-stranded RNA viruses.

[0096] In one embodiment of the present invention, single-stranded RNA viruses include positive-strand RNA viruses (+ssRNA), negative-strand RNA viruses (-ssRNA), and retroviruses. The +ssRNA viruses include picornaviruses (rhinovirus, poliovirus, hepatitis A virus), coronaviruses (MERS), flaviviruses (yellow fever, Japanese encephalitis, dengue fever, West Nile virus, Zika virus), A viruses (chikungunya), and hepatitis C virus, etc. The -ssRNA viruses include Bonavir virus, paramyxoviruses (measles, mumps, parainfluenza, respiratory syncytial virus (excluded from paramyxovirus in 2016), Hennipa virus), rhabdoviruses (rabies virus), and filoviruses (Ebola virus, Marburg virus). The retroviruses include HIV, murine leukemia virus (MLV), etc.

[0097] The term "mRNA" refers to "messenger RNA" and relates to a "transcription" that can be produced using a DNA template and can encode peptides or proteins. Typically, mRNA comprises a 5′-UTR, a protein-coding region, and a 3′-UTR. In the context of this invention, mRNA is preferably produced from a DNA template via in vitro transcription. As described above, in vitro transcription methods are known to those skilled in the art, and a variety of commercially available in vitro transcription kits are available.

[0098] The term “modification” includes any modification to RNA that is not naturally present in the RNA (preferably ssRNA, such as mRNA).

[0099] Nanopore sequencing adapter complex

[0100] Nanopore sequencing adapter complexes comprise sequencing adapters and helicases. A typical nanopore sequencing adapter comprises four regions: a first region is a single-stranded nucleotide region for guiding the target nucleic acid into the nanopore; a second region is a polynucleotide-binding protein (nucleotide-binding protein or rate-controlling protein) binding region; a third region is an adaptor region that blocks the progression of the polynucleotide-binding protein; and a fourth region is for attaching the target polynucleotide (such as the sticky end used in this invention). The fourth region typically contains a sequence complementary to the adapter for helicase binding and ligation of the double-stranded nucleic acid sequence.

[0101] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0102] Example 1: Sequencing of single-stranded RNA

[0103] 1. Preparation of the connector composite

[0104] 1) Design and synthesize the connector sequence (SEQ ID NO:1), sequence 2 (SEQ ID NO:2), and sequence 3 ((SEQ ID NO:3)).

[0105] 2) Joint sequence annealing treatment

[0106] The adapter sequence, sequence 2, and sequence 3 were dissolved in annealing buffer (50 mM HEPES, pH 8.0 and 100 mM KCl) to prepare three 100 μM stock solutions: Stock Solution 1, Stock Solution 2, and Stock Solution 3. Stock Solutions 1, 2, and 3 were mixed in a 1:1.1:1.1 ratio and annealed to obtain the annealed adapter. Annealing program: The temperature was reduced from 95 °C to 22 °C at a rate of -2 °C / min.

[0107] 3) The annealed linker sequence cross-links with GG-Dda helicase.

[0108] (1) Treat GG-Dda helicase (SEQ ID NO:4) with DTT (dithiothreitol, final concentration 2mM) and incubate at room temperature for 30 minutes to open the disulfide bonds, which is beneficial for the binding of proteins and nucleic acids in subsequent experiments;

[0109] (2) The annealed linker sequence obtained in step 2) was incubated with the disulfide-opening GG-Dda helicase at a molar ratio of 1:15 at room temperature for 30 min to allow the helicase to bind to the linker.

[0110] (3) Replace the buffer in the sample from step (2) with a desalting column;

[0111] (4) Add N,N,N',N'-tetramethylazodicarbonamide (TMAD, final concentration 500 μM) to the sample and incubate at 35°C for 1 h to lock the helicase onto the nucleic acid chain;

[0112] (5) Add ATP (final concentration 1mM) and incubate at room temperature for 30 min to allow the GG-Dda helicase bound to other sites on the nucleic acid chain to move and detach under the action of ATP;

[0113] (6) Purify the sample using molecular sieves, and collect the adapter complex that binds only one GG-Dda helicase according to the molecular weight; store it in a -20℃ freezer for subsequent experiments.

[0114] 2. Preparation of Tn5 transposable complex

[0115] Tn5 recognition sequences SEQ ID NO:5 and SEQ ID NO:6 were designed and synthesized. The two sequences were dissolved in annealing buffer (50 mM HEPES, pH 8.0 and 100 mM KCl) to prepare a 100 μM stock solution. The sequences were mixed in an equimolar ratio and annealed to obtain Tn5-Me. The annealing program could be: initially raise the temperature to 95℃ / 98℃, then lower the temperature by 1-2℃ per minute until it reaches 23-26℃. M-Tn5 transposase (SEQ ID NO:13) and Tn5-Me were gently mixed in an equimolar ratio and incubated at room temperature for 1 hour to obtain the Tn5 transposon complex.

[0116] 3. Total RNA extraction from Escherichia coli

[0117] Pick 3-5 single colonies of DH5α and place them in 2-8 mL of antibiotic-free LB broth. Incubate at 37°C with a shaker for 2-4 hours. The OD600 of the bacterial culture is measured to be 0.7 using a spectrophotometer. 1.2 mL of the bacterial culture is used for RNA extraction. In this example, RNA extraction was performed using the MolPure Bacterial RNA Kit (YE SEN), and the specific procedures were followed according to the kit's instructions. The concentration of the extracted E. coli RNA sample was measured to be 940 ng / μl using a nanodrop analyzer, with A260 / A280 = 2.02 and A260 / A230 = 2.09. The sample was also analyzed using an Agilent 4150 analyzer. Detailed quality control results can be found in [link to quality control information]. Figure 3 RNA samples that pass quality control are used for subsequent library construction.

[0118] 4. E. coli RNA fragmentation

[0119] The *E. coli* RNA obtained in step 3, the M-Tn5 transposon complex from step 2, and the reaction buffer (10 mM Tris-HCl, 5 mM MgCl2, 8% PEG8000, pH 7.5) were gently mixed and reacted in a PCR instrument at 16°C or 37°C for 15 minutes. The reaction was then immediately terminated by stopping the reaction with Stop Buffer (0.2% SDS) in a PCR instrument at 55°C for 10 minutes. The fragmentation results were characterized by agarose gel electrophoresis. Figure 2 As shown. Finally, the GeneJET PCR Purification Kit (Thermo Scientific) was used to analyze the results. Figure 2 The fragmented RNA was purified at 16°C and used for subsequent ligation reactions.

[0120] 5. Ligation of the adapter complex with fragmented E. coli RNA

[0121] The adapter complex from step 1 and the fragmented RNA from step 4 were incubated in ligation buffer (330 mM Tris-HCl, 50 mM MgCl2, 5 mM ATP, 30% Polyethylene glycol (PEG 6000), pH 8.0) and T4 DNA ligase at room temperature for 30 minutes, followed by incubation at 4°C for 20 minutes to complete the ligation reaction. The ligated samples were then purified using AMPure XPbeads. The purified samples were ready for sequencing.

[0122] 6. Sequencing of E. coli RNA samples

[0123] Electrical signal measurements were obtained from MspA nanopores embedded in the phospholipid bilayer of DPhPC (CAS No.: 207131-40-6, Chinese name: diaphytophosphatidylcholine) in a buffer solution (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), pH 8.0).

[0124] After achieving single-pore insertion of the phospholipid bilayer, residual excess nanopores were removed by flowing 2 ml of buffer (600 mM KCl, 75 mM K3[Fe(CN)6, 25 mM K4[Fe(CN)6]·3H2O, 100 mM HEPES, pH 8.0) through the system. The test sample from step 5, ATP (final concentration 2 mM), and MgCl2 (final concentration 10 mM) were then fed into the single-nanopore experimental system (total volume 100 μL), and the signal was measured for 1 h at a constant voltage of +180 mV (including a potential 2 s -180 mV voltage reversal).

[0125] Figure 3 This demonstrates that RNA can be directly constructed using the M-Tn5 transposon complex without reverse transcription. Furthermore, this direct RNA library construction method allows for more direct recording of methylation and other modification features present in the RNA itself.

[0126] Example 3: Sequencing of PhiX174 virion DNA single-stranded nucleic acid DNA

[0127] 1. Preparation of the joint compound

[0128] The preparation is consistent with the joint composite in step 1 of Example 1.

[0129] 2. Preparation of M-Tn5 transposase complex

[0130] This is consistent with the preparation of the M-Tn5 transposase complex in step 2 of Example 1.

[0131] 3. Preparation of PhiX174 virion DNA

[0132] The PhiX174 virion DNA (NEB, product number: N3023S), the M-Tn5 transposon complex from step 2, and the reaction buffer [10mM Tris-HCl, 5mM MgCl2, 8% PEG8000 (CAS No. 25322-68-3, Sigma No. P2139), pH 7.5] were gently mixed and reacted in a PCR instrument at 55°C for 10-12 minutes. The reaction was then immediately terminated with Stop Buffer (0.2% SDS, sodium dodecyl sulfate) and reacted in a PCR instrument at 55°C for 10 minutes. Finally, the fragmented DNA was purified using the GeneJET PCR Purification Kit (Thermo Scientific, Thermo product number K0701) for subsequent ligation reactions.

[0133] 4. Ligation of the adapter complex with fragmented DNA

[0134] The adapter complex from step 1 and the fragmented DNA from step 4 were incubated in ligation buffer (330 mM Tris-HCl, 50 mM MgCl2, 5 mM ATP, 30% Polyethylene glycol (PEG 6000, Sigma catalog number 81260), pH 8.0) and T4 DNA ligase at room temperature for 30 minutes, followed by incubation at 4°C for 20 minutes to complete the ligation reaction. The ligated sample was then purified using AMPure XP beads, and the purified sample could be directly used for sequencing.

[0135] 5. Sequencing of single-stranded DNA samples

[0136] The sequencing method is the same as that in Example 1. Figure 4 A typical nucleic acid perforation signal pattern is shown. Analysis of the perforation signal in this sample revealed that this library preparation method can detect and assemble the complete sequence information of PhiX174 virion DNA. This provides a novel method for sequencing DNA viruses, significantly reducing library preparation time.

[0137] Example 3: Sequencing of single-stranded RNA based on the M-MuA transposase system

[0138] 1. Preparation of the joint compound

[0139] The preparation of the joint compound in this embodiment is consistent with the preparation of the joint compound in step 1 of Example 1.

[0140] 2. Preparation of M-MuA transposase complex

[0141] The upstream primer SEQ ID NO:11 and the downstream primer SEQ ID NO:12 of M-MuA were designed and synthesized. The two primers were dissolved in annealing buffer (50 mM HEPES, pH 8.0 and 100 mM KCl) to prepare a 100 μM stock solution. The upstream and downstream primers were mixed in an equimolar ratio and then annealed to obtain MuA-Me. The annealing program could be: first raise the temperature to 95℃ / 98℃, then lower the temperature by 1-2℃ per minute until it reaches 23-26℃. The M-MuA transposase (SEQ ID NO:15) and MuA-Me were gently mixed in an equimolar ratio and incubated at 30℃ for 1 hour to obtain the MuA transposon complex.

[0142] 3. RNA preparation

[0143] Using lambda DNA (NEB) as a template and SEQ ID NO:8 and SEQ ID NO:9 as primers, PCR amplification was performed to obtain a 2Kb DNA sequence containing a T7 RNA Polymerase binding site. The obtained DNA sequence was purified and used as a template for subsequent transcription reactions using HiScribe. TM Transcription was performed using the T7 High Yield RNA Synthesis Kit (NEB) to obtain lambda 2K nt RNA, which was then purified using the TRIzol method. DNA template in the mRNA was removed using DNase I (GenScript), and the mRNA was treated at 37°C for 30 min, followed by further purification using the TRIzol method. The obtained lambda 2K nt RNA was stored at -80°C.

[0144] 4. RNA fragmentation

[0145] The purified RNA obtained in step 3, the MuA transposon complex from step 2, and the reaction buffer (5 mM Tris-HCl, 55 mM NaCl, 10 mM MgCl2, pH 8) were gently mixed and reacted in a PCR instrument at 37°C for 15 minutes. The reaction was then immediately terminated by reacting with Stop Buffer (0.2% SDS) in a PCR instrument at 55°C for 10 minutes. The fragmentation results were characterized by agarose gel electrophoresis. Figure 5As shown in the figure. Finally, the fragmented RNA was purified using the GeneJET PCR Purification Kit (Thermo Scientific) for subsequent ligation reactions.

[0146] 5. Ligation of the adapter complex with fragmented DNA

[0147] The adapter complex from step 1 and the fragmented DNA from step 4 were incubated in ligation buffer (330 mM Tris-HCl, 50 mM MgCl2, 5 mM ATP, 30% polyethylene glycol (PEG 6000), pH 8.0) and T4 DNA ligase at room temperature for 30 minutes, followed by incubation at 4°C for 20 minutes to complete the ligation reaction. The ligated samples were then purified using AMPure XPbeads. The purified samples were ready for sequencing.

[0148] 6. Sequencing of single-stranded DNA samples

[0149] The sequencing method was the same as that in Example 1. The perforation signal collection and analysis of this sample were performed as follows: Figure 5 As shown, under suitable reaction conditions, MuA transposase can also cleave single-stranded RNA, thereby enabling direct RNA library construction.

[0150] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

[0151] Sequence information:

[0152] SEQ ID NO:1: Connector sequence

[0153] SEQ ID NO:2: Sequence 2

[0154]

[0155] SEQ ID NO:3: Sequence 3

[0156]

[0157] SEQ ID NO:4: GG-Dda

[0158]

[0159] SEQ ID NO:5: Tn5 transposase recognition sequence

[0160] SEQ ID NO:6: Tn5 transposase recognition sequence

[0161] SEQ ID NO:7:

[0162]

[0163] SEQ ID NO:8: lambda DNA primer

[0164] SEQ ID NO:9: lambda DNA primer

[0165] SEQ ID NO:10: Wild-type Escherichia phage MuA transposase amino acid sequence

[0166] SEQ ID NO:11: (MuA-Me chain 1)

[0167]

[0168] SEQ ID NO:12: (MuA-Me chain 2)

[0169]

[0170] SEQ ID NO:13:M-Tn5

[0171]

[0172] SEQ ID NO:14: Truncation sequence of wild-type Escherichia phage MuA transposase

[0173] SEQ ID NO:15: M-MuA

[0174]

Claims

1. A method for labeling and fragmenting single-stranded nucleic acids, characterized in that: This includes using transposases to cleave the single-stranded nucleic acid into a double-stranded molecular sequence formed by internal base pairing, thereby completing the fragmentation and labeling of the single-stranded nucleic acid. The single-stranded nucleic acid is single-stranded DNA (ssDNA) and / or single-stranded RNA (ssRNA), and the transposase is a MuA transposase mutant, the amino acid sequence of which is shown in SEQ ID NO:

15.

2. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 1, characterized in that: The single-stranded RNA includes mRNA, repressive ssRNA, tRNA, and nucleic acid sequences from single-stranded RNA viruses.

3. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 1, characterized in that: The ssRNA includes any modified ssRNA.

4. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 3, characterized in that: The modifications include 2'-O-methyl (2'-O-Me), 2'-O-(2-methoxyethyl)(2'-O-moe), 2'-fluorine (2'-F), thiophosphate (PS) bond substitution, and m6A.

5. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 1, characterized in that: The single-stranded RNA contains its own complementary sequence, which allows a portion of the single-stranded RNA to fold and pair with itself to form a double-stranded molecular sequence.

6. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 5, characterized in that: The complementary sequences of the single-stranded nucleic acid are linked together by hydrogen bonds to form a double-stranded molecular sequence.

7. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 5, characterized in that: The double-stranded molecular sequence has local stability.

8. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 5, characterized in that: The double-stranded molecular sequence is located at any position of the single-stranded DNA and / or single-stranded RNA, and can be located at the 3' end, 5' end, or any intermediate position.

9. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 5, characterized in that: The double-stranded molecular sequence is located at multiple randomly distributed positions.

10. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 1, characterized in that: The internal bases can be adjacent bases or non-adjacent bases.

11. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 1, characterized in that: The transposase exists in the form of a transposase complex, which includes the transposase and a transposase recognition sequence.

12. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 11, characterized in that: The transposase recognition sequence optionally includes a ligase-recognized linker, aptamer, antibody, biotin, digoxigenin or digoxigenin ligand, bioorthogonal reactive group, methylation fragment, fluorescently labeled fragment, capture fragment, adapter sequence, primer sequence, promoter sequence or tag sequence, thereby making the fragmented nucleic acid molecule suitable for a variety of detection or analysis applications.

13. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 12, characterized in that: The transposase recognizes a Tn5-Me or MuA-Me sequence.

14. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 11, characterized in that: The transposase complex randomly binds to any position in the double-stranded molecular sequence, thereby cleaving the single-stranded nucleic acid into multiple single-stranded fragments and introducing the transposase recognition sequence into the single-stranded fragments.

15. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 14, characterized in that: The cleavage site is located at any position in the double-stranded molecule sequence.

16. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 14, characterized in that: The reaction conditions for the cutting are: reaction temperature 15~55℃, reaction time 15~60min.

17. The method for labeling and fragmenting single-stranded nucleic acids as described in claim 16, characterized in that: The reaction temperature is 37°C.

18. A library construction method for direct sequencing of single-stranded nucleic acids, characterized in that: This includes providing a sequence fragment to be tested; the method for preparing the sequence fragment to be tested is the method described in any one of claims 1-17.

19. The library construction method for direct sequencing of single-stranded nucleic acids as described in claim 18, characterized in that: include: 1) Using the method of any one of claims 1-17, a single-stranded nucleic acid is fragmented and a linker end of a transposase recognition sequence is introduced; 2) The fragmented single-stranded nucleic acids are ligated into sequencing adapter complexes to obtain sequencing libraries.

20. The library construction method for direct sequencing of single-stranded nucleic acids as described in claim 19, characterized in that: The sequencing adapter complex is an adapter complex used for nanopore sequencing.

21. The library construction method for direct sequencing of single-stranded nucleic acids as described in claim 19, characterized in that: The sequencing adapter complex includes regions for ligating the nucleic acid fragments to be analyzed.

22. The library construction method for direct sequencing of single-stranded nucleic acids as described in claim 21, characterized in that: The region contains a complementary sequence for helicase binding and ligation of double-stranded DNA fragments.

23. The library construction method for direct sequencing of single-stranded nucleic acids as described in claim 19, characterized in that: The transposase recognition sequence and the region for ligating the nucleic acid fragment to be analyzed have complementary sticky ends, and the ligation is achieved by a nucleic acid ligase.

24. The library construction method for direct sequencing of single-stranded nucleic acids as described in claim 23, characterized in that: The ligation is achieved using T4 DNA ligase.

25. The library construction method for direct sequencing of single-stranded nucleic acids as described in claim 19, characterized in that: The complementary sequences of the transfer strand in the transposase recognition sequence and the complementary sequences of the nucleic acid fragment linkage region to be analyzed in the sequencing adapter have complementary overhangs.

26. A sequencing library prepared by the library preparation method according to any one of claims 18-25.

27. A sequencing method, characterized in that: The sequencing method includes the steps of fragmenting and labeling single-stranded nucleic acids using the method described in any one of claims 1-17, or the steps of using the library construction method described in any one of claims 18-25, or the steps of sequencing the sequencing library described in claim 26.

28. A composition for constructing single-stranded nucleic acid nanopore sequencing libraries, characterized in that: The composition comprises: 1) Transposase and transposase recognition sequence; 2) A sequencing adapter complex, wherein the sequencing adapter includes a region for ligating the nucleic acid fragment to be analyzed; 3) The transposase recognition sequence and the region used for linking the nucleic acid fragment to be analyzed have complementary sticky ends; 4) Nucleotide ligase; The transposase is a MuA transposase mutant, and its amino acid sequence is shown in SEQ ID NO:

15.

29. Transposase mutants, including MuA transposase, which contains the amino acid sequence shown in SEQ ID NO:

15.

30. A nucleic acid sequence, wherein the nucleic acid sequence is: 1) a sequence encoding the transposase mutant of claim 29; and 2) a sequence complementary to 1).

31. A biological material expressing the transposase mutant of claim 29 or comprising the nucleic acid sequence of claim 30.

32. The application of the transposase mutant of claim 29, the nucleic acid sequence of claim 30, or the biological material of claim 31 in single-stranded nucleic acid molecular fragmentation or sequencing library construction.

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