Method for synthesizing single-stranded DNA, kit and application

Abstract

The invention discloses a method for synthesizing single-stranded DNA, a kit and application, and belongs to the technical field of biology. The donor DNA sequence and the receptor DNA sequence required during synthesis of the single-stranded DNA provided by the invention can be specifically combined with the target DNA under the catalytic action of the DNA ligase to synthesize the single-stranded DNA, and the synthesis efficiency of the single-stranded DNA is improved. Besides, the method for synthesizing the single-stranded DNA provided by the invention can be used for removing RNA in the reaction process to obtain the high-purity single-stranded DNA; the method and the corresponding kit can be used for construction of a DNA coding compound library, and have application value.

Description

Technical Field

[0001] The invention relates to a method, a kit and application for synthesizing single-stranded DNA, and belongs to the field of biotechnology. Background Art

[0002] DNA is composed of four bases. Different base arrangements determine different coded information. Therefore, DNA has a wide range of uses in the fields of genetic information storage, chemical information storage, image information storage, etc. For example, DNA can encode the information of compounds, and then establish a DNA-encoded compound library. In this method, compounds are built through the concept of "combinatorial chemistry". The DNA sequence can record the chemical synthesis information of each step. As the number of reactions increases, the chemical information increases, so the DNA sequence that records the chemical information also needs to be extended. Therefore, a large amount of DNA connection technology is required in the process of building this information library.

[0003] At present, DNA libraries can be divided into single-stranded DNA libraries and double-stranded DNA libraries according to DNA chains. Double-stranded DNA libraries are mainly connected through sticky ends using T4 DNA ligase. However, in double-stranded DNA, the molecular weight is large and the two chains are completely complementary, so dynamic short-chain DNA cannot be used for dynamic modification, which limits the development of this technology. Single-stranded DNA has a relatively small molecular weight and can dynamically complement with other short-chain DNAs, thereby increasing the diversity of the library. However, in the construction of single-stranded libraries, the connection of single-stranded DNA is a huge challenge. If T4 DNA ligase is used directly, the connection between single-stranded DNA is non-specific and specific sequence connection cannot be achieved.

[0004] Therefore, how to achieve efficient and accurate connection of single-stranded DNA is an urgent problem to be solved. Summary of the invention

[0005] Problem that the invention aims to solve

[0006] In order to overcome the defects of the prior art such as high cost and low efficiency in single-stranded DNA connection, the present invention provides a method for synthesizing single-stranded DNA guided by RNA and a kit for synthesizing single-stranded DNA, which solves the problems of high cost and limited efficiency in single-stranded DNA connection library construction.

[0007] Solutions for solving problems

[0008] [1]. A method for synthesizing single-stranded DNA, characterized in that the method for synthesizing single-stranded DNA comprises the following steps:

[0009] (1) The RNA fragment recognizes and binds to the complementary acceptor DNA fragment and donor DNA fragment;

[0010] (2) connecting the acceptor DNA fragment and the donor DNA fragment through DNA ligase mutants;

[0011] (3) removing RNA fragments in the reaction system to obtain single-stranded DNA;

[0012] The RNA fragment consists of at least 14 bases and comprises (a) and / or (b):

[0013] (a) a base fragment complementary to at least 6 consecutive bases at the 5' end of the donor DNA fragment,

[0014] (b) a base fragment complementary to at least 8 consecutive bases at the 3' end of the receptor DNA fragment,

[0015] Wherein, (a) and (b) have no overlapping parts, and the base complementary to the 3' end of the acceptor DNA fragment and the base complementary to the 5' end of the donor DNA fragment are adjacent;

[0016] Preferably, the first base from the 5' end of the donor DNA fragment is A or C, and the second base is T, G or C;

[0017] Preferably, the first base at the 3' end of the receptor DNA fragment is G, C or T, the second base is G or C, and the third base is G, C or T.

[0018] [2] The method for synthesizing single-stranded DNA according to [1] is characterized in that in step (1), the acceptor DNA, donor DNA and RNA fragment are mixed in a molar ratio of 1:1:(1-3), the mixed sample is reacted at not less than 80°C, preferably not less than 90°C, for 2-5 minutes, and then the sample is cooled to room temperature.

[0019] [3] The method for synthesizing single-stranded DNA according to [1] or [2], characterized in that the DNA ligase in step (2) is DNA ligase PBCV or a mutant thereof;

[0020] Optionally, the DNA ligase PBCV comprises the sequence as shown in SEQ ID NO:3, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:3;

[0021] Optionally, the DNA ligase PBCV mutant comprises the sequence as shown in SEQ ID NO: 1, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO: 1.

[0022] [4] The method for synthesizing single-stranded DNA according to [3], characterized in that the DNA ligase PBCV or its mutant is reacted in a system containing 30-60 mM Tris-HCl, 5-15 mM MgCl2, 0.5-2 mM ATP, 5-15 mM DTT and a pH of 7.0-8.0, and the reaction time is not less than 10 min.

[0023] Optionally, after the reaction is completed, the DNA ligase is inactivated at a temperature not lower than 90°C.

[0024] [5] The method for synthesizing single-stranded DNA according to any one of [1] to [4], characterized in that step (3) comprises the following steps: adding ribonuclease to the reaction solution obtained in step (2), reacting in a system containing 60-80 mM KCl, 40-60 mM Tris-HCl, 2-4 mM MgCl2, 2-15 mM DTT and a pH of 8.0-8.5 for not less than 25 min, adding ethanol to precipitate DNA after the reaction, centrifuging to obtain the precipitate, and adding water to dissolve the precipitate to obtain a solution containing single-stranded DNA;

[0025] Optionally, the ribonuclease is RNase H.

[0026] [6]. A kit for synthesizing single-stranded DNA, characterized in that the kit comprises a DNA ligase, an RNA fragment, an acceptor DNA fragment and a donor DNA fragment, wherein the RNA fragment consists of at least 14 bases and comprises (a) and / or (b):

[0027] (a) a base fragment complementary to at least 6 consecutive bases at the 5' end of the donor DNA fragment,

[0028] (b) a base fragment complementary to at least 8 consecutive bases at the 3' end of the receptor DNA fragment,

[0029] Wherein, (a) and (b) have no overlapping parts, and the base complementary to the 3' end of the acceptor DNA fragment and the base complementary to the 5' end of the donor DNA fragment are adjacent;

[0030] Preferably, the first base from the 5' end of the donor DNA fragment is A or C, and the second base is T, G or C;

[0031] Preferably, the first base at the 3' end of the acceptor DNA fragment is G, C or T, the second base is G or C, and the third base is G, C or T.

[0032] [7] The kit for synthesizing single-stranded DNA according to [6], characterized in that the DNA ligase is DNA ligase PBCV or a mutant thereof;

[0033] Optionally, the DNA ligase PBCV comprises the sequence as shown in SEQ ID NO:3, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:3;

[0034] Optionally, the DNA ligase PBCV mutant comprises the sequence as shown in SEQ ID NO: 1, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO: 1.

[0035] [8] The kit for synthesizing single-stranded DNA according to [6] or [7], characterized in that the kit further comprises a reaction solution of the ligase; optionally, the reaction solution comprises Tris-HCl, MgCl2, ATP and / or DTT;

[0036] Optionally, the kit comprises ribonuclease.

[0037] [9]. A single-stranded DNA encoded compound library, characterized in that the single-stranded DNA encoded compound library is constructed by the method for synthesizing single-stranded DNA as described in any one of [1] to [5], or the kit for synthesizing single-stranded DNA as described in any one of [6] to [8].

[0038]

[10] . Use of the single-stranded DNA synthesis kit described in any one of [6] to [8] in synthesizing single-stranded DNA or constructing a DNA-encoded compound library.

[0039] Effects of the Invention

[0040] In some embodiments, the catalytic ability of the PBCV ligase mutant provided by the present invention is significantly enhanced compared with the wild type, thereby improving the synthesis efficiency of single-stranded DNA;

[0041] In some embodiments, the acceptor DNA and donor DNA sequences provided by the present invention can improve the synthesis efficiency of single-stranded DNA;

[0042] In some embodiments, the method for synthesizing single-stranded DNA provided by the present invention can specifically bind to the target DNA and synthesize single-stranded DNA;

[0043] In some embodiments, the method for synthesizing single-stranded DNA provided by the present invention removes RNA during the reaction process to obtain high-purity single-stranded DNA;

[0044] In some embodiments, the PBCV ligase mutant, kit, and method for synthesizing single-stranded DNA provided by the present invention can be used in the construction of a DNA-encoded compound library and have application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram of RNA-guided single-stranded DNA ligation;

[0046] Figure 2 The figure is the purification result of PBCV1-DNA ligase mutation;

[0047] Figure 3 The graphs are reaction results of different ratios of acceptor DNA, donor DNA and RNA;

[0048] Figure 4 The figure is a comparison of the ligation efficiency of the wild-type and mutant enzymes;

[0049] Figure 5 This is the optimization result diagram of bases at different positions of donor DNA;

[0050] Figure 6 This is the optimization result diagram of bases at different positions of the receptor DNA;

[0051] Figure 7 Specificity detection diagram of RNA-guided DNA ligation reaction. DETAILED DESCRIPTION

[0052] Various exemplary embodiments, features and aspects of the present invention will be described in detail below. The word "exemplary" used here means "used as an example, embodiment or illustrative". Any embodiment described here as "exemplary" is not necessarily interpreted as being superior or better than other embodiments.

[0053] In addition, in order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. It should be understood by those skilled in the art that the present invention can be implemented without certain specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present invention.

[0054] Unless otherwise stated, the units used in this specification are all international standard units, and the numerical values ​​and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0055] In this specification, the word "may" means both performing a certain process and not performing a certain process.

[0056] As used in the claims and the specification, the words "comprise", "have", "include" or "contain" are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product or device comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to the process, method, product or device.

[0057] As used herein, the term "about" means that a value includes the standard deviation of error of the device or method used to determine the value. Exemplarily, the standard deviation is generally within the range of 20-30% of the original value.

[0058] Although the disclosure supports the definition of the term "or" as only alternatives and "and / or", the term "or" in the claims means "and / or" unless it is explicitly stated that it is only alternatives or the alternatives are mutually exclusive. In this specification, when the term "and / or" is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0059] In this specification, the references to "some specific / preferred embodiments", "other specific / preferred embodiments", "embodiments", etc., mean that the specific elements (e.g., features, structures, properties and / or characteristics) described in connection with the embodiments are included in at least one embodiment described herein, and may or may not exist in other embodiments. In addition, it should be understood that the elements may be combined in various embodiments in any suitable manner.

[0060] In this specification, the "donor DNA" and "acceptor DNA" mentioned are both single-stranded DNA fragments, wherein the donor DNA fragment consists of at least 6 bases and the acceptor DNA fragment consists of at least 8 bases; the 5'-P end of the donor DNA fragment is connected to the 3'-OH end of the acceptor DNA fragment to obtain a new single-stranded DNA fragment, which is a DNA fragment in the DNA-encoded compound library.

[0061] In this specification, the term "DNA ligase" is also called "DNA adhesive enzyme" which connects the 3'-OH end of a base in a DNA chain with the 5'-P end of its adjacent base to form a phosphodiester bond between the two, thereby connecting two adjacent bases. DNA ligases are divided into two major categories: one is ATP-dependent DNA ligase that uses the energy of ATP to catalyze the formation of a phosphodiester bond between two nucleotide chains, and the other is nicotinamide adenine dinucleotide (NAD + ) catalyzes the formation of a phosphodiester bond between two nucleotide chains. +Dependent DNA ligase. In some embodiments of the present invention, the DNA ligase is PBCV ligase, also known as SplintR ligase or Chorella virus DNA ligase, which can efficiently catalyze the connection of adjacent DNA nucleotides. This connection process requires a complementary RNA to act as a "splint" or "bridge" between two DNA single strands.

[0062] In this specification, the term "amino acid" may include natural amino acids, non-natural amino acids, amino acid analogs and all their D and L stereoisomers. The amino acids and their abbreviations and English abbreviations in the present invention are as follows:

[0063] Histidine (His, H); serine (Ser, S); glutamic acid (Glu, E); glutamine (Gln, Q); glycine (Gly, G); threonine (Thr, T); phenylalanine (Phe, F); aspartic acid (Asp, D); tyrosine (Tyr, Y); leucine (Leu, L); isoleucine (Ile, I); arginine (Arg, R); alanine (Ala, A); valine (Val, V); tryptophan (Trp, W); methionine (Met, M); asparagine (Asn, N); cysteine ​​(Cys, C); lysine (Lys, K); proline (Pro, P).

[0064] In this specification, the term "mutant" refers to a polynucleotide or polypeptide containing changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions relative to a "wild type" or "compared" polynucleotide or polypeptide, wherein substitution refers to replacing a nucleotide or amino acid occupying a position with a different nucleotide or amino acid. Deletion refers to the removal of a nucleotide or amino acid occupying a position. Insertion refers to the addition of a nucleotide or amino acid adjacent to and immediately following the nucleotide or amino acid occupying the position.

[0065] In this specification, the term "amino acid mutation" or "nucleotide mutation" includes "substitution, duplication, deletion or addition of one or more amino acids or nucleotides". In this specification, the term "mutation" refers to a change in a nucleotide sequence or an amino acid sequence. In a specific embodiment, the term "mutation" refers to "substitution".

[0066] In this specification, "mutation" may also include the addition, deletion or substitution of amino acids that do not affect the activity of PBCV ligase at one or more positions of the sequence shown in SEQ ID NO: 1. It is well known that changing a few amino acid residues in certain regions of a polypeptide, such as non-important regions, will not substantially change the biological activity, for example, a sequence obtained by appropriately replacing, adding or deleting certain amino acids will not affect its activity.

[0067] In this specification, the term "polynucleotide" refers to a polymer composed of nucleotides. A polynucleotide can be in the form of a separate fragment or a component of a larger nucleotide sequence structure, which is derived from a nucleotide sequence separated at least once in quantity or concentration, and can be identified, manipulated, and restored by standard molecular biology methods (e.g., using a cloning vector) and its component nucleotide sequences. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e., A, U, G, C), in which "U" replaces "T". In other words, "polynucleotide" refers to a nucleotide polymer removed from other nucleotides (separate fragments or entire fragments), or can be a component or ingredient of a larger nucleotide structure, such as an expression vector or a polycistronic sequence.

[0068] In this specification, the term "sequence identity" or "percent identity" in the comparison of two nucleic acids or polypeptides means that they are identical or have a specific percentage of identical sequences when compared and aligned with maximum correspondence using a nucleotide or amino acid residue sequence comparison algorithm or measured by visual inspection. In other words, the identity of a nucleotide or amino acid sequence can be defined by the following ratio, which is the ratio of the number of nucleotides or amino acids that are consistent when two or more nucleotide or amino acid sequences are aligned in a manner that maximizes the number of consistent nucleotides or amino acids and adds gaps as needed, to the total number of nucleotides or amino acids in the aligned portion.

[0069] In the present specification, the method for determining "sequence identity" or "percent identity" involved includes but is not limited to: Computational Molecular Biology, Lesk, AM, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., M Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Biology, 1996. Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining the identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0070] In this specification, the term "ribonuclease" or "RNase" refers to an enzyme that can hydrolyze the phosphodiester bond of RNA. It is a nuclease that catalyzes the hydrolysis of RNA, and the reaction products are mononucleotides and oligonucleotides. The oligonucleotide composition of the products catalyzed by different ribonucleases is different. In some specific embodiments of the present invention, the ribonuclease is RNase H, which is an endoribonuclease that can specifically decompose the RNA chain in the RNA-DNA hybrid to produce a product with free 3'-OH and 5'-phosphate ends.

[0071] In this specification, the term "DNA encoded libraries" (DELs) is a library of small molecule compounds covalently linked to specific DNA sequences. By amplifying and sequencing the DNA sequence of the bound compound, the compound can be effectively identified, thereby performing rapid and large-scale high-throughput screening.

[0072] First aspect

[0073] The first aspect of the present invention provides a method for synthesizing single-stranded DNA, which guides the adjacent binding of donor DNA and acceptor DNA through RNA, and then connects the 3'-OH end of the base of the acceptor DNA and the 5'-P end of the base of the adjacent donor DNA under the catalytic action of DNA ligase, thereby realizing the synthesis of single-stranded DNA with high specificity and high synthesis efficiency.

[0074] In some embodiments, the method for synthesizing single-stranded DNA comprises the following steps:

[0075] (1) The RNA fragment recognizes and binds to the complementary acceptor DNA fragment and donor DNA fragment;

[0076] (2) DNA ligase mediates the connection between the acceptor DNA fragment and the donor DNA fragment;

[0077] (3) Remove RNA fragments from the reaction system to obtain single-stranded DNA.

[0078] In some specific embodiments, the RNA fragment consists of at least 14 bases and comprises (a) and / or (b):

[0079] (a) a base fragment complementary to at least 6 consecutive bases at the 5' end of the donor DNA fragment,

[0080] (b) a base fragment complementary to at least 8 consecutive bases at the 3' end of the receptor DNA fragment,

[0081] There is no overlap between (a) and (b), and the base complementary to the 3' end of the acceptor DNA fragment and the base complementary to the 5' end of the donor DNA fragment are adjacent.

[0082] In the present invention, the "non-overlapping portion between (a) and (b)" means that the base fragment from the base complementary to the 5' end of the donor DNA fragment toward the 3' end of the donor DNA fragment does not overlap with the (b); or, the base fragment from the base complementary to the 3' end of the acceptor DNA fragment toward the 5' end of the acceptor DNA fragment does not overlap with the (a).

[0083] In some preferred embodiments, the first base at the 5' end of the donor DNA fragment is A or C, and the second base is T, G or C.

[0084] In some preferred embodiments, the first base at the 3' end of the acceptor DNA fragment is G, C or T, the second base is G or C, and the third base is G, C or T.

[0085] In some embodiments, the step (1) is to mix the acceptor DNA, donor DNA and RNA fragment in a molar ratio of 1:1:(1-3), react the mixed sample at no less than 80°C, react for 2-5 minutes, and then cool the sample to room temperature.

[0086] In some specific embodiments, the recipient DNA, donor DNA and RNA are mixed in a molar ratio of 1:1:1, 1:1:1.5, 1:1:2 or 1:1:3, preferably 1:1:2 or 1:1:3, more preferably 1:1:3.

[0087] In some specific embodiments, the mixed sample is reacted at no less than 90°C, preferably in a metal bath at 90°C for 3 minutes to denature the RNA and DNA, and then the denatured RNA and DNA are cooled to room temperature for annealing to achieve the binding of the RNA fragments to the receptor DNA fragments and the donor DNA fragments, respectively.

[0088] In some specific embodiments, the ligase in step (2) is DNA ligase PBCV or a mutant thereof.

[0089] In some optional embodiments, the DNA ligase PBCV comprises the sequence shown in SEQ ID NO:3, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:3.

[0090] In other optional embodiments, the DNA ligase PBCV mutant comprises a sequence as shown in SEQ ID NO:1, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:1; preferably, the DNA ligase PBCV mutant is encoded by a polynucleotide as shown in SEQ ID NO:2.

[0091] In some optional embodiments, the PBCV ligase mutant is reacted in a system containing 30-60 mM Tris-HCl, 5-15 mM MgCl2, 0.5-2 mM ATP, 5-15 mM DTT and a pH of 7.0-8.0.

[0092] In some optional embodiments, after the ligation reaction catalyzed by the ligase is completed, urea loading buffer is added to the reaction solution to inactivate the ligase at a temperature not lower than 90°C.

[0093] In some embodiments, step (3) is to add RNase enzyme to the reaction solution obtained in step (2) to remove RNA in the reaction solution to obtain DNA with higher purity, and the reaction system contains substances suitable for RNase enzyme to act.

[0094] In some preferred embodiments, the RNase enzyme is RNase H; the reaction system comprises 60-80 mM KCl, 40-60 mM Tris-HCl, 2-4 mM MgCl2 and 2-15 mM DTT; preferably 60-80 mM KCl, 40-60 mM Tris-HCl, 2-4 mM MgCl2 and 9-11 mM DTT; more preferably 75-80 mM KCl, 50-60 mM Tris-HCl, 3-4 mM MgCl2 and 10-11 mM DTT.

[0095] In some specific embodiments, the reaction is carried out at a pH of 8.0 to 8.5, preferably 8.0 to 8.3; the reaction time is not less than 25 min, preferably not less than 30 min.

[0096] Further, after the above reaction is completed, ethanol is added to the reaction solution to precipitate the DNA, the precipitate is centrifuged, and water is added to the precipitate to dissolve it to obtain a solution containing single-stranded DNA. In some more specific embodiments, after the above reaction is completed, 5M NaCl is added in an amount of 10% of the volume of the reaction solution, 2.5 times the volume of 100% pre-cooled ethanol, mixed, and allowed to stand at -80°C for 1 hour or overnight. After standing, take it out, and after melting, centrifuge it at 12000g in an EP tube for 15 minutes. After centrifugation, remove the supernatant, place it in a metal bath at 45-50°C for incubation, remove residual ethanol, and dissolve the solid obtained after removing ethanol with ddH2O to obtain a solution containing single-stranded DNA.

[0097] Second aspect

[0098] The second aspect of the present invention provides a kit for synthesizing single-stranded DNA, the kit comprising DNA ligase, RNA fragments, single-stranded DNA fragments and DNA ligase; the single-stranded DNA fragments comprise donor DNA fragments and acceptor DNA fragments.

[0099] In some specific embodiments, the RNA fragment consists of at least 14 bases and comprises (a) and / or (b):

[0100] (a) a base fragment complementary to at least 6 consecutive bases at the 5' end of the donor DNA fragment,

[0101] (b) a base segment complementary to at least 8 consecutive bases at the 3' end of the acceptor DNA segment, wherein (a) and (b) have no overlapping parts, and the bases complementary to the 3' end of the acceptor DNA segment and the bases complementary to the 5' end of the donor DNA segment are adjacent;

[0102] In some preferred embodiments, the first base at the 5' end of the donor DNA fragment is A or C, and the second base is T, G or C.

[0103] In some preferred embodiments, the first base at the 3' end of the acceptor DNA fragment is G, C or T, the second base is G or C, and the third base is G, C or T.

[0104] In other specific embodiments, the kit further comprises a reaction solution of the DNA ligase, and optionally, the reaction solution comprises Tris-HCl, MgCl2, ATP and / or DTT.

[0105] Furthermore, the working concentrations of Tris-HCl, MgCl2, ATP and DTT are 30-60mM, 5-15mM, 0.5-2mM and 5-15mM, respectively; preferably 50-60mM, 10-15mM, 1-2mM and 10-15mM; more preferably 50mM, 10mM, 1mM and 10mM.

[0106] In some specific embodiments, the kit further comprises ribonuclease. In some optional embodiments, the ribonuclease is RNase H, and the kit may further comprise a reaction solution of the RNase H. Optionally, the reaction solution comprises KCl, Tris-HCl, MgCl2 and / or DTT, and the working concentrations thereof are 60-80mM, 40-60mM, 2-4mM, 9-11mM, respectively; preferably 75-80mM, 50-60mM, 3-4mM, 10-11mM; more preferably 75mM, 50mM, 3mM, 10mM.

[0107] In some optional embodiments, the above kit contains a substance for precipitating DNA, and the substance for precipitating DNA includes sodium ions and ethanol; optionally, the sodium ions are provided by sodium chloride or sodium acetate.

[0108] The third aspect

[0109] The third aspect of the present invention provides a single-stranded DNA encoded compound library, which is constructed by the method for synthesizing single-stranded DNA as described in the first aspect, or the kit for synthesizing single-stranded DNA as described in the second aspect.

[0110] The fourth aspect

[0111] The fourth aspect of the present invention provides the use of the single-stranded DNA synthesis kit as described in the second aspect in synthesizing single-stranded DNA and constructing a DNA-encoded compound library.

[0112] Example

[0113] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0114] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0115] The experimental methods in the following examples without specifying specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions.

[0116] In the present invention, conventional reagents such as Tris-Base, BSA, NaCl, Tris-HCl, MgSO4, imidazole and glycerol were purchased from Thermo Fisher; the synthesis of donor DNA, acceptor DNA and RNA was completed by Nanjing GenScript Company; and the Ni-NTA nickel column (20502ES10) was purchased from Yisheng Biotechnology Company.

[0117] Example 1: Expression and purification of PBCV DNase mutants

[0118] In this example, the coding gene of PBCV DNA ligase mutant 1 (amino acid sequence as shown in SEQ ID NO: 1) was codon optimized to obtain a nucleotide sequence as shown in SEQ ID NO: 2, and the nucleotide sequence obtained by codon optimization was cloned into pET-28a plasmid to construct a recombinant plasmid for fusion expression of PBCV DNA ligase mutant 1 and His-tag (this step was completed by Nanjing GenScript Co., Ltd.), and the recombinant plasmid with His-tag was expressed in Escherichia coli BL21 (DE3), affinity purified using a nickel column, eluted using different concentrations of imidazole, and desalted after elution for ligation experiments.

[0119] result:

[0120] like Figure 2 As shown, PBCV DNA ligase can be soluble expressed in E. coli, bound to a nickel column, and eluted with an imidazole solution to achieve affinity purification.

[0121] According to the above method, the wild type PBCV DNA ligase, mutant 2 PBCV DNA ligase, and mutant 3 PBCV DNA ligase were expressed and purified; the amino acid sequence of the wild type PBCV DNA ligase is shown in SEQ ID NO:3, and the corresponding nucleotide sequence of the coding gene after codon optimization is shown in SEQ ID NO:4; the amino acid sequence of mutant 2 PBCV DNA ligase is shown in SEQ ID NO:5, and the corresponding nucleotide sequence of the coding gene after codon optimization is shown in SEQ ID NO:6; the amino acid sequence of mutant 3 PBCV DNA ligase is shown in SEQ ID NO:7, and the corresponding nucleotide sequence of the coding gene after codon optimization is shown in SEQ ID NO:8.

[0122] Example 2: RNA-guided single-stranded DNA ligation reaction

[0123] In this example, RNA fragments, acceptor DNA fragments, donor DNA fragments, and the PBCV DNA enzyme obtained in Example 1 above were used to perform single-stranded DNA ligation reaction.

[0124] RNA: AGAUUACUTAUACC (SEQ ID NO:9),

[0125] Receptor DNA: TCTCCAGTTACTTCTAATGT (SEQ ID NO: 10),

[0126] Donor DNA: ATATGGGAGGATGTTACATCCGATCGCAG (SEQ ID NO: 11).

[0127] (1) Single-stranded DNA ligation reaction with different ratios of acceptor DNA, donor DNA and RNA

[0128] Take 10 μM of the above RNA fragment, acceptor DNA fragment and donor DNA fragment, 10 μL each, and mix them according to the molar ratio of acceptor DNA: donor DNA: RNA of 1:1:1, 1:1:1.5, 1:1:2, 1:1:3, and denature the mixed sample in a metal bath at 90°C for 3 minutes, then turn off the metal bath and let the mixed sample cool naturally to room temperature (annealing). After the mixed sample is annealed, centrifuge, take 3 μL of the supernatant, add 5 μL of DEPC water, 1 μL of PBCV-1 reaction solution (the working concentrations of each substance in the reaction solution are 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5), 1 μL of PBCV DNA ligase mutant 1 to 10 μL system, centrifuge and mix, and connect at room temperature for 15 minutes. Then, 10 μL of urea loading buffer (C506046, Sangon Biotechnology Co., Ltd.) was added, mixed and incubated at 95°C for 3 minutes to denature the protein and terminate the reaction.

[0129] The single-stranded DNA was connected by PBCV ligase, and then 1 μL RNase H (12906ES60, Yisheng Biotechnology Co., Ltd.) was added to the RNase H reaction solution (the solution contains 75mM KCl, 50mM Tris-HCl, 3mM MgCl2, 10mM DTT, pH 8.3) for 30 minutes. After the reaction, 5M NaCl was added at 10% of the volume of the reaction solution, and 100% ethanol pre-cooled in a -80°C refrigerator with a volume of 2.5 times the volume of the reaction solution was mixed by pipetting, and allowed to stand at -80°C for 1 hour or overnight. After standing, take it out, and after melting, centrifuge it at 12000g in an EP tube for 15 minutes. After centrifugation, remove the supernatant, place it in a metal bath at 50°C for incubation, and remove the residual ethanol. The residual solid was dissolved with ddH2O, and the DNA concentration was measured using Nanodrop for quantification.

[0130] result:

[0131] Figure 3 Using PBCV DNA ligase mutant 1, donor DNA, acceptor DNA and RNA are combined to form a complete DNA, and its efficiency is related to the ratio of each other.

[0132] (2) Single-stranded DNA ligation reaction with different PBCV DNA enzymes

[0133] Take 10 μM of the above RNA fragment, acceptor DNA fragment and donor DNA fragment, 10 μL each, mix them in a ratio of acceptor DNA: donor DNA: RNA of 1:1:3, denature the mixed sample in a metal bath at 90°C for 3 minutes, then close the metal bath and let the mixed sample cool naturally to room temperature (annealing). After annealing, centrifuge the mixed sample, take 3 μL of the supernatant, add 5 μL of DEPC water, 1 μL of PBCV-1 reaction solution (containing 50 mM Tris-HCl, 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5), 1 μg of PBCV DNA ligase (PBCV DNA ligase wild type, PBCV DNA ligase mutant 1, PBCV DNA ligase mutant 2 or PBCV DNA ligase mutant 3) to the 10 μL system, centrifuge and mix, and connect at room temperature for 15 minutes. Then, 10 μL of urea loading buffer (C506046, Sangon Biotechnology Co., Ltd.) was added, mixed and incubated at 95°C for 3 minutes to denature the protein and terminate the reaction.

[0134] The single-stranded DNA was connected by PBCV ligase, and then 1 μL RNase H (12906ES60, Yisheng Biotechnology Co., Ltd.) was added to the RNase H reaction solution (the solution contains 75mM KCl, 5mM Tris-HCl, 3mM MgCl2, 10mM DTT, pH 8.3) for 30 minutes. After the reaction, 5M NaCl was added at 10% of the volume of the reaction solution, and 100% ethanol pre-cooled in a -80°C refrigerator with a volume of 2.5 times the reaction solution was mixed by pipetting, and allowed to stand at -80°C for 1 hour or overnight. After standing, take it out, and after melting, centrifuge it at 12000g in an EP tube for 15 minutes. After centrifugation, remove the supernatant, place it in a metal bath at 50°C for incubation, and remove residual ethanol. The residual solid was dissolved with ddH2O, and the DNA concentration was measured using Nanodrop for quantification.

[0135] like Figure 4 As shown, the catalytic abilities of the wild-type PBCV DNA ligase, PBCV DNA ligase mutant 1, PBCV DNA ligase mutant 2 and PBCV DNA ligase mutant 3 are different. The ligation efficiency of the ligation reaction using PBCV DNA ligase mutant 1 is significantly improved compared with the wild-type, that is, the catalytic ability is significantly improved relative to the wild-type.

[0136]

[0137] Example 3: Optimization of single-stranded DNA linker sequence

[0138] Ligase is sensitive to the nucleotides in the binding region. In this example, the nucleotide sequence in the binding region is screened to improve the sensitivity of PBCV DNA ligase and increase the efficiency of ligation.

[0139] First, the donor DNA part was studied: an RNA containing 14 bases (as shown in SEQ ID NO:9) was used as a guide sequence, in which 6 nucleic acids were complementary to the donor DNA and 8 nucleic acids were complementary to the recipient DNA; then, saturation mutation was performed on the bases at each position of the donor DNA (as shown in SEQ ID NO:11) or the recipient DNA (as shown in SEQ ID NO:10) to verify the effect of each site on the ligation reaction.

[0140] The specific reaction steps refer to Example 2, using PBCV DNA ligase mutant 1, and the reaction was carried out at a molar ratio of acceptor DNA:donor DNA:RNA of 1:1:3.

[0141] result:

[0142] like Figure 5 and Figure 6 As shown, Figure 5 To optimize the bases at different positions of the donor DNA (1 to 6 in the figure are the 1st to 6th bases from the 5' end to the 3' end). For the donor DNA fragment, the first two bases have a greater impact, and the most important base is p(A / C)(T / G / C) at the 5' end; Figure 6 To optimize the bases at different positions of the receptor DNA (1 to 8 in the figure are the 1st to 8th bases from the 3' end to the 5' end). The three bases at the 3' end of the receptor DNA fragment have a greater impact on the ligation reaction. The best bases are (G / C / T)(G / C)(G / C / T).

[0143] Example 4: Verification of the specificity of different RNAs for single-stranded DNA

[0144] The single-stranded DNA ligation reaction has high requirements for specificity. Only with strong specificity can the connection and information recording be accurate. We use acceptor DNA1, donor DNA1, RNA1, acceptor DNA2, donor DNA2 and RNA2 to cross-carry out enzyme ligation reaction.

[0145] The specific reaction steps refer to Example 2, using PBCV DNA ligase mutant 1, and the reaction was carried out at a molar ratio of acceptor DNA:donor DNA:RNA of 1:1:3.

[0146] Receptor DNA 1: TATAACTTTACTGCCAATGT (SEQ ID NO: 12),

[0147] Donor DNA 1: ATACCGGACCTACAATGTACCAGAAGCGTC (SEQ ID NO: 13),

[0148] RNA1: CGGUAUACAUUGGC (SEQ ID NO: 14),

[0149] Receptor DNA2: TATAACTTTACTCCGCAGGT (SEQ ID NO: 15),

[0150] Donor DNA2: ATTGCCGACCTACAATGTACCAGAAGCGTC (SEQ ID NO: 16),

[0151] RNA2: GGCAAUACCUGCGC (SEQ ID NO: 17).

[0152] result:

[0153] like Figure 7 As shown, mismatched RNA cannot guide the connection of DNA, indicating that the method for synthesizing single-stranded DNA provided by the present invention has good specificity.

[0154] >Amino acid sequence of PBCV ligase mutant 1 (SEQ ID NO: 1)

[0155] MAITKPILAATLENIEDVQFPCLATPKYDGIRSVKQTQMLSRTFKPIRNSVMNRLLTELLPEGSDSEIVIEGATFQDTTSAVMTGHKMYNAKFSLFWFDYVTDDPLKKYIDRVEDMKNYITVHPHILEHAQVKIIPLIPVEINNITELL QYERDVLSKGFEGLMLRKPDGKYKFGRSTLKEGLILKMKQFKDAEATIISMTALFKNTNTKTKDNFGYSKRSTHKSGKVEEDVMGSIEVDYDGVVFSIGTGFDADQRRDFWQNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRHEEDR*

[0156] >Nucleotide sequence of the gene encoding PBCV ligase mutant 1 (SEQ ID NO: 2)

[0157] ATGGCAATTACTAAGCCGATTTTGGCAGCCACGCTTGAAAACATCGAGGACGTACA

[0158] ATTTCCGTGCTTAGCCACGCCAAAATACGACGGAATCCGCTCTGTTAAACAAACTCA

[0159] GATGCTTTCTCGTACCTTTAAACCAATTCGTAATTCAGTGATGAACCGCTTATTAACC

[0160] GAGTTACTTCCGGAGGGGAGCGATTCCGAAATCGTAATTGAGGGTGCAACATTCCA

[0161] GGATACCACATCTGCAGTTATGACGGGACACAAAATGTACAATGCCAAGTTCTCGCT

[0162] TTTTTGGTTTGACTACGTTACGGACGATCCTTTGAAGAAATACATTGATCGCGTGGA

[0163] GGATATGAAGAATTACATCACAGTACACCCCCATATTTTAGAACACGCGCAGGTGAA

[0164] AATTATTCCGTTAATCCCCGTAGAGATCAACAATATCACGGAGCTGCTTCAGTACGA

[0165] ACGTGATGTCTTATCTAAGGGTTTTGAAGGTTTGATGCTGCGCAAGCCAGATGGCAA

[0166] ATATAAGTTCGGGCGCAGTACATTAAAGGAAGGTTTGATCTTAAAGATGAAACAATT

[0167] TAAAGATGCGGAGGCAACTATTATCAGTATGACTGCGCTTTTCAAGAATACAAATAC

[0168] CAAGACAAAGGACAATTTCGGGTACTCCAAACGCAGTACGCATAAATCCGGGAAGG

[0169] TGGAAGAAGACGTAATGGGGAGCATTGAAGTAGACTACGACGGGGTGGTGTTTAGT

[0170] ATCGGAACAGGATTCGATGCCGATCAACGCCGTGACTTCTGGCAAAATAAAGAGAG

[0171] TTACATTGGAAAGATGGTCAAGTTTAAATACTTCGAAATGGGGTCAAAGGATTGCCC

[0172] ACGTTTCCCTGTGTTCATCGGTATCCGCCACGAGGAAGATCGTTAA

[0173] >Amino acid sequence of wild-type PBCV ligase (SEQ ID NO:3)

[0174] MAITKPLLAATLENIEDVQFPCLATPKIAGIRSVKQTQMLSRTFKPIRNSVMNRLLTELL

[0175] PEGSDGEISIEGATFQDTTSAVMTGHKMYNAKFSYYWFDYVTDDPLKKYIDRVEDMK

[0176] NYITVHPHILEHAQVKIIPLIPVEINNITELLQYERDVLSKGFEGVMIRKPDGKYKFGRST

[0177] LKEGILLKMKQFKDAEATIISMTALFKNTNTKTKDNFGYSKRSTHKSGKVEEDVMGSI

[0178] EVDYDGVVFSIGTGFDADQRRDFWQNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRHEEDR*

[0179] >Nucleotide sequence of the coding gene of wild-type PBCV ligase (SEQ ID NO:4)

[0180] ATGGCAATTACTAAGCCGTTGTTGGCAGCCACGCTTGAAAACATCGAGGACGTACA

[0181] ATTTCCGTGCTTAGCCACGCCAAAAATCGCAGGAATCCGCTCTGTTAAACAAACTC

[0182] AGATGCTTTCTCGTACCTTTAAACCAATTCGTAATTCAGTGATGAACCGCTTATTAAC

[0183] CGAGTTACTTCCGGAGGGGAGCGATGGAGAAATCTCAAATTGAGGGTGCAACATTCC

[0184] AGGATACCACATCTGCAGTTATGACGGGACACAAAATGTACAATGCCAAGTTCTCGT

[0185] ACTACTGGTTTGACTACGTTACGGACGATCCTTTGAAGAAATACATTGATCGCGTGG

[0186] AGGATATGAAGAATTACATCACAGTACACCCCCATATTTTAGAACACGCGCAGGTGA

[0187] AAATTATTCCGTTAATCCCCGTAGAGATCAACAATATCACGGAGCTGCTTCAGTACG

[0188] AACGTGATGTCTTATCTAAGGGTTTTGAAGGTGTGATGATCCGCAAGCCAGATGGCA

[0189] AATATAAGTTCGGGCGCAGTACATTAAAAGGAAGGTATCTTGTTAAAGATGAAACAAT

[0190] TTAAAGATGCGGAGGCAACTATTATCAGTATGACTGCGCTTTTCAAGAATACAAATA

[0191] CCAAGACAAAGGACAATTTCGGGTACTCCAAACGCAGTACGCATAAATCCGGGAAG

[0192] GTGGAAGAAGACGTAATGGGGAGCATTGAAGTAGACTACGACGGGGTGGTGTTTA

[0193] GTATCGGAACAGGATTCGATGCCGATCAACGCCGTGACTTCTGGCAAAAATAAGAG

[0194] AGTTACATTGGAAAGATGGTCAAGTTTAAATACTTCGAAATGGGGTCAAAGGATTGC

[0195] CCACGTTTCCCTGTGTTCATCGGTATCCGCCACGAGGAAGATCGTTAA

[0196] >Amino acid sequence of PBCV ligase mutant 2 (SEQ ID NO:5)

[0197] MAITKPLVAATLENIEDVQFPCLATPKIAGIRSVKQTQMLSRTFKPIRNSVMNRLLTELLP

[0198] EGSNGEISIEGATFQDTTSAVMTGHKMYNAKFSYYWFGYVTDDPLKKYIDRVEDMKN

[0199] YITVHPHILEHAQVKIIPLIPVEINNITELLQYERDVLSKGFEGVMIRKPDGKYKFGRSTL

[0200] KEGILLKMKQFKDAEATIISMTALFKNTNTKTKDNFGYSKRSTHKSGRVEEDVMGSIE

[0201] VDYDGVVFSIGTGFDADQRRDFWQNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRHEEDR*

[0202] >Nucleotide sequence of the coding gene of PBCV ligase mutant 2 (SEQ ID NO:6)

[0203] ATGGCAATTACTAAGCCGTTGGTGGCAGCCACGCTTGAAAACATCGAGGACGTACA

[0204] ATTTCCGTGCTTAGCCACGCCAAAAATCGCAGGAATCCGCTCTGTTAAACAAACTC

[0205] AGATGCTTTCTCGTACCTTTAAACCAATTCGTAATTCAGTGATGAACCGCTTATTAAC

[0206] CGAGTTACTTCCGGAGGGGAGCAATGGAGAAATCTCAAATTGAGGGTGCAACATTCC

[0207] AGGATACCACATCTGCAGTTATGACGGGACACAAAATGTACAATGCCAAGTTCTCGT

[0208] ACTACTGGTTTGGCTACGTTACGGACGATCCTTTGAAGAAATACATTGATCGCGTGG

[0209] AGGATATGAAGAATTACATCACAGTACACCCCCATATTTTAGAACACGCGCAGGTGA

[0210] AAATTATTCCGTTAATCCCCGTAGAGATCAACAATATCACGGAGCTGCTTCAGTACG

[0211] AACGTGATGTCTTATCTAAGGGTTTTGAAGGTGTGATGATCCGCAAGCCAGATGGCA

[0212] AATATAAGTTCGGGCGCAGTACATTAAAAGGAAGGTATCTTGTTAAAGATGAAACAAT

[0213] TTAAAGATGCGGAGGCAACTATTATCAGTATGACTGCGCTTTTCAAGAATACAAATA

[0214] CCAAGACAAAGGACAATTTCGGGTACTCCAAACGCAGTACGCATAAATCCGGGCGG

[0215] GTGGAAGAAGACGTAATGGGGAGCATTGAAGTAGACTACGACGGGGTGGTGTTTA

[0216] GTATCGGAACAGGATTCGATGCCGATCAACGCCGTGACTTCTGGCAAAAATAAGAG

[0217] AGTTACATTGGAAAGATGGTCCAAGTTTAAATACTTCGAAATGGGGTCAAAGGATTGC

[0218] CCACGTTTCCCTGTGTTCATCGGTATCCGCCACGAGGAAGATCGTTAA

[0219] >Amino acid sequence of PBCV ligase mutant 3 (SEQ ID NO:7)

[0220] MAITKPLLAATLENIEDVQFPCLATPKIAGIDSVKQTQMLSRTFKPIRNSVMNRLLTELL

[0221] PEGSDGEISIEGATFQDTTSAVMTGHKMYNAKFSYYWFRYVTDDPLKKYIDRVEDMK

[0222] NYITVHPHILEHAQVKIIPLIPVEINNITRLLQYERDVLSKGFEGVMIRKPDGKYKFGRST

[0223] LKEGILLKMKQFKDAEATIISMTALFKNTNTKTKDNFGYSKRSTHKSGKVKRRVMGSI

[0224] EVDYDGVVFSIGTGFDADQDDDFWQNKESYIGKMVKFKYFEMGSKDCPRFPVFIGIRHEEDR*

[0225] >Nucleotide sequence of the coding gene of PBCV ligase mutant 3 (SEQ ID NO:8)

[0226] ATGGCAATTACTAAGCCGTTGTTGGCAGCCACGCTTGAAAACATCGAGGACGTACA

[0227] ATTTCCGTGCTTAGCCACGCCAAAAATCGCAGGAATCGACTCTGTTAAACAAACTC

[0228] AGATGCTTTCTCGTACCTTTAAACCAATTCGTAATTCAGTGATGAACCGCTTATTAAC

[0229] CGAGTTACTTCCGGAGGGGAGCGATGGAGAAATCTCAATTGAGGGTGCAACATTCC

[0230] AGGATACCACATCTGCAGTTATGACGGGACACAAAATGTACAATGCCAAGTTCTCGT

[0231] ACTACTGGTTTCGCTACGTTACGGACGATCCTTTGAAGAAATACATTGATCGCGTGG

[0232] AGGATATGAAGAATTACATCACAGTACACCCCCATATTTTAGAACACGCGCAGGTGA

[0233] AAATTATTCCGTTAATCCCCGTAGAGATCAACAATATCACGCGGCTGCTTCAGTACG

[0234] AACGTGATGTCTTATCTAAGGGTTTTGAAGGTGTGATGATCCGCAAGCCAGATGGCA

[0235] AATATAAGTTCGGGCGCAGTACATTAAAAGGAAGGTATCTTGTTAAAGATGAAACAAT

[0236] TTAAAGATGCGGAGGCAACTATTATCAGTATGACTGCGCTTTTCAAGAATACAAATA

[0237] CCAAGACAAAGGACAATTTCGGGTACTCCAAACGCAGTACGCATAAATCCGGGAAG

[0238] GTGAAACGGCCGTAATGGGGAGCATTGAAGTAGACTACGACGGGGTGGTGTTTAG

[0239] TATCGGAACAGGATTCGATGCCGATCAAGATGATGACTTCTGGAAAATAAAGAGA

[0240] GTTACATTGGAAAGATGGTCAAGTTTAAATACTTCGAAATGGGGTCAAAGGATTGCC

[0241] CACGTTTCCCTGTGTTCATCGGTATCCGCCACGAGGAAGATCGTTAA

[0242] It should be noted that, although the technical solution of the present invention is introduced with specific examples, those skilled in the art will appreciate that the present invention should not be limited thereto.

[0243] The embodiments of the present invention have been described above, and the above description is exemplary, not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or technical improvements in the market, or to enable other persons of ordinary skill in the art to understand the embodiments disclosed herein.

Claims

1. A method for synthesizing single-stranded DNA, characterized in that: The method for synthesizing single-stranded DNA comprises the following steps: (1) The RNA fragment recognizes and binds to the complementary acceptor DNA fragment and donor DNA fragment; (2) DNA ligase mediates the connection between the acceptor DNA fragment and the donor DNA fragment; (3) removing RNA fragments in the reaction system to obtain single-stranded DNA; The RNA fragment consists of at least 14 bases and comprises (a) and / or (b): (a) a base fragment complementary to at least 6 consecutive bases at the 5' end of the donor DNA fragment, (b) a base fragment complementary to at least 8 consecutive bases at the 3' end of the receptor DNA fragment, Wherein, (a) and (b) have no overlapping parts, and the base complementary to the 3' end of the acceptor DNA fragment and the base complementary to the 5' end of the donor DNA fragment are adjacent; Preferably, the first base from the 5' end of the donor DNA fragment is A or C, and the second base is T, G or C; Preferably, the first base at the 3' end of the receptor DNA fragment is G, C or T, the second base is G or C, and the third base is G, C or T.

2. The method for synthesizing single-stranded DNA according to claim 1, characterized in that In the step (1), the acceptor DNA, donor DNA and RNA fragment are mixed in a molar ratio of 1:1:(1-3), the mixed sample is reacted at not less than 80° C., preferably not less than 90° C., for 2-5 minutes, and then the sample is cooled to room temperature.

3. The method for synthesizing single-stranded DNA according to claim 1 or 2, characterized in that: The DNA ligase in step (2) is DNA ligase PBCV or a mutant thereof; Optionally, the DNA ligase PBCV comprises the sequence as shown in SEQ ID NO:3, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:3; Optionally, the DNA ligase PBCV mutant comprises the sequence as shown in SEQ ID NO: 1, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:

1.

4. The method for synthesizing single-stranded DNA according to claim 3, characterized in that: The DNA ligase PBCV or its mutant is reacted in a system containing 30-60 mM Tris-HCl, 5-15 mM MgCl2, 0.5-2 mM ATP, 5-15 mM DTT and a pH of 7.0-8.0, and the reaction time is not less than 10 minutes. Optionally, after the reaction is completed, the DNA ligase is inactivated at a temperature not lower than 90°C.

5. The method for synthesizing single-stranded DNA according to any one of claims 1 to 4, characterized in that: The step (3) comprises the following steps: adding ribonuclease to the reaction solution obtained in step (2), reacting for not less than 25 minutes in a system containing 60-80 mM KCl, 40-60 mM Tris-HCl, 2-4 mM MgCl2, 2-15 mM DTT and a pH of 8.0-8.5, adding ethanol to precipitate DNA after the reaction, centrifuging to obtain the precipitate, adding water to dissolve the precipitate to obtain a solution containing single-stranded DNA, Optionally, the ribonuclease is RNase H.

6. A kit for synthesizing single-stranded DNA, characterized in that: The kit comprises a DNA ligase, an RNA fragment, a receptor DNA fragment and a donor DNA fragment, wherein the RNA fragment consists of at least 14 bases and comprises (a) and / or (b): (a) a base fragment complementary to at least 6 consecutive bases at the 5' end of the donor DNA fragment, (b) a base fragment complementary to at least 8 consecutive bases at the 3' end of the receptor DNA fragment, Wherein, (a) and (b) have no overlapping parts, and the base complementary to the 3' end of the acceptor DNA fragment and the base complementary to the 5' end of the donor DNA fragment are adjacent; Preferably, the first base from the 5' end of the donor DNA fragment is A or C, and the second base is T, G or C; Preferably, the first base at the 3' end of the acceptor DNA fragment is G, C or T, the second base is G or C, and the third base is G, C or T.

7. The single-stranded DNA synthesis kit according to claim 6, characterized in that: The DNA ligase is DNA ligase PBCV or a mutant thereof; Optionally, the DNA ligase PBCV comprises the sequence as shown in SEQ ID NO:3, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:3; Optionally, the DNA ligase PBCV mutant comprises the sequence as shown in SEQ ID NO: 1, or a variant thereof having at least 95%, 96%, 97%, 98% or 99% or higher sequence identity with SEQ ID NO:

1.

8. The single-stranded DNA synthesis kit according to claim 6 or 7, characterized in that: The kit further comprises a reaction solution of the DNA ligase; optionally, the reaction solution comprises Tris-HCl, MgCl2, ATP and / or DTT; Optionally, the kit comprises ribonuclease.

9. A single-stranded DNA encoded compound library, characterized in that: The single-stranded DNA encoded compound library is constructed by the method for synthesizing single-stranded DNA according to any one of claims 1 to 5, or the kit for synthesizing single-stranded DNA according to any one of claims 6 to 8.

10. Use of the single-stranded DNA synthesis kit according to any one of claims 6 to 8 in synthesizing single-stranded DNA or constructing a DNA-encoded compound library.

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