Method for producing double-stranded DNA

Abstract

The present invention provides a method of producing a double stranded DNA (dsDNA) molecule. Specifically, the present invention provides a method of forming a dsDNA complex using a plurality of single-stranded DNA (ssDNA) molecules, where the ssDNA molecules produce dsDNA molecules by ligation.

Description

Technical Field

[0001] The present invention belongs to the field of synthetic biology and relates to a method for producing double-stranded DNA (dsDNA) molecules, such as long dsDNA molecules, such as containing at least about 1000bp. Specifically, the present invention provides a method for forming a dsDNA complex using multiple single-stranded DNA (ssDNA) molecules, wherein the ssDNA molecules are connected to produce dsDNA molecules. Some ssDNA molecules may be extended before connection. The ssDNA molecules used in the present invention may be products of enzymatic synthesis, such as products of enzyme-mediated rolling circle amplification (RCA) reactions. The present invention also provides the use of multiple ssDNA molecules in a ligase-mediated reaction to produce dsDNA molecules. Double-stranded DNA molecules obtained by the method are also provided. In addition, a dsDNA molecule library is also provided, which contains multiple different (e.g., functionalized or modified) dsDNA molecules obtained by the method. Background Art

[0002] Synthetic biology is widely recognized as an important and rapidly developing field of science. Although synthetic biology is a multidisciplinary field of study, its key aspects are related to genetic engineering, including the synthesis of new genes and the modification of existing genes. Significant advances in the past two decades have made gene synthesis more affordable, which in turn has led to advances in many fields and the development of new applications.

[0003] At present, gene synthesis (i.e., DNA synthesis) is based on the assembly of adjacent short oligonucleotides, which are usually synthesized by solid phase synthesis, especially phosphoramidite chemistry. Typically, these ssDNA segments are 60 to 80 nucleotides (nt) long, covering the two chains of the DNA duplex to be produced. In order to reduce the number of oligonucleotides required to generate the desired sequence (e.g., gene sequence), each ssDNA segment is designed to have an annealing sequence 15 to 25 nucleotides away from the ssDNA segment present in the opposite chain. Then, a thermostable DNA polymerase randomly extends the assembled oligonucleotides to generate products of different lengths, which are then used as templates for PCR reactions, which are driven by the outermost layer of excess, assembled oligonucleotides. This is called polymerase cycle assembly (PCA) or assembly PCR.

[0004] Importantly, both the synthetic and assembly aspects of the above methods impose strict constraints on the accurate production of dsDNA (e.g., gene synthesis).

[0005] First, solid phase chemical synthesis (e.g., phosphoramidite synthesis) is inefficient and error-prone. Second, the number of errors increases with the length of the oligonucleotide, while the product yield decreases. The error rate of producing polynucleotides by solid phase synthesis is significantly higher than the error rate of polymerases observed in nature, and the error rate increases sharply with the length of the polynucleotide, resulting in a purity of only 70% common in commercial polynucleotides of about 50 nucleotides in length. This error rate makes solid phase synthesis unsuitable for the production of longer polynucleotides. For example, the error rate of solid phase synthesis is typically greater than 0.5%, which means that the production of ssDNA molecules containing 100 nucleotides will produce a mixture of ssDNA molecules, of which more than 40% of the ssDNA molecules contain at least one error, i.e., the purity of the synthesized product is less than 60%. Finally, the process of assembling ssDNA segments (oligonucleotides) to form a target gene is prone to errors, especially when there are multiple oligonucleotides with similar sequences, which is at least in part because the typical overlap between the relative chains is short.

[0006] Therefore, errors in the starting product are exacerbated by further errors in the assembly process and lead to errors in the final assembled product, which need to be eliminated. Therefore, it is very challenging, time-consuming, labor-intensive and expensive to synthesize long DNA sequences (such as genes) efficiently and accurately using existing methods. This is due to the demanding post-synthesis quality control procedures that require identification and selection of products with the correct sequence, or more commonly, sequencing / error correction / re-cloning cycles. In addition, even with these post-assembly processes, molecules with the correct sequence cannot necessarily be produced, especially molecules with long sequences and / or complex or repetitive sequences.

[0007] In view of the problems with existing methods, there is a need for alternative methods for producing dsDNA molecules, particularly methods for efficiently producing long dsDNA molecules (eg, about 1 kb or longer) with high yield and purity. Summary of the invention

[0008] In work prior to the present invention, the inventors unexpectedly discovered that long dsDNA molecules can be accurately and efficiently produced using ligase-mediated assembly of ssDNA molecules. In particular, the methods and uses provided herein do not involve PCR methods that typically produce byproducts, especially when long and complex sequences are required. In particular, the inventors have discovered that long and pure ssDNA building blocks (e.g., each building block contains 150 to 10,000 nucleotides) can be used to accurately assemble stable dsDNA complexes that can be efficiently joined to produce the desired dsDNA molecules.

[0009] While not wishing to be bound by theory, it is believed that the purity of the starting material (e.g., enzymatically synthesized ssDNA molecules) combined with the length of the annealing sequence (e.g., at least 50 bp) (which greatly reduces instances of errors due to misannealing events) allows for accurate assembly of ultrapure dsDNA molecules (e.g., genes or gene fragments). Advantageously, this minimizes (e.g., eliminates) the need for post-assembly processes, enabling assembly of any sequence regardless of length and complexity. Surprisingly, the examples demonstrate that dsDNA molecules can be produced in a one-pot reaction that includes both synthesis of ssDNA molecules and assembly into the desired dsDNA molecules. This is particularly advantageous because it further simplifies the method.

[0010] Figure 1 Representative strategies for assembling dsDNA complexes that can be joined to form desired dsDNA molecules are shown. These strategies can be characterized as "complete overlap" ( Figure 1 A), "partial overlap" ( Figure 1 B) and "minimum overlap" ( Figure 1 C) strategy, as described below.

[0011] Complete overlap: The positive strand of the target dsDNA (e.g., a gene) is provided by four segments, A, B, C, and D (e.g., each segment contains 1000 nucleotides); the negative strand is provided by five segments, E, F, G, H, and I (e.g., three segments (F, G, and H) each contain 1000 nucleotides, and two segments (E and I) each contain 500 nt. The smaller segments (E and I) are arranged to form the ends (3' and 5' ends, respectively) of the negative strand of the target dsDNA, so that the longer segments (F, G, and H) contain large regions that are complementary (overlapping) to the segments in the positive strand. There are no gaps between adjacent segments (i.e., the 5' and 3' ends of the segments are directly adjacent), so that the overlapping segments of the opposite strands can be used as templates for the ligation reaction, and the segments are ligated in a ligase-mediated ligation reaction, i.e., segment A is used as a ligation template for segments E and F, segment F is used as a ligation template for segments A and B, and so on.

[0012] Partial overlap: The positive strand of the target dsDNA (e.g., gene) is provided by three segments, A, J, and D (e.g., each segment contains 1000 nucleotides), wherein segments A and D form the ends of the positive strand of the target dsDNA (5' and 3' ends, respectively), and segment J contains the central part of the positive strand of the target dsDNA (e.g., it contains nucleotides 1501 to 2500 of the positive strand). The negative strand is provided by four segments, E, K, L, and I (e.g., two segments (K and L) each contain 1000 nucleotides, and two segments (E and I) each contain 500nt). As with the above-mentioned complete overlap method, small segments E and I are arranged to form the ends of the negative strand of the target dsDNA (3' and 5' ends, respectively). These segments are arranged in such a way that there are gaps between adjacent segments (i.e., the 5' and 3' ends are indirectly adjacent), wherein the longer segments only partially overlap, for example, contain a complementary region of about 250bp. The DNA complex formed by hybridization of complementary sequences allows the inner 3' ends of the segments to be extended in a template-mediated polymerase extension reaction using the overlapping segments of the opposing strands as templates for extension reactions, i.e., segment A serves as an extension template for segment K, segment K serves as an extension template for segment A, and so on. Then, similar to the complete overlap method, the overlapping segments of the opposing strands can be used as templates for ligation reactions to join the extended segments in a ligase-mediated ligation reaction, i.e., segment A serves as a ligation template for segments E and K, segment K serves as a ligation template for segments A and J, and so on.

[0013] Minimum overlap: The positive chain is the same as the complete overlap strategy, using segments A, B, C and D. The negative chain is provided by four smaller segments M, N, O and P (for example, each segment contains 200 nucleotides). Only one of the segments provides the end of the negative chain, that is, segment P provides the 5' end of the negative chain. The other negative chain segments (M, N and O) partially overlap with the positive chain segments, for example, containing a complementary region of about 100 bp. Similar to the above-mentioned partial overlap strategy, the DNA complex formed by hybridization of complementary sequences allows the use of the overlapping segments of the positive chain as templates for the extension reaction, and the internal 3' end of the negative chain segment is extended in the template-mediated polymerase extension reaction, that is, segment A is used as an extension template for segment M, segment B is used as an extension template for segment N, and so on. Then, similar to the complete overlap method, the overlapping segments of the opposite chain can be used as templates for the ligation reaction, and the extended segments are connected in the ligase-mediated ligation reaction, that is, segment B is used as a ligation template for segments M and N, and segment M is used as a ligation template for segments A and B, and so on.

[0014] Those skilled in the art will appreciate that the above strategies can be applied to increase the number of segments, such as producing longer dsDNA molecules. Similarly, the length of the segments can be modified. For example, all segments of the positive strand and / or negative strand do not have to have the same length. In particular, the segments can be designed to ensure that the position of the connection point (i.e., the position in the dsDNA complex directly adjacent to the 5' end and 3' end of the segment) does not overlap with a repetitive sequence and / or a sequence capable of forming a secondary structure, so as to minimize mismatches within the DNA complex.

[0015] It is also obvious that multiple versions (e.g., sequence variants) of one or more ssDNA segments can be provided to generate a mixture of dsDNA molecules, i.e., to generate a gene library, such as for phage display methods, etc. For example, referring to the above strategies and Figure 1 , multiple versions (e.g., sequence variants) of segment K can be provided in the reaction mixture, these versions having a common complementary region and different sequences in the region opposite to the gap in the opposite chain (i.e., the gap between segments A and J). As shown in Example 4, a library of ssDNA variants can be generated by including manganese ions in the DNA synthesis reaction. The product of the extension and ligation reactions will be a mixture of dsDNA molecules, which contain different sequences in the region provided (and encoded) by multiple versions of segment K. It is worth noting that different versions of ssDNA segments do not need to be sequence variants. For example, multiple ssDNA molecules can be provided, each ssDNA molecule containing modified nucleotides (e.g., functionalized nucleotides, such as methylated nucleotides), as shown in Example 5. Different versions may contain modifications at different positions and / or different modifications.

[0016] In addition, it should be understood that one or more of the plurality of ssDNA molecules may contain one or more modified nucleotides (e.g., functionalized nucleotides, such as methylated nucleotides), such as site-specific modifications, such that the modified nucleotides are incorporated into the generated dsDNA molecules. For example, one or more of the ssDNA molecules may contain one or more methylated cytosines to generate methylated dsDNA molecules (see Example 5), which may have particular applications in gene silencing and epigenetic techniques.

[0017] It is apparent that the above-described partial overlap strategy can be modified so that the segment providing the 3' end of the negative strand (segment E) can be omitted, since segment K can be extended using segment A as a template to provide the 3' end of the negative strand of the dsDNA molecule. In other words, the segment providing the 3' end of the negative strand (e.g., the second strand) in the partial overlap strategy is optional, similar to the minimal overlap strategy.

[0018] Similarly, the segment providing the 3' end of the positive strand (segment D) can be truncated so that it does not include the 3' end of the positive strand of the target dsDNA, because segment D can be extended using segment I as a template to provide the 3' end of the positive strand. Therefore, in the partial overlap strategy, the segment of the positive strand and / or the segment of the negative strand can provide the 3' end of the target dsDNA molecule through a templated extension reaction (i.e., a polymerase-mediated extension reaction).

[0019] Thus, provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-mediated ligation reaction to produce a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, wherein:

[0020] (i) each of the first plurality of single-stranded DNA (ssDNA) molecules comprises (e.g., consists of) non-overlapping portions of a first strand of a dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule, and the second ssDNA: (a) comprises the 3' end of the first strand of the dsDNA molecule, or (b) is capable of providing the 3' end of the first strand of the dsDNA molecule by a polymerase-mediated extension reaction using the ssDNA molecule comprising the 5' end of the second strand as a template; and

[0021] (ii) each of the second plurality of ssDNA molecules comprises (e.g., consists of) non-overlapping portions of the second strand of the dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule and, optionally, the second ssDNA comprises the 3' end of the second strand of the dsDNA molecule,

[0022] in:

[0023] (1) At least two of the plurality of ssDNA molecules in (ii) each comprise a first region that is completely complementary to a terminal region of a certain ssDNA molecule in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i);

[0024] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms adjacent portions of the first strand of the dsDNA molecule;

[0025] (3) the complementary region contains at least 50 nucleotides;

[0026] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0027] (5) ssDNA molecules are provided in equimolar amounts;

[0028] And wherein the first and second plurality of ssDNA molecules, when contacted under conditions suitable for DNA molecular hybridization, form a dsDNA complex comprising all of the ssDNA molecules that can be ligated in a ligase-mediated reaction to produce a dsDNA molecule.

[0029] Thus, provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-mediated ligation reaction to produce a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, wherein:

[0030] (i) each of the first plurality of single-stranded DNA (ssDNA) molecules comprises (e.g., consists of) non-overlapping portions of a first strand of a dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule and the second ssDNA comprises the 3' end of the first strand of the dsDNA molecule; and

[0031] (ii) each of the second plurality of ssDNA molecules comprises (e.g., consists of) non-overlapping portions of the second strand of the dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule and, optionally, the second ssDNA comprises the 3' end of the second strand of the dsDNA molecule,

[0032] in:

[0033] (1) At least two of the plurality of ssDNA molecules in (ii) each comprise a first region that is completely complementary to a terminal region of a certain ssDNA molecule in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i);

[0034] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms adjacent portions of the first strand of the dsDNA molecule;

[0035] (3) the complementary region contains at least 50 nucleotides;

[0036] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0037] (5) ssDNA molecules are provided in equimolar amounts;

[0038] And wherein the first and second plurality of ssDNA molecules, when contacted under conditions suitable for DNA molecular hybridization, form a dsDNA complex comprising all of the ssDNA molecules that can be ligated in a ligase-mediated reaction to produce a dsDNA molecule.

[0039] In addition, the present invention provides the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-mediated ligation reaction to produce a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, wherein:

[0040] (i) the first plurality of ssDNA molecules comprises at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule and the second ssDNA comprises the 3' end of the first strand of the dsDNA molecule; and

[0041] (ii) a second plurality of ssDNA molecules comprises at least four ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is completely complementary to the region of the second ssDNA in (i); the second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule that is completely complementary to the region of the first ssDNA molecule in (i),

[0042] in:

[0043] (1) At least two of the at least four ssDNA molecules in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i);

[0044] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms directly adjacent portions of the first strand of the dsDNA molecule;

[0045] (3) the complementary region contains at least 50 nucleotides;

[0046] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0047] (5) ssDNA molecules are provided in equimolar amounts;

[0048] And wherein the first and second plurality of ssDNA molecules, when contacted under conditions suitable for DNA molecular hybridization, form a dsDNA complex comprising all of the ssDNA molecules that can be ligated in a ligase-mediated ligation reaction to produce a dsDNA molecule.

[0049] Also provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-mediated ligation reaction to produce a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, wherein:

[0050] (i) the first plurality of ssDNA molecules comprises at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; and the second ssDNA molecule: (a) comprises the 3' end of the first strand of the dsDNA molecule, or (b) is capable of providing the 3' end of the first strand of the dsDNA molecule by a polymerase-mediated extension reaction using the ssDNA molecule comprising the 5' end of the second strand as a template; and

[0051] (ii) a second plurality of ssDNA molecules comprises at least three (e.g., at least four) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is fully complementary to a region of the second ssDNA in (i); optionally, the second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule that is fully complementary to a region of the first ssDNA molecule in (i),

[0052] in:

[0053] (1) At least three ssDNA molecules (e.g., at least two of the at least four ssDNA molecules) in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i);

[0054] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming an indirectly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule;

[0055] (3) the complementary region contains at least 50 nucleotides;

[0056] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0057] (5) ssDNA molecules are provided in equimolar amounts;

[0058] and wherein the first and second plurality of ssDNA molecules form a partial dsDNA complex comprising all of the ssDNA molecules when contacted under conditions suitable for DNA molecule hybridization, wherein the inner 3' ends of the ssDNA molecules in the partial dsDNA complex can be extended in a polymerase-mediated extension reaction to produce a complete dsDNA complex, and the complete dsDNA complex can be ligated in a ligase-mediated ligation reaction to produce a dsDNA molecule.

[0059] Also provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-mediated ligation reaction to produce a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, wherein:

[0060] (i) the first plurality of ssDNA molecules comprises at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; the second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and

[0061] (ii) a second plurality of ssDNA molecules comprises at least four ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is completely complementary to the region of the second ssDNA in (i); the second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule that is completely complementary to the region of the first ssDNA molecule in (i),

[0062] in:

[0063] (1) At least two of the at least four ssDNA molecules in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i);

[0064] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming an indirectly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule;

[0065] (3) the complementary region contains at least 50 nucleotides;

[0066] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0067] (5) ssDNA molecules are provided in equimolar amounts;

[0068] and wherein the first and second plurality of ssDNA molecules form a partial dsDNA complex comprising all of the ssDNA molecules when contacted under conditions suitable for DNA molecule hybridization, wherein the inner 3' ends of the ssDNA molecules in the partial dsDNA complex can be extended in a polymerase-mediated extension reaction to produce a complete dsDNA complex, and the complete dsDNA complex can be ligated in a ligase-mediated ligation reaction to produce a dsDNA molecule.

[0069] Also provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-mediated ligation reaction to produce a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, wherein:

[0070] (i) the first plurality of ssDNA molecules comprises at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; the second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and

[0071] (ii) a second plurality of ssDNA molecules comprises at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises a 5' end of the second strand of the dsDNA molecule that is fully complementary to a region of the second ssDNA molecule in (i),

[0072] in:

[0073] (1) At least two of the at least three ssDNA molecules in (ii) do not include the 5' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to a terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i);

[0074] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming a directly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule;

[0075] (3) the complementary region contains at least 50 nucleotides;

[0076] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0077] (5) ssDNA molecules are provided in equimolar amounts;

[0078] And wherein the first and second plurality of ssDNA molecules form a partial dsDNA complex comprising all ssDNA molecules when contacted under conditions suitable for DNA molecule hybridization, wherein the inner 3' ends of the ssDNA molecules in the partial dsDNA complex (the inner 3' ends of the second plurality of ssDNA molecules) can be extended in a polymerase-mediated extension reaction to produce a complete dsDNA complex, and the complete dsDNA complex can be ligated in a ligase-mediated ligation reaction to produce a dsDNA molecule.

[0079] Alternatively, the present invention provides a method for producing a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising:

[0080] (a) Provide:

[0081] (i) a plurality of single-stranded DNA (ssDNA) molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule, and the second ssDNA: (a) comprises the 3' end of the first strand of the dsDNA molecule, or (b) is capable of providing the 3' end of the first strand of the dsDNA molecule by a polymerase-mediated extension reaction using the ssDNA molecule comprising the 5' end of the second strand as a template; and

[0082] (ii) a plurality of ssDNA molecules, each ssDNA molecule comprising (e.g. consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule and, optionally, the second ssDNA comprises the 3' end of the second strand of the dsDNA molecule,

[0083] in:

[0084] (1) At least two of the plurality of ssDNA molecules in (ii) each comprise a first region that is completely complementary to a terminal region of a certain ssDNA molecule in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i);

[0085] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms adjacent portions of the first strand of the dsDNA molecule;

[0086] (3) the complementary region contains at least 50 nucleotides;

[0087] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0088] (5) ssDNA molecules are provided in equimolar amounts;

[0089] (b) hybridizing ssDNA molecules containing complementary regions to produce a dsDNA complex comprising all of the ssDNA molecules in (a); and

[0090] (c) Ligation of adjacent ssDNA molecules to generate dsDNA molecules.

[0091] Also provided herein is a method for producing a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising:

[0092] (a) Provide:

[0093] (i) a plurality of single-stranded DNA (ssDNA) molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule and the second ssDNA comprises the 3' end of the first strand of the dsDNA molecule; and

[0094] (ii) a plurality of ssDNA molecules, each ssDNA molecule comprising (e.g. consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule and, optionally, the second ssDNA comprises the 3' end of the second strand of the dsDNA molecule,

[0095] in:

[0096] (1) At least two of the plurality of ssDNA molecules of (ii) each comprise a first region that is completely complementary to a terminal region of a certain ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule of (i);

[0097] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms adjacent portions of the first strand of the dsDNA molecule;

[0098] (3) the complementary region contains at least 50 nucleotides;

[0099] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0100] (5) ssDNA molecules are provided in equimolar amounts;

[0101] (b) hybridizing ssDNA molecules containing complementary regions to produce a dsDNA complex comprising all of the ssDNA molecules in (a); and

[0102] (c) Ligation of adjacent ssDNA molecules to generate dsDNA molecules.

[0103] Also provided herein is a method for producing a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising:

[0104] (a) Provide:

[0105] (i) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule and the second ssDNA comprises the 3' end of the first strand of the dsDNA molecule; and

[0106] (ii) at least four ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is completely complementary to the region of the second ssDNA in (i); the second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule that is completely complementary to the region of the first ssDNA molecule in (i),

[0107] in:

[0108] (1) At least two of the at least four ssDNA molecules in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i);

[0109] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms directly adjacent portions of the first strand of the dsDNA molecule;

[0110] (3) the complementary region contains at least 50 nucleotides;

[0111] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0112] (5) ssDNA molecules are provided in equimolar amounts;

[0113] (b) hybridizing ssDNA molecules containing complementary regions to produce a complete dsDNA complex comprising all of the ssDNA molecules in (a); and

[0114] (c) Ligation of directly adjacent ssDNA molecules to produce a dsDNA molecule.

[0115] Also provided herein is a method for producing a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising:

[0116] (a) Provide:

[0117] (i) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; and the second ssDNA molecule: (a) comprises the 3' end of the first strand of the dsDNA molecule, or (b) is capable of providing the 3' end of the first strand of the dsDNA molecule by a polymerase-mediated extension reaction using the ssDNA molecule comprising the 5' end of the second strand as a template; and

[0118] (ii) at least three (e.g., at least four) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is fully complementary to the region of the second ssDNA in (i); optionally, the second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule that is fully complementary to the region of the first ssDNA molecule in (i),

[0119] in:

[0120] (1) At least three ssDNA molecules (e.g., at least two of the at least four ssDNA molecules) in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i);

[0121] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming an indirectly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule;

[0122] (3) the complementary region contains at least 50 nucleotides;

[0123] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0124] (5) ssDNA molecules are provided in equimolar amounts;

[0125] (b) hybridizing ssDNA molecules containing complementary regions to produce a partial dsDNA complex comprising all of the ssDNA molecules in (a);

[0126] (c) extending the 3' end of the ssDNA molecule to produce a complete dsDNA complex; and

[0127] (d) Ligation of directly adjacent ssDNA molecules to produce a dsDNA molecule.

[0128] Also provided herein is a method for producing a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising:

[0129] (a) Provide:

[0130] (i) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; the second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and

[0131] (ii) at least four ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is completely complementary to the region of the second ssDNA in (i); the second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule that is completely complementary to the region of the first ssDNA molecule in (i),

[0132] in:

[0133] (1) At least two of the at least four ssDNA molecules in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i);

[0134] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming an indirectly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule;

[0135] (3) the complementary region contains at least 50 nucleotides;

[0136] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0137] (5) ssDNA molecules are provided in equimolar amounts;

[0138] (b) hybridizing ssDNA molecules containing complementary regions to produce a partial dsDNA complex comprising all of the ssDNA molecules in (a);

[0139] (c) extending the 3' end of the ssDNA molecule to produce a complete dsDNA complex; and

[0140] (d) Ligation of directly adjacent ssDNA molecules to produce a dsDNA molecule.

[0141] Also provided herein is a method for producing a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising:

[0142] (a) Provide:

[0143] (i) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; the second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and

[0144] (ii) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is fully complementary to a region of the second ssDNA molecule in (i),

[0145] in:

[0146] (1) At least two of the at least three ssDNA molecules in (ii) do not include the 5' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to a terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i);

[0147] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming a directly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule;

[0148] (3) the complementary region contains at least 50 nucleotides;

[0149] (4) all ssDNA molecules contain at least 150 nucleotides; and

[0150] (5) ssDNA molecules are provided in equimolar amounts;

[0151] (b) hybridizing ssDNA molecules containing complementary regions to produce a partial dsDNA complex comprising all of the ssDNA molecules in (a);

[0152] (c) extending the 3' end of the ssDNA molecule in (ii) to produce a complete dsDNA complex; and

[0153] (d) Ligation of directly adjacent ssDNA molecules to produce a dsDNA molecule.

[0154] Although the methods and uses provided herein are particularly useful in producing (i.e., synthesizing) long dsDNA molecules (e.g., comprising at least 1500 bp), it will be appreciated by those skilled in the art that the methods and uses provided herein may also be particularly advantageous for producing dsDNA molecules containing repetitive sequences and / or sequences capable of forming secondary structures. In view of the challenges faced in producing smaller dsDNA molecules containing repetitive sequences and / or sequences capable of forming secondary structures, when the methods and uses provided herein are applied to the production of dsDNA molecules containing repetitive sequences and / or sequences capable of forming secondary structures, the above size requirements do not necessarily apply. Thus, for example, when the dsDNA molecule contains repetitive sequences and / or sequences capable of forming secondary structures, the methods and uses provided herein may be used to produce dsDNA molecules containing 500 bp or more (e.g., 750 bp or 1000 bp or more).

[0155] Also provided herein are dsDNA molecules produced by the methods and uses provided herein.

[0156] Also provided herein is a dsDNA molecule library, which comprises a plurality of different (eg, functionalized or modified) dsDNA molecules obtained by the methods and uses provided herein. DETAILED DESCRIPTION

[0157] The terms "double-stranded DNA (dsDNA)" and "dsDNA molecule" are used interchangeably herein and refer to a nucleic acid molecule composed of two antiparallel polynucleotides comprising deoxyribonucleotides (dNTPs) held together by hydrogen bonds through Watson-Crick type or analogous base pair interactions. The two antiparallel polynucleotides of a dsDNA molecule may be referred to as DNA "strands" and may be distinguished as "first and second strands" or "positive and negative strands."

[0158] In view of the fact that synthetic and / or functionalized nucleotides (i.e., modified nucleotides) can participate in Watson-Crick type or similar base pair interactions, it is clear that dsDNA does not need to be composed entirely of standard or conventional dNTPs, i.e., dsDNA can contain synthetic and / or functionalized nucleotides. For example, the 5' and / or 3' nucleotides in the dsDNA molecule can be modified, such as post-synthesis modification. In addition, during the synthesis of DNA molecules, such as during the synthesis of single-stranded DNA (ssDNA) molecules for the production of double-stranded molecules described herein, synthetic and / or functionalized nucleotides can be incorporated into DNA molecules. Therefore, the dsDNA produced according to the methods and uses provided herein is expected to contain one or more functionalized, modified or non-standard nucleotides, such as methylated nucleotides, such as methylated cytosine. Therefore, the dsDNA molecules provided by the methods and uses described herein can be modified (e.g., functionalized) dsDNA molecules, such as methylated dsDNA molecules. Specifically, the dsDNA molecules provided by the methods and uses described herein can contain one or more (e.g., multiple) site-specific modifications. For example, the dsDNA molecule may contain one or more site-specific modifications relative to the corresponding natural dsDNA molecule. However, the majority of the nucleotides in the dsDNA molecule will be conventional dNTPs, for example, at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the nucleotides in the dsDNA molecule will be conventional dNTPs. Therefore, the dsDNA molecules produced according to the methods and uses provided herein can be composed of conventional dNTPs.

[0159] Alternatively, one or more of the plurality of ssDNA molecules used in the methods and uses may comprise one or more modified nucleotides (e.g., functionalized nucleotides, such as methylated nucleotides), for example site-specifically modified, i.e., such that the modified nucleotides are incorporated into the generated dsDNA molecules.

[0160] The terms "conventional nucleotides" and "standard nucleotides" are used interchangeably herein and refer to deoxynucleotides that contain one of the four bases found in DNA: adenine, guanine, cytosine, and thymine. Thus, the term "conventional nucleotides" includes, for example, dATP, dGTP, dCTP, and dTTP. Although uracil does not normally occur naturally in DNA, dUTP can be easily used instead of dTTP, or in addition to dTTP. Thus, in the context of the present invention, dUTP can be considered a "conventional" nucleotide.

[0161] The term "functionalized nucleotide" or "functionalized dNTP" refers to a nucleotide comprising a modification relative to an unmodified conventional nucleotide, wherein the modification provides the functionalized nucleotide and / or a polynucleotide comprising at least one functionalized nucleotide, and the functionalized nucleotide (i.e., relative to the corresponding conventional nucleotide) has additional or alternative properties or characteristics. Therefore, functionalized nucleotides may also be referred to as synthetic nucleotides, modified nucleotides, or non-standard nucleotides. For example, the modification may make the nucleotide detectable, such as by introducing a label, or may be able to interact and / or react with another component, i.e., a component with which the corresponding conventional nucleotide does not interact or react. The modification may make the polynucleotide containing the nucleotide resistant to degradation, such as chemical and / or enzymatic degradation (e.g., nuclease degradation), or may change the metabolism of the nucleotide. The modification may be a naturally occurring modification in vivo, such as methylation (e.g., a modified nucleotide may be 5-methylcytosine). Examples of functionalized nucleotides are provided in WO2020 / 161187, which is incorporated herein by reference.

[0162] Two antiparallel polynucleotides hybridize to form a dsDNA molecule because they contain sufficiently complementary nucleotide sequences to form a duplex through Watson-Crick base pairing or similar base pair interactions. Two nucleotide sequences are "complementary" to each other when the molecules share homology in base pair configuration. Under appropriate hybridization conditions, "complementary" nucleotide sequences will specifically bind to form a stable duplex. For example, when a segment of a first sequence can bind to a segment of a second sequence in an antiparallel manner, the two sequences are complementary, wherein the 3' end of each sequence binds to the 5' end of the other sequence, and then each A, T (U), G, and C of one sequence are aligned with the T (U), A, C, and G of the other sequence, respectively. Therefore, two sequences do not need to be completely homologous to be "complementary". Polynucleotides containing incompletely homologous sequences can be described as having partial complementarity (i.e., partial complementarity) and hybridizing to form partial dsDNA molecules, i.e., containing regions of mismatched base pairs, thereby forming regions of single-stranded molecules. Thus, polynucleotides comprising completely homologous sequences can be described as having perfect complementarity (ie, fully complementary) and hybridizing to form a perfect dsDNA molecule, ie, containing no regions of mismatched base pairs.

[0163] It should be understood that a partial dsDNA molecule may also refer to a DNA molecule in which there are no mismatched base pairs, i.e., a molecule comprising a dsDNA region and a ssDNA region in which there are no opposing strands (such that there is a gap on one of the DNA strands). Such molecules may be referred to herein as partial dsDNA complexes. As described above, strategies for generating partial dsDNA complexes can be used to generate mixtures of dsDNA molecules (e.g., libraries of dsDNA molecules, such as gene libraries) by using a plurality of ssDNA molecules having a common complementary region that is adjacent to (i.e., flanked by) intervening variant sequences.

[0164] The dsDNA molecules produced according to the methods and uses provided herein can be fully complementary, i.e., fully double-stranded, which is preferred. However, it is contemplated that in some cases it is desirable to produce partially complementary dsDNA molecules, for example, when at least about 90% (most preferably at least about 95%, 96%, 97%, 98%, or 99%) of the nucleotides share a base pair configuration over the entire length of the molecule. Thus, for example, a dsDNA molecule can have fully complementary regions separated by one or more regions of partial complementarity or no complementarity, such that overall at least about 90% of the nucleotides form base pairs with complementary nucleotides on the opposite strand.

[0165] The methods or uses provided herein are particularly useful in producing long dsDNA molecules. Long dsDNA refers to dsDNA molecules of any length that cannot be effectively and / or accurately synthesized using the above-mentioned conventional methods (such as PCA). Since the efficiency and accuracy of the synthesis reaction may vary depending on the complexity of the dsDNA to be produced, long dsDNA molecules may include dsDNA molecules with a length of less than 1 kbp (e.g., 500 to 1000 bp), wherein the molecule cannot be effectively and / or accurately synthesized using conventional methods (such as PCA). For example, the methods or uses provided herein can be used to synthesize any dsDNA molecule (regardless of length) that cannot be produced by conventional methods (such as PCR assembly methods), with an accuracy of more than 70%, i.e., 30% or more of the synthesized products contain incorrect sequences.

[0166] Preferably, the dsDNA molecules produced by the methods or uses provided herein may comprise at least about 1500 bp (base pairs), such as at least about 1750 bp, 2000 bp, 2250 bp, 2500 bp, 3000 bp, 3500 bp or 4000 bp. The dsDNA molecules produced by the methods or uses provided herein may be very long, for example, they may comprise 5 kbp, 6 kbp, 7 kbp, 8 kbp, 9 kbp, 10 kbp or more, such as 15 kbp, 20 kbp, 25 kbp, 30 kbp, 35 kbp, 40 kbp or more. For example, the dsDNA may comprise about 1 to 50 kbp, such as about 1.5 to 50 kbp, 2 to 45 kbp, 3 to 40 kbp, 4 to 35 kbp or 5 to 30 kbp.

[0167] The methods and uses provided herein can be advantageously used to produce dsDNA molecules comprising any sequence. The dsDNA molecule can comprise a coding sequence (i.e., an RNA coding sequence) and / or a non-coding sequence (e.g., a promoter). The coding sequence can encode any cellular or viral RNA. Thus, it can be mRNA, tRNA, rRNA, viral RNA, small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), small interfering RNA (siRNA), piwi interacting RNA, or a combination thereof. (piRNA), ribozyme RNA, antisense RNA or non-coding RNA (e.g., long non-coding RNA). Therefore, dsDN A can encode mRNA, and therefore can be considered to include a polypeptide coding sequence. Non-coding sequences can be any non-coding DNA, such as expression control sequences (e.g., promoters, enhancers, terminators, or functional parts thereof) or recombinant DNA cloning media or vectors. For example, dsDNA can include non-coding sequences (e.g., promoters) operably (operably) connected to coding sequences (e.g., polypeptide coding sequences). Therefore, dsDNA can include genes. It should be understood that dsDNA can include any combination of coding sequences and non-coding sequences, including combinations not present in nature, such as promoter sequences operably connected to heterologous polypeptide coding sequences. The dsDNA produced by the methods and uses provided herein can be considered as "target dsDNA" or "desired dsDNA", and these terms are used interchangeably herein.

[0168] The methods and uses provided herein can be particularly beneficial for producing dsDNA molecules containing repetitive sequences and / or sequences capable of forming secondary structures. In this regard, the use of single-stranded polynucleotides (e.g., ssDNA) containing repetitive sequences and / or sequences capable of forming secondary structures to form dsDNA complexes generally produces a mixture of different complexes (intermolecular complexes and intramolecular complexes) because each polynucleotide can be complementary to multiple polynucleotides in the mixture. Therefore, using conventional methods (e.g., using PCA) to form dsDNA molecules from these dsDNA complexes produces a mixture of different dsDNA molecules, wherein only part of the dsDNA molecules can contain the desired sequence. However, it is assumed that the use of single-stranded polynucleotides containing long complementary regions (e.g., at least 50bp) can promote the formation of dsDNA complexes containing the desired (i.e., correct) sequence, so that these complexes are present in excess. In addition, the use of ligase-mediated ligation reactions (optionally followed by polymerase-mediated extension reactions) rather than PCR-based methods (e.g., PCA) to form dsDNA molecules from these complexes is considered to improve the yield of dsDNA molecules with the desired sequence. Thus, the methods or uses provided herein can be used to produce dsDNA molecules comprising repetitive sequences and / or sequences capable of forming secondary structures, optionally wherein the dsDNA comprises at least about 500 bp, such as at least about 750 bp, 1000 bp, 1250 bp or 1500 bp.

[0169] Thus, the methods or uses provided herein do not involve polymerase chain reaction (PCR), such as PCA or assembly PCR, to produce dsDNA. Alternatively, the step of forming or producing dsDNA molecules does not use polymerase chain reaction (PCR), such as PCA or assembly PCR. As described above, the step of producing dsDNA molecules from dsDNA complexes involves a ligase-mediated ligation reaction, optionally preceded by a polymerase-mediated extension reaction. It is noteworthy that the ssDNA molecules used in the methods provided herein can be produced by methods involving PCR (e.g., asymmetric PCR). Thus, it should be understood that the step of forming dsDNA molecules does not involve PCR.

[0170] A repetitive sequence refers to a nucleotide sequence that occurs more than once in a polynucleotide. For example, a polynucleotide region (e.g., 2 or more nucleotides, typically 3 to 10 nucleotides or more, such as 10 to 60 nucleotides) containing a single type of nucleotide (e.g., dATP) can be considered a repetitive sequence. Similarly, a more complex sequence (i.e., containing more than one type of nucleotide) that is repeated once or multiple times in a polynucleotide can be considered a repetitive sequence. Repetitive sequences can be tandem (i.e., directly adjacent to each other) or interspersed (i.e., separated by non-repetitive sequences). Therefore, a repetitive sequence can include a sequence of at least 3 nucleotides (e.g., at least 4, 5, or 6 nucleotides, such as about 10, 15, or 20 nucleotides), which occurs more than once in a polynucleotide, for example, at least 2, 3, 4, or more times, such as 5, 6, 7, 8, 9, 10, or more times.

[0171] A sequence capable of forming a secondary structure refers to a nucleotide sequence having a region of self-complementarity (completely or partially complementary), so that a polynucleotide comprising the sequence forms one or more secondary structures, such as one or more intramolecular double-stranded regions, under conditions suitable for hybridization of complementary sequences. The intramolecular double-stranded regions may be separated by single-stranded regions, so that the polynucleotide contains one or more so-called stem-loop regions.

[0172] The methods and uses provided herein use multiple single-stranded DNA (ssDNA) molecules (polynucleotides) to form each chain of a dsDNA molecule. Therefore, each ssDNA molecule provides a "non-overlapping portion" of the chain of the dsDNA molecule. Therefore, the term "non-overlapping portion" means that each ssDNA molecule provides a unique (discrete or different) portion (fragment, segment, etc.) of a chain of the dsDNA that is not contained in any other ssDNA molecule. Although more than one ssDNA that provides a portion of a chain of a dsDNA molecule may contain the same sequence, these sequences are present in different parts of the chain of the dsDNA molecule, for example due to sequence repetitions in the chain of the dsDNA molecule. Therefore, when compared over its entire length, each ssDNA molecule in a plurality of ssDNA molecules has a different nucleotide sequence.

[0173] Therefore, when the multiple ssDNA molecules provided by the method and use are aligned end-to-end in the correct order, these ssDNA molecules form at least a portion of each strand of the dsDNA molecule, including at least the 5' end of the first strand and the second strand, preferably the 5' end and 3' end of the first strand and at least the 5' end of the second strand.

[0174] As described above, each ssDNA molecule used in the methods and uses provided herein can provide multiple versions, i.e., different versions (e.g., sequence variants) of equivalent non-overlapping portions of the strands of the dsDNA molecule, such that the product of the ligase-mediated ligation reaction is a mixture of dsDNA molecules. The dsDNA molecules in the mixture differ depending on the version of the ssDNA molecule incorporated into the dsDNA complex. If the different versions of the ssDNA molecules differ due to the functional groups on the nucleotides (e.g., the position and / or type of the functional groups), the sequences of the different versions can be the same.

[0175] The term "sequence variant" refers to nucleotide sequences that share some sequence identity but are not 100% identical. Thus, in the context of the present methods, sequence variants refer to ssDNA molecules containing identical sequences (i.e., sequences that form complementary regions) that are adjacent (flanked) by non-identical intervening sequences. Intervening sequences can be similar to each other (e.g., all intervening sequences in a plurality of sequences can have at least 60%, 70%, 80%, 90% or more sequence identity, such as 95%, 96%, 97%, 98% or 99% sequence identity), or can be different from each other (e.g., all intervening sequences in a plurality of sequences can have less than 60%, 50% or 40% sequence identity, such as 30%, 25%, 20% or less sequence identity). It should be understood that the above sequence identity values ​​can apply to the entire sequence while also taking into account identical end sequences. Sequence identity can be determined by any suitable method known in the art, such as using the BLAST alignment algorithm.

[0176] In the context of different non-overlapping portions of a strand of a dsDNA molecule, the term "different" refers to a nucleotide sequence in which the ssDNA molecule comprises one or more different nucleotides. Thus, different ssDNA molecules may comprise a nucleotide sequence of one or more different nucleotides, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides, such as 20, 30, 40, 50, 60, 70, 80, 90 or more nucleotides. The difference may be in the length and / or sequence of the ssDNA molecules. Alternatively, different ssDNA molecules have less than 100% sequence identity to each other, such as less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50% sequence identity.

[0177] For example, in the above-mentioned "complete overlap" strategy, the end-to-end alignment of non-overlapping ssDNA molecules provides the complete sequence of both strands of dsDNA, i.e., there are no "gaps" in the sequence. Therefore, the ssDNA molecules used in the "complete overlap" strategy can be regarded as continuous ssDNA molecules.

[0178] In the above-mentioned "partial overlap" strategy, the end-to-end alignment of non-overlapping ssDNA molecules provides incomplete sequences of the two strands of dsDNA, that is, there are "gaps" in the sequences of the two strands. Therefore, the ssDNA molecules provided in the "partial overlap" strategy can be regarded as non-continuous ssDNA molecules.

[0179] As used herein, the term "gap" refers to the portion of the nucleotide sequence of a chain of a dsDNA molecule that is not directly provided by a plurality of ssDNA molecules. As discussed in more detail below, the gap is "filled" using the sequence of the opposite chain as a template for a polymerase-mediated extension reaction. Therefore, it can be considered that these portions of the nucleotide sequence of a chain of a dsDNA molecule are provided indirectly. For example, in the above-mentioned "partial overlap" strategy, the 3' end of the first chain and / or the second chain of the dsDNA molecule can be provided indirectly.

[0180] In the above-mentioned "minimum overlap" strategy, the end-to-end alignment of non-overlapping ssDNA molecules provides a complete sequence of the first strand and an incomplete sequence of the second strand of the dsDNA. Therefore, the multiple ssDNA molecules providing the first strand in the "minimum overlap" strategy can be regarded as continuous ssDNA molecules, while the multiple ssDNA molecules providing the second strand in the "minimum overlap" strategy can be regarded as non-continuous ssDNA molecules.

[0181] It should be understood that the above strategies can be used in combination, so that one or both strands can be provided by a mixture of continuous and non-continuous ssDNA molecules, so that only a portion of the ssDNA molecules need to be extended in the polymerase-mediated extension reaction. Therefore, in some aspects, the plurality of ssDNA molecules providing one strand of the dsDNA are a mixture of continuous and non-continuous ssDNA molecules and / or the plurality of ssDNA molecules providing the other strand of the dsDNA are a mixture of continuous and non-continuous ssDNA molecules.

[0182] However, in some aspects, the plurality of ssDNA molecules provided by the method and use are all continuous ssDNA molecules. In some aspects, the plurality of ssDNA molecules provided by the method and use are all non-continuous ssDNA molecules. In some aspects, the plurality of ssDNA molecules providing one strand of a dsDNA molecule are all continuous ssDNA molecules, while the plurality of ssDNA molecules providing another strand of a dsDNA molecule are all non-continuous ssDNA molecules.

[0183] The size of the ssDNA molecule (polynucleotide) can be any size suitable for producing a dsDNA molecule of the above size and providing a complementary region of at least 50 base pairs (bp) to its opposite strand, as further defined below. It is apparent that the size of each ssDNA molecule in each plurality of ssDNA molecules depends on the number of molecules, and the strategy used to produce each strand of the dsDNA molecule (e.g., complete overlap, partial overlap, etc.). Typically, continuous ssDNA molecules are longer than non-continuous ssDNA molecules. Therefore, the size of each ssDNA in the plurality of ssDNA molecules may be different. However, in some embodiments, the size of each ssDNA molecule in the plurality of ssDNA molecules used to provide a strand of a dsDNA molecule may be substantially the same, for example within about 20% (e.g., within about 15% or 10%) of the total number of nucleotides of each other, i.e., ±10% (e.g., ±7.5% or 5%) of the total number of nucleotides of each other. Thus, in a representative example, each ssDNA in the plurality of ssDNA molecules used to provide a strand of dsDNA may consist of about 450 to 550 nucleotides (i.e., 500 nucleotides ± 50 nucleotides). However, it is apparent that the ssDNA molecules providing the 5' end and 3' end of one strand of the dsDNA molecule (e.g., the second strand as defined above) may be shorter than other ssDNA molecules in the plurality of ssDNA molecules. For example, in a complete overlap strategy, the ssDNA molecules providing the 5' end and 3' end of the second strand of the dsDNA molecule are shorter than other ssDNA molecules in the plurality of ssDNA molecules.

[0184] The length of each ssDNA molecule in the plurality of ssDNA molecules can be about 150 to 10,000 nucleotides, e.g., about 200 to about 9,000 nucleotides, about 250 to about 8,000 nucleotides, about 350 to about 7,000 nucleotides, about 400 to about 6,000 nucleotides, about 450 to about 5,000 nucleotides, about 500 to about 4,000 nucleotides, about 600 to about 3,500 nucleotides, about 700 to about 3,000 nucleotides, about 800 to about 2,500 nucleotides, about 900 to about 2,000 nucleotides, about 1,000 to about 1,500 nucleotides, etc.

[0185] Alternatively, each ssDNA molecule in the plurality of ssDNA molecules can comprise at least about 200, 300, 400, 500 nucleotides, such as at least about 600, 700, 800, 900, 1000 nucleotides, such as 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more nucleotides.

[0186] As described above, continuous ssDNA molecules are generally longer than non-continuous ssDNA molecules. Thus, for example, continuous ssDNA molecules can contain at least about 500 nucleotides, such as at least about 600, 700, 800, 900, 1000 nucleotides, such as 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more nucleotides. In methods using a mixture of continuous and non-continuous ssDNA, non-continuous ssDNA molecules are generally shorter than continuous ssDNA molecules, such as at least about 150, 200, 300, 400 or 500 nucleotides.

[0187] Furthermore, even in methods and uses in which all ssDNA molecules are continuous, the ssDNA molecules providing the 5' and 3' ends of one strand of the dsDNA can be shorter than other ssDNA molecules, e.g., at least shorter than the length of the complementary region, e.g., at least 50 nucleotides shorter, such as 75, 100, 125, 150 nucleotides or more.

[0188] Thus, in the context of the methods and uses provided above (i.e., fully overlapping and partially overlapping strategies), the ssDNA molecule in (a)(i) may comprise at least about 500 nucleotides, such as at least about 600, 700, 800, 900, or 1000 nucleotides; at least two of the ssDNA molecules in (a)(ii) may comprise at least about 500 nucleotides, such as at least about 600, 700, 800, 900, or 1000 nucleotides.

[0189] In the context of the other methods and uses provided above (i.e., the minimal overlap strategy), the ssDNA molecule in (a)(i) may comprise at least about 500 nucleotides, e.g., at least about 600, 700, 800, 900, or 1000 nucleotides; the ssDNA molecule in (a)(ii) may be shorter than the ssDNA molecule in (a)(i), e.g., at least about 150, 200, 300, 400, or 500 nucleotides.

[0190] In addition to providing a plurality of ssDNA molecules of sufficient size to generate dsDNA complexes and subsequently dsDNA molecules, it is understood that each ssDNA molecule having a non-terminal 5' end (i.e., an ssDNA molecule that does not provide the 5' end of its respective chain) must be phosphorylated in order to be ligated. While it is advantageous that each ssDNA molecule having a non-terminal 5' end has a phosphorylated 5' end, this is not required, as the 5' end can be phosphorylated after the dsDNA complex is assembled. Therefore, the method may include a further step of phosphorylating the 5' end of the ssDNA molecule, for example using a kinase, such as T4 polynucleotide kinase.

[0191] Similarly, it will be appreciated that each ssDNA molecule having a non-terminal 3' end (i.e., an ssDNA molecule that does not directly contribute the 3' end of its respective strand) must be capable of being extended in a polymerase-mediated extension reaction and / or ligated in a ligase-mediated ligation reaction, e.g., comprises an extendable and / or ligatable 3' end (i.e., comprises a functional hydroxyl group).

[0192] In some aspects, it may also be useful to provide one or two ssDNA molecules that provide the 5' end of the dsDNA molecule without a phosphorylated 5' end, e.g., to minimize intermolecular and intramolecular ligation events between dsDNA complexes. For example, the 5' end of one or two ssDNA molecules that provide the 5' end of the dsDNA molecule may be modified. In this regard, the dsDNA produced by the methods and uses provided herein may be a linear dsDNA molecule.

[0193] Similarly, in some aspects, it may be useful to provide one or two ssDNA molecules that provide the 3' end of a dsDNA molecule (i.e., directly provided) without an extendable and / or ligatable 3' end, e.g., to minimize intermolecular ligation events between dsDNA complexes and / or to minimize the generation of unwanted extension products. For example, the 3' end of one or both ssDNA molecules that provide the 3' end of a dsDNA molecule may be modified. As discussed further below, the dsDNA molecules may be subjected to further steps, such as post-synthetic modifications, such as phosphorylation of the 5' end to enhance the functionality of the dsDNA molecule, e.g., to facilitate its ligation to other DNA molecules (e.g., vectors such as plasmids).

[0194] It should be understood that in the context of single-stranded DNA molecules, the term "single-stranded" refers to a polynucleotide that is single-stranded under denaturing conditions (e.g., after application of heat or a suitable chemical denaturant), i.e., a polynucleotide that has only one continuous backbone (one chain). As described above, this does not exclude the formation of secondary or tertiary structures by single-stranded DNA molecules. For example, single-stranded DNA molecules may contain self-complementary regions and are therefore able to form hairpin or stem-loop structures, mediated by hybridization of one region of a single-stranded DNA molecule with a complementary region elsewhere in the same single-stranded DNA molecule. The terms "single-stranded DNA (ssDNA) molecule," "ssDNA" and "polynucleotide" are used interchangeably herein.

[0195] As used herein, the term "plurality" refers to three or more, such as at least 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30 or more, such as 40, 50, 75, 100 or more, depending on the context of the invention. For example, a plurality of ssDNA molecules, each comprising (e.g., consisting of) non-overlapping portions of a strand of a dsDNA molecule, may contain at least 3 ssDNA molecules, such as 4, 5, 6, 7, 8, 9, 10, 12, 20, 25, 30 or more ssDNA molecules, such as 3 to 50, 4 to 45, 5 to 40, 6 to 35, 7 to 30, 8 to 25, 9 to 20, or 10 to 15 ssDNA molecules. It should be understood that in some strategies (i.e., complete overlap and some partial overlap strategies), one strand of the dsDNA is provided by more ssDNA molecules than the other strand, i.e., at least one more ssDNA molecule. In a representative example, the first strand is provided by four ssDNA molecules and the second strand is provided by five ssDNA molecules. Thus, the number of ssDNA molecules provided in each plurality of ssDNA molecules may be different. However, in some strategies (i.e., the minimum overlap strategy or some partial overlap strategies), the number of ssDNA molecules provided in each plurality of ssDNA molecules is the same.

[0196] It can be seen that if multiple copies (i.e., a plurality) of each plurality of ssDNA molecules are provided in the methods and uses described herein, this will result in the production of a plurality of dsDNA complexes and subsequently dsDNA molecules, for example, 10 of each dsDNA molecule. 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more copies. Thus, the methods and uses provided herein can be viewed as producing a plurality of dsDNA molecules. In this regard, each copy of the ssDNA molecule providing the non-overlapping portion of the strand of the dsDNA molecule can be identical, i.e., contain the same sequence. If the ssDNA molecule contains modified nucleotides, the modified nucleotides in each copy can be identical, i.e., the number, type, and position of the functional groups in each copy of the ssDNA molecule are identical.

[0197] Similarly, when different versions (e.g., sequence variants) of a ssDNA molecule are provided, the term "plurality" may refer to at least 3 different versions (e.g., 3 sequence variants), such as 4, 5, 6, 7, 8, 9, 10, 12, 20, 25, 30 or more different versions, wherein multiple copies of each version may be provided (e.g., 10 copies of each version). 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 When different versions are used to make a library of dsDNA molecules into a gene library (e.g., for phage display), the term "plurality" may refer to at least 100 versions (e.g., 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more versions), where multiple copies of each version may be provided (e.g., 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 or more copies).

[0198] As described above, it is assumed that the use of very pure and correct ssDNA molecules contributes to the effectiveness of the methods and uses provided herein. In this regard, it is well known in the art that polynucleotides produced using solid phase chemical synthesis methods (e.g., phosphoramidite synthesis) often contain impurities (e.g., byproducts of the chemical synthesis reaction) and incorrect sequences, especially polynucleotides containing more than 50 nucleotides. In contrast, polynucleotides produced using enzymatic synthesis methods, especially using so-called high-fidelity DNA polymerases, can be easily highly purified with minimal sequence errors.

[0199] Thus, the ssDNA molecules provided in the methods and uses described herein are not produced by chemical synthesis methods, such as solid phase chemical synthesis methods, such as phosphoramidite synthesis.

[0200] Alternatively, each ssDNA molecule provided in the methods and uses described herein is produced by enzymatic synthesis, i.e., an enzymatically produced ssDNA molecule. Many methods for enzymatically producing ssDNA molecules are known in the art, such as asymmetric PCR (e.g., LATE-PCR), monoclonal stoichiometric (MOSIC) polynucleotide production (Ducani et al., 2013, Nature Methods, 647-652, incorporated herein by reference), terminal deoxynucleotidyl transferase (TdT) synthesis, etc. It should be understood that any suitable enzymatic synthesis method can be used to produce ssDNA molecules for the methods and uses provided herein. Therefore, the ssDNA molecules used in the methods and uses provided herein can be produced by asymmetric PCR.

[0201] Although it is known that PCR-based synthesis methods may produce polynucleotides containing errors, the probability of errors occurring in polynucleotides containing about 1000 nucleotides or less is low. Therefore, when ssDNA molecules are produced by asymmetric PCR, they may contain 1000 nucleotides or less. However, it should be understood that longer ssDNA molecules (e.g., containing more than 1000 nucleotides) can be accurately synthesized using high-fidelity polymerases, such as Thermococcus litoralis DNA polymerase (Vent) described in Perler et al., Proc. Natl. Acad. Sci. USA (1992) 89: 5577-5581; Pyrococcus GB-D (Deep Vent); Pyrococcus furiosus DNA polymerase (Pfu) described in Lundberg et al., Gene (1991) 108: 1-6; Pyrococcus wollastonii (Pwo), etc.

[0202] Therefore, enzymatic synthesis methods (e.g., asymmetric PCR) can use high-fidelity polymerases, i.e., those with an error rate of 2.0 × 10 -5 or lower, such as 1.0×10 -5 or lower, 9.0×10 -6 or lower, 8.0×10 -6 or lower or 7.0×10 -6 or lower polymerase.

[0203] The “error rate” (f) was calculated as f = n / S (target size × d), where n is the number of mutations observed in all sequenced polynucleotides and (target size × d) for each cloned polynucleotide, where (d) is the average fold increase for each PCR reaction, where the fold increase is calculated according to Equation 2 d =(ng after PCR DNA / input ng DNA) computing.

[0204] Although any enzymatic synthesis method can be used to produce ssDNA molecules for the methods provided herein, it is preferred to use ssDNA molecules produced by methods that do not involve PCR to avoid the introduction of sequence errors, especially the production of long ssDNA molecules (e.g., containing at least about 500 nucleotides, such as 1000 nucleotides or more). For example, ssDNA molecules can be produced using a rolling circle amplification (RCA)-based method, particularly the MOSIC method. Methods for producing ssDNA molecules using the MOSIC method are described in Ducani et al. (supra), WO2020 / 161187, and WO2021 / 170656 (all incorporated herein by reference).

[0205] Thus, the ssDNA molecules used in the methods described herein can be produced by a method comprising:

[0206] (a) providing a circular DNA molecule, the circular DNA molecule comprising a nucleotide sequence encoding a ssDNA molecule adjacent to a cleavage domain;

[0207] (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule in (a) as a template; and

[0208] (c) cleaving (e.g., enzymatically cleaving) the product of the RCA reaction at the cleavage domain to release multiple copies of the ssDNA molecule; and optionally

[0209] (d) isolating or purifying the ssDNA molecules produced in (c).

[0210] The step of providing a circular DNA molecule can be achieved by any suitable means and can depend on the structure of the circular DNA molecule. For example, the circular DNA molecule can be provided by a minicircle plasmid described in WO2021 / 170656 (incorporated herein by reference). In this regard, the circular DNA molecule can be a single-stranded DNA molecule or a double-stranded DNA molecule.

[0211] In embodiments where the circular DNA molecule is double stranded, it will obviously have to be treated to provide an RCA template. Therefore, in some embodiments, the method comprises the additional step of cleaving a single strand of the circular DNA molecule prior to performing the RCA reaction to provide an RCA template.

[0212] Thus, step (a) of the methods provided herein may include the above-described methods for producing ssDNA molecules in a plurality of ssDNA molecules. Step (a) of the methods provided herein may include the above-described methods for producing all of the plurality of ssDNA molecules used in the methods provided herein. It should be understood that each of the plurality of ssDNA molecules can be produced in a separate reaction and then pooled or combined to form a plurality of ssDNA molecules of one or two chains. Alternatively, in a so-called multiplex reaction, a plurality of ssDNA molecules of one or two chains can be produced in a single reaction mixture. For example, as shown in the Examples, each ssDNA molecule can be encoded by a separate circular DNA molecule, which can be combined in a single reaction mixture to simultaneously produce ssDNA molecules.

[0213] It should be understood that the circular DNA molecule may comprise a plurality of nucleotide sequences adjacent to the cleavage domain, each nucleotide sequence encoding a ssDNA molecule provided in (a), such that more than one ssDNA molecule of a plurality of ssDNA molecules, such as all of a plurality of ssDNA molecules of a chain of a dsDNA molecule, may be provided in a single step.

[0214] One or two strands of ssDNA molecules can be generated by combining the above multiplex reaction strategy with a circular DNA molecule comprising a plurality of nucleotide sequences, each encoding a ssDNA molecule.

[0215] It should be understood that each ssDNA molecule used in the methods and uses provided herein can be provided separately and therefore can be produced by different methods. Thus, for example, some ssDNA molecules can be produced by asymmetric PCR and / or TdT synthesis, and some ssDNA molecules can be produced by the MOSIC method, which are then combined (i.e., mixed) to provide multiple ssDNA molecules. However, in order to avoid additional steps, it may be preferred to produce all ssDNA molecules by the same method, such as the MOSIC method described above.

[0216] As shown in the Examples, it has been unexpectedly demonstrated that all ssDNA molecules of both strands of a dsDNA molecule can be produced and assembled in a single reaction mixture, a so-called "one-pot" reaction. This is particularly advantageous because it reduces the complexity of the method and the consumables used in the process, thereby reducing time and cost. Thus, all steps of the method, including the step of producing ssDNA molecules, can be performed in a single reaction mixture and / or container. In other words, the methods provided herein can be one-pot reactions.

[0217] As used herein, the term "cleavage domain" generally refers to a domain within a circular DNA molecule that results in a domain within an RCA product that can be specifically cleaved to release a ssDNA molecule. Thus, a cleavage domain in a circular DNA molecule can directly cleave, or it can simply encode a cleavage domain that only functions in the RCA product or under specific conditions (e.g., when in contact with an auxiliary factor).

[0218] In some embodiments, the cleavage domain comprises or consists of a sequence capable of forming a hairpin structure. A hairpin structure may also be referred to as a hairpin loop or a stem loop, and these terms are used interchangeably herein. A hairpin is an intramolecular base pairing pattern that may occur in single-stranded DNA or RNA molecules. A hairpin occurs when two regions of the same strand (usually complementary nucleotide sequences when read in opposite directions) form a double-stranded stem (duplex) and an unpaired (i.e., single-stranded) loop through base pairing (hybridization). The resulting structure can be described as a lollipop shape.

[0219] Therefore, in some embodiments where the cleavage domain comprises or consists of a sequence capable of forming a hairpin structure, the cleavage domain comprises a self-complementary sequence. As the RCA product is extended, these self-complementary regions hybridize to produce a hairpin structure, wherein the double-stranded portion of the hairpin structure comprises a sequence recognized by a cleavage enzyme. Therefore, the cleavage of the double-stranded portion of the hairpin structure in the RCA product results in the release of the ssDNA molecule and the hairpin structure (i.e., the oligonucleotide forming the hairpin structure).

[0220] "Cleavage" includes any means of breaking a covalent bond. Thus, in the context of the present invention, cleavage includes cleavage of covalent bonds in a nucleotide chain (ie, chain cleavage or chain breakage), for example by cleavage of a phosphodiester bond.

[0221] Thus, in some embodiments, the cleavage domain may comprise a sequence recognized by one or more enzymes capable of cleaving nucleic acid molecules, i.e., capable of destroying the phosphodiester bond between two or more nucleotides. For example, the cleavage domain may comprise a restriction endonuclease (restriction enzyme) recognition sequence. Restriction enzymes cut double-stranded or single-stranded DNA at specific recognition nucleic acid sequences called restriction enzyme cutting sites, and suitable enzymes are well known in the art. For example, it may be particularly advantageous to use rare-cutting restriction enzymes, i.e., enzymes with long recognition sites (at least 8 base pairs in length), to facilitate the design of polynucleotide sequences in circular DNA molecules, for example, to avoid including cleavage recognition sites within the sequence of ssDNA molecules.

[0222] The cleavage domain may comprise a sequence recognized by a type II restriction endonuclease, more preferably a type IIs restriction endonuclease. Although any suitable cleavage domain and cleavage enzyme may be used, the cleavage enzyme that recognizes the cleavage domain adjacent to the polynucleotide sequence may be BseGI, BtsCI, or an isoschizomer thereof, such as BstF5I or FokI. Other representative enzymes that may be used include BsrDI, BtsI, BtsIMutI, MlyI or their isoschizomers.

[0223] As described above, the methods provided herein can utilize long ssDNA molecules, for example, at least about 500, 750, or 1000 nucleotides in length. It should be understood that longer sequences are more likely to contain sequences recognized by endonucleases, particularly type IIs restriction endonucleases. Thus, the cleavage domain adjacent to the polynucleotide can be a homing endonuclease cleavage domain, i.e., the cleavage enzyme used in the cleavage step can be a homing endonuclease. For example, the cleavage domain adjacent to the polynucleotide can be a meganuclease cleavage domain, i.e., the cleavage enzyme used in the cleavage step can be a meganuclease.

[0224] The term "meganuclease" refers to an endonuclease characterized by a large recognition site, such as a double-stranded DNA sequence of 12 to 40 base pairs. Therefore, many homing endonucleases, such as I-SceI, can be considered as meganucleases. Chimeric meganucleases can be produced by fusing the nucleic acid binding domains and endonuclease cleavage domains of different proteins. For example, any protein domain capable of site-specific recognition (binding) of a DNA sequence as described above can be fused to the cleavage domain of an endonuclease, and the cleavage domain is cleaved outside the sequence recognized by the endonuclease, such as cleaved at a specific distance of the recognition sequence. Any suitable meganuclease known in the art can be used in the methods described herein, such as in the step of cleaving an RCA product.

[0225] Thus, the step of cleaving the RCA reaction product may comprise contacting the RCA product with a cleavage enzyme under suitable conditions to selectively cleave the RCA product in the cleavage domain.

[0226] After the RCA product is cracked, the ssDNA polynucleotide is released. The released polynucleotide may consist of only a polynucleotide sequence (i.e., not containing additional nucleotides), or may include one or more additional nucleotides from a cleavage domain adjacent to the polynucleotide sequence at one or both ends. Therefore, the ssDNA polynucleotide may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides from a cleavage domain at one or both ends. Preferably, the sequence of the circular DNA molecule is designed so that when the RCA product is cracked, the released polynucleotide does not include any additional nucleotides from the cleavage domain. Alternatively, the cleavage domain adjacent to the polynucleotide sequence may be arranged in the circular DNA molecule so that after it is cracked in the RCA product, an ssDNA polynucleotide that does not include any additional nucleotides from the cleavage domain is released.

[0227] Since the cleavage enzyme can cleave the nucleic acid molecule at a position other than the cleavage enzyme recognition sequence, one or more nucleotides may be present between the cleavage domain and the polynucleotide sequence. Alternatively, the cleavage domain may also comprise a nucleotide sequence in addition to the cleavage enzyme recognition sequence to ensure that the intact ssDNA polynucleotide is released after cleavage, preferably without any additional nucleotides (e.g., nucleotides that form part of the cleavage domain).

[0228] Therefore, in the context of the polynucleotide sequence being adjacent to the cleavage domain, the term "adjacent" refers to that the cleavage domain is directly adjacent to or indirectly adjacent to the polynucleotide sequence. Alternatively, the cleavage domain is located at either end of the polynucleotide sequence, i.e., the cleavage domain is located at the upstream and downstream (5' end and 3' end) of the polynucleotide sequence. The cleavage site of the cleavage domain (the site where the cleavage domain is cracked by a lytic enzyme) is directly adjacent to the end of the polynucleotide sequence adjacent to it. However, the polynucleotide sequence and the cleavage domain sequence can overlap, i.e., the end of the polynucleotide sequence can form a partial cleavage domain, such as when the cleavage site is an internal site in the cleavage domain, i.e., the cleavage domain can form the end or part of the end of the polynucleotide sequence. Therefore, one or more nucleotides may be present between the cleavage domain and the polynucleotide sequence (i.e., between the sequence ends), such as 1,2,3,4,5,6,7,8,9 or 10 or more nucleotides. However, the cleavage domain and the polynucleotide sequence may overlap one or more nucleotides, such as 1,2,3,4,5,6,7,8,9 or 10 or more nucleotides.

[0229] The size of the cleavage domain in the circular DNA molecule is not particularly limited, depending on the type of cleavage domain, as described above. Design or select the relative length of the polynucleotide sequence and the cleavage domain so that they are different from each other, so that after the RCA product is cracked at the cleavage domain, the ssDNA polynucleotide is easy to purify. Therefore, a cleavage domain shorter than the polynucleotide sequence in the circular DNA molecule can be selected. If the circular DNA molecule comprises a plurality of polynucleotide sequences of different lengths, a cleavage domain shorter than the shortest polynucleotide sequence in the circular DNA molecule can be selected.

[0230] The length of the cleavage domain may be about 4 to 50 nucleotides, such as about 5 to 45, 6 to 40, 7 to 35 or 8 to 30 nucleotides. In some embodiments, the length of the cleavage domain may be about 10 to 25 nucleotides, including about 12 to 22 or about 14 to 20 nucleotides. However, it is apparent that any suitable length of the cleavage domain can be used in the present invention as long as the above-mentioned functional requirements are met. Therefore, longer cleavage domains are contemplated, such as lengths of about 22 to 70 nucleotides, including about 25 to 60 or about 30 to 55 nucleotides. Alternatively, the length of the cleavage domain may be about 35 to 80 nucleotides, including about 40 to 70 or about 45 to 60 nucleotides.

[0231] The cleavage domain adjacent to the polynucleotide sequence can be identical or different from each other. Advantageously, the cleavage domain adjacent to the polynucleotide sequence is identical so that a single cleavage step is sufficient to release all single-stranded DNA polynucleotides. However, as described above, some lytic enzymes can cleave nucleic acid molecules at positions outside the lytic enzyme recognition sequence, or can recognize more than one sequence (e.g., allowing the variation in the enzyme recognition sequence). Therefore, the entire sequence of the cleavage domain need not be identical so that it is cleaved by the same enzyme. For example, the step of cracking the RCA product may include contacting the RCA product with a single lytic enzyme under the conditions of the cleavage domain suitable for cracking the RCA product.

[0232] Suitable cleavage conditions for the cleavage domain in the RCA product depend on the means used to achieve cleavage. For example, when cleavage is achieved using a cleavage enzyme such as a restriction endonuclease or a homing endonuclease, the conditions will vary depending on the enzyme selected, and suitable conditions are well known in the art, for example, the cleavage step can be performed according to the manufacturer's instructions.

[0233] For example, a cleavage enzyme, such as a restriction endonuclease or a homing endonuclease, can specifically bind to its cleavage recognition site and selectively (e.g., specifically) cleave nucleic acids in a variety of buffers, such as phosphate buffered saline (PBS), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), HEPES buffer (HBS), and Tris buffer (TBS), all containing or not containing EDTA. Cleavage can occur in a pH range of about 3.0 to 10.0, such as 4.0 to 9.0, 5.0 to 8.0, and in a wide temperature range, such as 0 to 70°C. Other suitable conditions can be easily determined by those skilled in the art.

[0234] Therefore, the method for producing ssDNA molecules may include a step of rolling circle amplification using a circular DNA molecule as a template. Rolling circle amplification (RCA) is well known in the art and is described in Dean et al., 2001 ("Rapid amplification of plasmid and phage DNA using phi29 DNA polymerase and multi-primer rolling circle amplification", Genome Research, 11, pp. 1095-1099), the disclosure of which is incorporated herein by reference. In short, RCA involves the use of a circular single-stranded nucleic acid molecule (e.g., a circular or circular oligonucleotide) as a rolling circle template (RCA template) and the use of a strand displacement polymerase to extend a primer hybridized to the template to synthesize a nucleic acid molecule. Adding a polymerase and nucleotides starts the synthesis reaction, i.e., polymerization. Since the rolling circle template is circular, the resulting product is a long single-stranded nucleic acid molecule consisting of a tandem repeat sequence complementary to the rolling circle template.

[0235] A typical RCA reaction mixture includes a circular DNA molecule as a template, and one or more primers for a primer extension reaction, for example, RCA can be templated by a single primer to generate a single tandem product, or by a plurality of primers, each primer annealing to a different region of the circular template, so that each circular template produces a plurality of tandem products. The oligonucleotide primer that can be contacted with the circular DNA molecule should have a sufficient length to hybridize with the circular DNA molecule under annealing conditions. It should be understood that the primer can be provided by cleaving (e.g., nicking) a single strand of the double-stranded circular DNA molecule provided in step (a).

[0236] The RCA reaction mixture may also include an aqueous buffer medium comprising a source of monovalent ions, a source of divalent cations, and a buffering agent. Any convenient source of monovalent ions may be used, such as potassium chloride, potassium acetate, ammonium acetate, potassium glutamate, ammonium chloride, ammonium sulfate, and the like. The divalent cation may be magnesium, manganese, zinc, and the like, wherein the cation is typically magnesium. Any convenient source of magnesium cations may be used, including magnesium chloride, magnesium acetate, and the like. The Mg present in the buffer may be present in the buffer. 2+The amount of the buffer may be 0.5 to 10 mM, but is preferably about 3 to 6 mM, ideally about 5 mM. Representative buffers or salts that may be present in the buffer include Tris, Tricine, HEPES, MOPS, etc., wherein the amount of the buffer is generally about 5 to 150 mM, usually about 10 to 100 mM, more usually about 20 to 50 mM, wherein the amount of the buffer that may be present is sufficient to provide a pH of about 6.0 to 9.5. Other agents that may be present in the buffer medium include chelating agents, such as EDTA, EGTA, etc.

[0237] In addition to the above components, the reaction mixture typically includes a polymerase (as further defined below, such as phi29 DNA polymerase), one or more nucleotides and other components required for a DNA polymerase reaction as described below. The required polymerase activity may be provided by one or more different polymerases.

[0238] The RCA reaction ultimately produces a polynucleotide product that contains adjacent (tandem) repeats of the complementary sequence of the circular DNA molecule. The product may be referred to as a concatemer, an RCA product, or an "RCP". Thus, the RCA product contains a linear sequence consisting of a polynucleotide sequence adjacent to the cleavage domain (or more specifically, the reverse complement of the polynucleotide sequence of the circular DNA molecule template).

[0239] Any DNA polymerase with at least partial strand displacement activity can be used for RCA reaction, and the RCA reaction is used to produce ssDNA molecules for the method provided herein. The strand displacement activity ensures that the polymerase can displace the primer sequence and the extension product after extending around the circular DNA molecule, and continues to "roll" around the template. In the embodiment where the nicked strand of the DNA mini-circle provides a primer for RCA extension, the strand displacement activity ensures that the polymerase can displace the nicked strand. Suitable DNA polymerases with at least partial strand displacement activity include phi29 DNA polymerase, Escherichia coli DNA polymerase I, Bsu DNA polymerase (large fragment), Bst DNA polymerase (large fragment) and Klenow fragment. As used herein, the term "DNA polymerase" includes not only naturally occurring enzymes, but also all such modified derivatives, and also includes derivatives of naturally occurring DNA polymerases. For example, in some embodiments, the DNA polymerase can be modified to remove 5'-3' exonuclease activity.

[0240] Particularly preferred DNA polymerases for use in the RCA reaction include phi29 DNA polymerase, Bst DNA polymerase, and derivatives thereof, such as sequence-modified derivatives or mutants thereof.

[0241] The sequence-modified derivatives or mutants of the DNA polymerase include at least a portion of the functional activity of the wild-type sequence, such as a mutant of DNA polymerase activity and at least a portion of strand displacement activity. The mutation may affect the activity characteristics of the enzyme under different reaction conditions (such as temperature, template concentration, primer concentration, etc.), such as increasing or decreasing the polymerization rate. The mutation or sequence modification may also affect the exonuclease activity and / or the thermostability of the enzyme.

[0242] As described above, the RCA reaction can be performed using conventional nucleotides, functionalized nucleotides, or a mixture of conventional nucleotides and functionalized nucleotides.

[0243] The term "release" is used herein to refer to cleavage of the RCA product at a cleavage domain adjacent to a polynucleotide sequence, thereby disengaging or separating the polynucleotide from the cleavage domain. Release of a given polynucleotide is expected to involve cleavage at two cleavage domains adjacent to the polynucleotide sequence.

[0244] In order to generate single-stranded DNA polynucleotides, all cleavage domains in the RCA product do not have to be cleaved. The cleavage of some cleavage domains will still lead to the release of some single-stranded DNA polynucleotides. Therefore, the step of cleaving the RCA product can result in the cleavage of at least about 30%, for example at least about 35%, 40%, 45%, 50%, 60%, 70% or 80% of the cleavage domains in the RCA product. However, the step of cleaving the RCA product can result in the cleavage of at least about 90%, for example 95% or more of the cleavage domains in the RCA product.

[0245] Alternatively, the step of cleaving the RCA product can result in the release of at least about 30%, for example at least about 35%, 40%, 45%, 50%, 60%, 70% or 80% of the single-stranded DNA polynucleotides contained in the RCA product. In addition, the step of cleaving the RCA product can result in the release of at least about 90%, for example 95% or more of the single-stranded DNA polynucleotides contained in the RCA product.

[0246] Once the single-stranded DNA polynucleotide is released, it may be necessary to separate, extract or purify the single-stranded DNA polynucleotide from the cleavage reaction mixture (e.g., reaction components and / or degradation products, such as cleavage domains, uncleaved RCA products, etc.) to provide ssDNA molecules for use in the methods and uses provided herein.

[0247] Therefore, the method for producing ssDNA molecules may further include the step of isolating, extracting or purifying the single-stranded DNA polynucleotide. Such isolation, extraction or purification can be performed by any suitable method known in the art.

[0248] After separation, extraction or purification steps, the single-stranded DNA polynucleotide is preferably substantially free of any contaminating components (e.g., reaction components and / or degradation products, such as cleavage domains, uncleaved RCA products, etc.) derived from materials or components used in the separation process or its preparation. The single-stranded DNA polynucleotide is purified to a purity exceeding about 70%, 80% or 90%, such as a purity exceeding about 95% or 99%, assessed by w / w (dry weight). This purity level may include degradation products of the single-stranded DNA polynucleotide.

[0249] For example, gel electrophoresis can be performed using agarose gel or polyacrylamide gel to separate the products of the cleavage reaction by size. The desired polynucleotide can then be separated from the gel and, if necessary, further purified according to methods known in the art. Other methods for purifying, separating or extracting the polynucleotides of the present invention utilize chromatography (e.g., HPLC, size exclusion, ion exchange, affinity, hydrophobic interaction, reversed phase) or capillary electrophoresis.

[0250] However, it should be understood that in some embodiments, such as in "one-pot" process embodiments, it is not necessary or desirable to separate, extract or purify the ssDNA molecules. Thus, in some embodiments, the ssDNA molecules are not separated, extracted or purified prior to the step of hybridizing the ssDNA molecules.

[0251] The methods and uses provided herein utilize complementary regions between ssDNA molecules in each plurality of ssDNA molecules to form DNA complexes. Each ssDNA molecule in a plurality of ssDNA molecules (i.e., a plurality of ssDNA molecules that provide all portions of a strand of a dsDNA molecule) comprises a region complementary to at least one ssDNA molecule in the other plurality of ssDNA molecules. The complementary region typically includes a "terminal region" of the ssDNA molecule, i.e., a region in the ssDNA molecule that includes the 5' end or the 3' end.

[0252] It should be understood that the terminal ssDNA molecule of one chain (i.e., the ssDNA molecule at the 5' end or 3' end of the chain containing the dsDNA molecule) will have a sequence that is complementary to only one ssDNA molecule of the other chain. However, the internal ssDNA molecule (i.e., the ssDNA molecule at the 5' end or 3' end of the chain not containing the dsDNA molecule) and the terminal ssDNA molecule of the other chain will have two complementary regions, i.e., a first region that is completely complementary to the terminal region of a certain ssDNA molecule of the opposite chain and a second region that is completely complementary to the terminal region of another ssDNA molecule of the opposite chain.

[0253] The two complementary regions of the ssDNA molecule (e.g., the inner ssDNA molecule) can be directly adjacent to each other, such that there is no intervening nucleotide sequence between the regions (e.g., in a complete overlap strategy). This means that the 5' and 3' ends of the ssDNA molecules of the opposite strands hybridized to the ssDNA molecule are directly adjacent to each other, i.e., there is no gap between the 5' and 3' ends.

[0254] However, in other strategies, two complementary regions of a ssDNA molecule (e.g., an internal ssDNA molecule) may be indirectly adjacent to each other such that there is an intervening nucleotide sequence between the regions (e.g., in a partially overlapping strategy). This means that the 5' and 3' ends of the ssDNA molecule of the opposite strand hybridized to the ssDNA molecule are indirectly adjacent to each other such that there is a gap between the 5' and 3' ends that must be filled before the ssDNA molecules can be ligated.

[0255] The ssDNA molecules in each plurality of ssDNA molecules are designed so that the complementary regions hybridize with ssDNA molecules of adjacent portions of the opposing strands, so that when the ssDNA molecules are joined, portions of each strand are joined in the correct order to produce a dsDNA molecule. As described above, ssDNA molecules can hybridize with ssDNA molecules of opposing strands such that the ssDNA molecules are directly adjacent (no gap) or indirectly adjacent (i.e., there is a gap). Directly adjacent ssDNA molecules can be considered to form a junction (e.g., a nicking site) between the two molecules, such that the molecules can join in a ligase-mediated ligation reaction.

[0256] The term "region" refers to a portion of a ssDNA molecule.

[0257] As stated above, it is preferred that the region of the ssDNA molecule which hybridizes to the ssDNA molecule of the opposite strand is fully complementary as defined above.

[0258] While not wishing to be bound by theory, it is hypothesized that the size of the complementary region contributes to the effectiveness of the methods and uses provided herein. Thus, the region of the ssDNA molecule that is complementary to the ssDNA molecule of the opposite strand must contain at least 50 nucleotides, such as at least about 60, 70, 80, 90, or 100 nucleotides. The complementary region may contain at least about 110, 125, 150, 200, 250, 300, 400, or 500 nucleotides. Thus, for example, the complementary region may contain 50 to 750, 60 to 700, 70 to 650, 80 to 600, 90 to 550, or 100 to 500 nucleotides, such as 150 to 500, 200 to 500, or 250 to 500 nucleotides.

[0259] The ssDNA molecules are provided in equimolar (i.e., stoichiometrically 1:1:1, etc.) amounts such that the number of each ssDNA molecule is approximately the same. It is believed that this can reduce the formation of undesirable dsDNA complexes. Thus, when different versions of an ssDNA molecule (e.g., sequence variants of an ssDNA molecule) are provided, the total concentration of the different versions is equimolar to the concentration of the other ssDNA molecules in the plurality of ssDNA molecules, i.e., each version of the ssDNA molecule is provided at a lower concentration than if only one version of the ssDNA molecule were provided.

[0260] The ssDNA molecules can be provided by any suitable method or means. For example, each plurality of ssDNA molecules can be combined individually to provide a mixture of two individual ssDNA molecules (i.e., one for each strand), which are then combined, i.e., capable of hybridization. Alternatively, all ssDNA molecules can be combined in a single mixture in any order under any suitable conditions, such as conditions suitable for maintaining the integrity of the ssDNA molecules, i.e., avoiding degradation of the ssDNA molecules, such as in a buffer solution, such as 20 to 100 mM Tris-HCl (pH 7 to 9), optionally containing a chelating agent such as EDTA or EGTA.

[0261] Thus, the methods and uses provided herein may involve a step of combining or mixing the ssDNA molecules, i.e., providing a mixture of ssDNA molecules. However, as described above, ssDNA molecules can be produced in a single reaction mixture, and in these embodiments, a step of combining or mixing the ssDNA molecules is not required.

[0262] The method and use involve hybridization of ssDNA molecules containing complementary regions to produce a dsDNA complex containing all ssDNA molecules. This step can be considered as a step of assembling the dsDNA complex. The hybridization step involves placing the mixture of ssDNA molecules under annealing conditions. It is obvious that the method and use provided herein involve a single hybridization step and a subsequent single connection step, that is, before hybridization and connection, all ssDNA molecules forming dsDNA molecules are combined or mixed (present in the mixture). Therefore, the method and use do not involve sequential hybridization and / or sequential connection steps or multi-step hybridization and / or connection steps, for example, wherein a first subset of ssDNA molecules is formed by hybridization and connection to form a partial dsDNA molecule, and the partial dsDNA molecule is used for subsequent hybridization and connection steps to form a complete dsDNA molecule.

[0263] The term "annealing conditions" refers to conditions under which two nucleic acid molecules comprising complementary nucleotide sequences will specifically hybridize to each other. Various parameters affect hybridization, including temperature, salt concentration, nucleic acid concentration, composition and length, and buffer composition. One skilled in the art can routinely and readily determine annealing conditions suitable for a particular combination of ssDNA molecules.

[0264] In a representative example, the step of hybridizing ssDNA molecules to produce a dsDNA complex comprises placing a mixture of ssDNA molecules (i.e., all ssDNA molecules) at a temperature suitable for denaturing the DNA molecules, such as heating the mixture to at least 94°C, such as 94 to 100°C (e.g., 94, 95, 96, 97, 98, or 99°C), followed by cooling the mixture to a temperature that enables the ssDNA molecules to specifically hybridize to each other, such as cooling the mixture to less than 40°C, such as 4 to 40°C (e.g., 10 to 35, 15 to 30, 20 to 25°C, such as room temperature). As described above, the methods and uses provided herein advantageously use long complementary regions. Therefore, it should be understood that ssDNA molecules can hybridize to each other at relatively high temperatures, such as up to about 85°C. Thus, cooling the mixture to a temperature at which the ssDNA molecules can specifically hybridize to each other may involve cooling the mixture to below about 90° C., such as about 40 to 90° C. (e.g., about 50 to 89° C., 60 to 88° C., 65 to 87° C., 70 to 86° C., or 75 to 85° C.). Subsequent extension and / or ligation steps may require further cooling, i.e., cooling to a temperature at which the polymerase and / or ligase is active (e.g., to avoid denaturing the enzyme). However, when the extension and / or ligation steps use a thermostable polymerase and / or ligase, such as Taq polymerase and / or Taq ligase (or a functional variant thereof), further cooling may not be required.

[0265] Thus, the methods and uses provided herein may involve steps of heating and cooling ssDNA molecules (ie, mixtures of ssDNA molecules) to enable ssDNA molecules containing complementary regions to hybridize, thereby generating dsDNA complexes.

[0266] As used herein, the term "hybridize" or "hybridization" refers to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex by Watson-Crick base pairing.

[0267] The term "dsDNA complex" refers to a dsDNA molecule in which each strand consists of more than one ssDNA. Alternatively, the term refers to multiple ssDNA molecules that are non-covalently bound (i.e., hybridized) to form a dsDNA molecule, wherein the phosphodiester backbone of one or both strands is not completely joined, i.e., one or both strands contain more than one 5' and 3' end available for reaction.

[0268] A dsDNA complex can be considered a "complete double-stranded" or "intact double-stranded" DNA complex when all ssDNA molecules on each strand are directly adjacent to each other, i.e., the ssDNA molecules on each strand form a ligatable junction. Thus, a complete double-stranded DNA complex can be considered a "nicked dsDNA," i.e., containing one or more breaks in the phosphodiester backbone.

[0269] A dsDNA complex can be considered a "partially double-stranded" DNA complex when two or more ssDNA molecules on a strand are indirectly adjacent to each other, ie, the ssDNA molecules on at least one strand form a gap in the strand.

[0270] Thus, the step of hybridizing ssDNA molecules can produce a complete dsDNA complex (eg, a complete overlap strategy) or a partial dsDNA complex (eg, a partial or minimal overlap strategy). The step of hybridizing ssDNA molecules can be viewed as assembling a dsDNA complex.

[0271] It is obvious that when a ssDNA molecule is non-covalently bound (i.e., hybridized) through a complementary region in a dsDNA complex, it is no longer completely single-stranded (it may be partially single-stranded). However, in the context of the methods and uses provided herein, it is convenient to refer to these ssDNA molecules as ssDNA molecules based on the discrete ssDNA portions of these ssDNA molecules that form dsDNA molecules under denaturing conditions.

[0272] When the step of hybridizing the ssDNA molecules results in the formation (i.e., assembly) of a partial dsDNA complex, the gaps between the ssDNA molecules must be "filled" before the ssDNA molecules can be ligated, i.e., joined in a ligase-mediated ligation reaction (to produce a complete dsDNA complex).

[0273] The gaps between the ssDNA molecules in the partial dsDNA complex are "filled" by the polymerase using the ssDNA molecules of the opposite chain as a template. Therefore, the methods provided herein may include a step of extending the 3' end of the ssDNA molecule to produce a complete dsDNA complex. It should be understood that the extension step involves the extension of the internal 3' end of the ssDNA molecule, i.e., the templated extension of the 3' end. In other words, the 3' end of the ssDNA molecule that forms the 3' end of the dsDNA molecule (i.e., the terminal 3' end) is not extended in the extension step. Since the nucleotide sequence of the ssDNA molecule opposite to the gap can be different, i.e., by providing multiple versions of ssDNA molecules in the reaction mixture, the polymerase-mediated extension reaction can promote the production of a mixture of dsDNA molecules (i.e., containing different sequences).

[0274] Any suitable DNA polymerase can be used for the extension step and can be easily selected by those skilled in the art as a rule. In this regard, it is obvious that the polymerase should not have strand displacement activity or 5' exonuclease activity to avoid displacement or degradation of the ssDNA molecule in the partial dsDNA complex downstream (3' end) of the ssDNA molecule that provides the 5' end of the dsDNA molecule. For example, the DNA polymerase can be T4 DNA polymerase, Klenow fragment of DNA polymerase I, Taq polymerase or a sequence-modified derivative or mutant thereof. The polymerase can be a thermostable polymerase and / or a high-fidelity polymerase, such as High-fidelity DNA polymerase (NEB). Thus, the polymerase-mediated extension step can be performed at a relatively high temperature, such as 55 to 80° C., such as 60 to 75° C. or 65 to 75° C. This can involve raising the temperature of the reaction mixture containing the dsDNA complex, i.e., performing a heating step after the hybridization step (i.e., after assembling the dsDNA complex). The extension step can be performed simultaneously with the hybridization step.

[0275] The sequence-modified derivative or mutant of the DNA polymerase comprises at least part of the functional activity of the wild-type sequence, for example a mutant of DNA polymerase activity. The sudden change may affect the activity characteristics of the enzyme under different reaction conditions (for example temperature, template concentration, primer concentration, etc.), for example, improve or reduce the polymerization rate. The sudden change or sequence modification may also affect the thermostability of exonuclease activity and / or enzyme.

[0276] After the formation of the complete dsDNA complex, i.e., after the hybridization and extension steps (if applicable), the ssDNA molecules are ligated to produce (synthesize, form, etc.) dsDNA molecules. Alternatively, the hybridization and extension steps (if applicable) result in the formation of a ligatable junction where the ssDNA molecules are directly adjacent to each other and are fixed by complementary regions on the ssDNA molecules of the opposite strands. The step of ligating adjacent ssDNA molecules can be achieved by any suitable enzymatic means, i.e., adding a ligase under appropriate conditions. As discussed further below, the ligase activity (e.g., a ligase) can be provided in a host cell containing the dsDNA complex, i.e., when the dsDNA complex is conjugated to a linearized nucleic acid vector (e.g., a plasmid) and introduced into a host cell (e.g., a bacterial cell).

[0277] Thus, the methods and uses provided herein do not include chemical ligation, in particular conjugation of adjacent nucleotides by click chemistry, i.e., CuAAC reaction between adjacent terminal cytidine and thymidine residues to produce 1,4-linked 1,2,3-triazoles. However, as described elsewhere herein, dsDNA molecules produced by this method can be subjected to click chemistry reactions in subsequent steps.

[0278] As known in the art, ligase catalyzes the formation of a phosphodiester bond between the juxtaposed 3'-hydroxyl and 5'-phosphate ends of two adjacent nucleic acids. Therefore, ligase can be considered as any enzyme capable of catalyzing the reaction. Any convenient ligase can be used, wherein representative ligases of interest include, but are not limited to, temperature-sensitive ligases and thermostable ligases. Temperature-sensitive ligases include, but are not limited to, bacteriophage T4 DNA ligase, bacteriophage T7 ligase, and E. coli ligase. Thermostable ligases include, but are not limited to, Taq ligase, Tth ligase, and Pfu ligase. Thermostable ligases can be obtained from thermophilic or hyperthermophilic organisms, including, but not limited to, prokaryotes, eukaryotes, or archaebacterial organisms. Enzymes suitable for the ligation step are known in the art, including, for example, T4 DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermococcus (strain 9°N) DNA ligase (9°N TM DNA ligase, New England Biolabs) and Ampligase TM (Epicentre Biotechnologies).

[0279] In this ligation step, a suitable ligase and any necessary and / or desired reagents are combined with the reaction mixture and maintained under conditions sufficient to allow the connection points in the dsDNA complex to be connected. As discussed further below, the ligation reaction can occur in a host cell, such as a bacterial cell. The ligation reaction conditions are well known to those skilled in the art. During the ligation process, in some embodiments, the reaction mixture can be maintained at a temperature of about 4°C to about 50°C, such as about 20°C to about 37°C, for about 5 seconds to about 16 hours, such as about 1 minute to about 1 hour. In other embodiments, the reaction mixture can be maintained at a temperature of about 35°C to about 75°C, such as about 37°C to about 75°C, such as about 40°C, 50°C, 60°C, 70°C or 75°C, for about 5 seconds to about 16 hours, such as about 1 minute to about 1 hour, including about 2 minutes to about 8 hours. In a representative example, the ligation reaction mixture comprises 50mM Tris (pH 7.5), 10mM MgCl 2 , 10 mM DTT, 1 mM ATP, 25 mg / mL BSA, and 0.125 units / mL T4 DNA ligase. In another representative example, 2.125 mM magnesium ions and 0.125 units / mL DNA ligase were used.

[0280] As described above, the ligation step generally results in the synthesis of a linear dsDNA molecule. However, when the dsDNA complex is contacted with a suitable linearized nucleic acid vector (e.g., a plasmid) prior to ligation, the ligation step can result in the synthesis of a circular dsDNA molecule comprising a desired dsDNA molecule or a target dsDNA molecule.

[0281] The dsDNA molecules obtained by the methods and uses provided herein provide another aspect of the invention.

[0282] It will be appreciated that the dsDNA molecules may be subjected to further processing steps following their synthesis, such as to isolate, modify and / or amplify the dsDNA molecules.

[0283] The ligation step of the methods and uses provided herein may not be completely effective, i.e., there may be some unreacted (unligated) ssDNA molecules and / or undesired byproducts. Therefore, it may be necessary to separate the dsDNA molecules from the unreacted components and / or other components (e.g., ligase) in the reaction mixture. Any suitable means may be used to separate the dsDNA molecules from the other components in the reaction mixture.

[0284] Thus, the method may include a further step of isolating or purifying the dsDNA molecules. For example, gel electrophoresis can be performed using an agarose gel or a polyacrylamide gel to separate the products of the ligation reaction by size. The desired dsDNA molecules can then be isolated from the gel and, if necessary, further purified according to methods known in the art. Other methods for purifying, isolating or extracting dsDNA molecules utilize chromatography (e.g., HPLC, size exclusion, ion exchange, affinity, hydrophobic interaction, reverse phase) or capillary electrophoresis.

[0285] As described above, the dsDNA molecules produced by the above methods may contain a reactive group that is capable of reacting with another chemical group, such as a chemical group on a molecule or component that is conjugated to the dsDNA molecule by methods such as click chemistry. For example, conjugating an additional molecule or component (which itself may contain or be considered a functional group) to the dsDNA molecule may be particularly useful for incorporating large or bulky groups, such as groups that may inhibit or reduce the efficiency of the present method if present in a ssDNA molecule.

[0286] Thus, the method may include the step of conjugating a molecule or component to the dsDNA molecule via a functional group (eg, a reactive group) in the dsDNA molecule, such as by click chemistry.

[0287] Alternatively, the method may include the step of modifying the dsDNA molecule, for example to include a functional group, such as a label.

[0288] Thus, the methods and uses provided herein can generate a mixture of dsDNA molecules, ie, a library of dsDNA molecules comprising a plurality of different (eg, functionalized or modified) dsDNA molecules.

[0289] As described above, a mixture of dsDNA molecules can be achieved by providing multiple versions (types) of one or more ssDNA molecules. The versions (types) of ssDNA molecules can have the same sequence and differ in the nucleotides containing the functional groups (e.g., in the position and / or number of the functional groups). Additionally or alternatively, the versions (types) of ssDNA molecules can have the same sequence and differ in the type of functional groups on the modified nucleotides. For example, each version (type) of ssDNA molecules can contain multiple functional groups.

[0290] It is also obvious to the skilled person that a mixture of dsDNA molecules can be achieved by providing multiple versions (types) of one or more ssDNA molecules, wherein the versions (types) of the ssDNA molecules may have different sequences (i.e., the versions may be sequence variants). Such sequence variants may be achieved by any suitable means known in the art. For example, site-specific variants may be produced separately and mixed to provide multiple versions (types) of ssDNA molecules. In another representative example, when the ssDNA molecules are enzymatically synthesized, sequence variations may be introduced randomly, for example, by conducting a synthesis reaction at a high salt concentration or in the presence of manganese ions to induce mutagenesis. The product of the synthesis reaction will be a mixture of ssDNA molecules comprising multiple versions of the ssDNA molecules (i.e., sequence variants of the ssDNA molecules). As described above, when the multiple versions of the ssDNA molecules are sequence variants, each of the multiple versions of the ssDNA molecules will contain a common complementary region.

[0291] Alternatively, mutations can be introduced into the synthesized strand by carrying out an extension reaction at high salt concentrations or in the presence of manganese ions, thereby obtaining a mixture of dsDNA molecules. Mutations can be introduced into the opposite strand in a subsequent processing step.

[0292] The method may also include the step of amplifying dsDNA molecules. Methods for amplifying dsDNA molecules are well known in the art and any suitable method may be used. However, as described above, PCR-based methods may result in the introduction of errors (mutations) in dsDNA products. Therefore, it is preferred that a method other than the PCR-based method be used to amplify dsDNA molecules. Alternatively, if a PCR-based method is used to amplify dsDNA molecules, it is preferred that the method use a high-fidelity polymerase as defined above.

[0293] The method may involve amplifying dsDNA using a host cell. For example, the method may include:

[0294] (i) inserting a dsDNA molecule into a nucleic acid vector (e.g., a DNA plasmid);

[0295] (ii) an amplification vector (e.g., a DNA plasmid);

[0296] (iii) excising the dsDNA molecule from the vector; and optionally

[0297] (iv) isolating or purifying dsDNA molecules, for example from vector nucleic acids.

[0298] Step (ii) may comprise transfecting the vector (eg, DNA plasmid) into bacteria and culturing the bacteria.

[0299] Suitable plasmid sequences are well known in the art. It is noteworthy that by inserting the dsDNA into a nucleic acid vector and amplifying the vector, the dsDNA sequence can be checked (e.g., by sequencing) and any errors in the sequence can be corrected, for example, by iterative sequencing and mutagenesis using any suitable method. Therefore, the method may include a step of checking the sequence of the dsDNA molecule, for example, after the above steps (i), (ii) or (iii), for example, sequencing the dsDNA molecule. The method may also include a step of correcting one or more errors in the dsDNA molecule sequence, and then the corrected dsDNA can be amplified, for example, by the above method, i.e., the above steps (ii) to (iv).

[0300] Inserting a dsDNA molecule into a plasmid also facilitates the production of a large number of copies of the dsDNA molecule. For example, a single bacterial colony containing a plasmid comprising a sequence-verified dsDNA molecule can be cultured to amplify the plasmid, which can then be purified from the bacteria by any suitable means known in the art.

[0301] The dsDNA molecule can be excised from the plasmid, for example using a restriction enzyme as described above. The excised linear dsDNA molecule can be purified by PAGE and gel extraction or other suitable methods. The purified dsDNA molecule can then be used as desired.

[0302] In addition to amplification, the process of transfecting dsDNA molecules containing plasmids into bacteria can also generate bacterial glycerol stocks. Bacteria containing the desired dsDNA molecule plasmid can be prepared in glycerol, frozen, and stored stably for long periods of time.

[0303] The inventors have found that the dsDNA complex formed in the hybridization step is very stable. Therefore, when it is necessary to insert the DNA molecule into a nucleic acid vector (e.g., a plasmid), the ligation step and the insertion step can advantageously be performed in a single reaction. Therefore, the ligation step may involve contacting (mixing) the dsDNA complex with a nucleic acid vector (e.g., a linearized plasmid) and a tool for connecting polynucleotides (e.g., a ligase), forming a dsDNA molecule under suitable conditions and connecting it to the nucleic acid vector. This step may have other advantages because any single-strand breaks (i.e., nicks) in the vector will be repaired when the vector is transferred into a host cell.

[0304] It will be appreciated by those skilled in the art that the ligation step can be achieved or performed in a host cell (see, e.g., Examples 1, 6, and 7). In this regard, the ligation step can involve contacting (mixing) the dsDNA complex with a nucleic acid vector (e.g., a linearized plasmid) and a suitable host cell (e.g., a bacterial cell, such as E. coli), allowing the host cell to take up the assembled dsDNA complex: nucleic acid vector under suitable conditions, wherein the ligase activity in the host cell will connect the molecules to form a complete nucleic acid vector comprising the target dsDNA molecule. Alternatively, when the assembled dsDNA complex: nucleic acid vector is transferred into a host cell, the host cell will repair the single-strand breaks (i.e., nicks) in the dsDNA complex and the vector.

[0305] It is apparent that any suitable host cell may be used, for example a host cell comprising an enzyme having ligase activity (eg, ligase). In a representative example, the host cell is a bacterial host cell, such as an E. coli cell.

[0306] The stability of the dsDNA complex also facilitates the use of a thermostable polymerase for an extension step after assembly of the dsDNA complex, i.e., the reaction mixture containing the assembled dsDNA complex can be contacted with a polymerase as described above (e.g., a thermostable polymerase) and heated to a temperature that is optimal for the polymerase activity of the enzyme, e.g., higher than the temperature to which the reaction mixture was cooled during the hybridization step.

[0307] Thus, in a representative example, provided herein is a method of producing a double-stranded DNA (dsDNA) molecule comprising at least 3000 bp (e.g., at least 4000 bp or 5000 bp), the method comprising:

[0308] (a) Provide:

[0309] (i) at least three (e.g., at least four or five) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule and the second ssDNA comprises the 3' end of the first strand of the dsDNA molecule; and

[0310] (ii) at least four (e.g., at least five or six) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is completely complementary to the region of the second ssDNA molecule in (i); the second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule that is completely complementary to the region of the first ssDNA molecule in (i),

[0311] in:

[0312] (1) At least two (e.g., at least three or four) of the at least four (e.g., at least five or six) ssDNA molecules in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to a terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i);

[0313] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms directly adjacent portions of the first strand of the dsDNA molecule;

[0314] (3) the complementary region comprises at least 150 (e.g., at least 200 or 250) nucleotides;

[0315] (4) all ssDNA molecules comprise at least 400 (e.g., at least 500) nucleotides, optionally wherein all ssDNA molecules in (i) and the ssDNA molecules excluding the 5' end and the 3' end of the second strand of the dsDNA molecule in (ii) comprise at least 800 (e.g., at least 900 or 1000) nucleotides; and

[0316] (5) ssDNA molecules are provided in equimolar amounts;

[0317] (b) hybridizing ssDNA molecules containing complementary regions to produce a complete dsDNA complex comprising all of the ssDNA molecules in (a); and

[0318] (c) Ligation of directly adjacent ssDNA molecules in a complete dsDNA complex to generate a dsDNA molecule.

[0319] In another representative example, provided herein is a method of producing a double-stranded DNA (dsDNA) molecule comprising at least 3000 bp (e.g., at least 4000 bp or 5000 bp), the method comprising:

[0320] (a) Provide:

[0321] (i) at least three (e.g., at least four or five) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; the second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and

[0322] (ii) at least four (e.g., at least five or six) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is completely complementary to the region of the second ssDNA molecule in (i); the second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule that is completely complementary to the region of the first ssDNA molecule in (i),

[0323] in:

[0324] (1) At least two (e.g., at least three or four) of the at least four (e.g., at least five or six) ssDNA molecules in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to a terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i);

[0325] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming an indirectly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule;

[0326] (3) the complementary region comprises at least 150 (e.g., at least 200 or 250) nucleotides;

[0327] (4) all ssDNA molecules comprise at least 400 (e.g., at least 500) nucleotides, optionally wherein all ssDNA molecules in (i) and the ssDNA molecules excluding the 5' end and the 3' end of the second strand of the dsDNA molecule in (ii) comprise at least 800 (e.g., at least 900 or 1000) nucleotides; and

[0328] (5) ssDNA molecules are provided in equimolar amounts;

[0329] (b) hybridizing ssDNA molecules containing complementary regions to produce a partial dsDNA complex comprising all of the ssDNA molecules in (a);

[0330] (c) extending the 3' end of the ssDNA molecule in the partial dsDNA complex to produce a complete dsDNA complex; and

[0331] (d) Ligation of directly adjacent ssDNA molecules in a complete dsDNA complex to generate a dsDNA molecule.

[0332] In another representative example, provided herein is a method for producing a double-stranded DNA (dsDNA) molecule comprising at least 3000 bp (e.g., at least 4000 bp or 5000 bp), the method comprising:

[0333] (a) Provide:

[0334] (i) at least three (e.g., at least four or five) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of a dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; the second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and

[0335] (ii) at least three (e.g., at least four or five) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: the first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule that is fully complementary to a region of the second ssDNA molecule in (i),

[0336] in:

[0337] (1) At least two (e.g., at least three or four) of the at least three (e.g., at least four or five) ssDNA molecules in (ii) do not include the 5' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to a terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i);

[0338] (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming a directly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule;

[0339] (3) the complementary region comprises at least 50 (e.g., at least 75 or 100) nucleotides;

[0340] (4) all ssDNA molecules in (i) contain at least 800 (e.g., at least 900 or 1000) nucleotides, and all ssDNA molecules in (ii) contain at least 150 (e.g., at least 175 or 200) nucleotides; and

[0341] (5) ssDNA molecules are provided in equimolar amounts;

[0342] (b) hybridizing ssDNA molecules containing complementary regions to produce a partial dsDNA complex comprising all of the ssDNA molecules in (a);

[0343] (c) extending the 3' end of the ssDNA molecule in (ii) in the partial dsDNA complex to produce a complete dsDNA complex; and

[0344] (d) Ligation of directly adjacent ssDNA molecules in a complete dsDNA complex to generate a dsDNA molecule.

[0345] The invention will now be described in more detail in the following non-limiting examples with reference to the following drawings:

[0346] Figure 1 Schematic diagram of the assembly strategy is shown: (A) complete overlap; (B) partial overlap; (C) minimal overlap, where the ssDNA molecules labeled A to P refer to SEQ ID NOs: 1 to 16, respectively.

[0347] Figure 2 A photograph of an agarose gel stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP is shown, wherein: (A) lane A shows the complete overlapping reaction product described in Example 1, and lane N shows the negative control reaction; (B) lane B shows the partial overlapping reaction product described in Example 1.

[0348] Figure 3 A photograph of an agarose gel stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP is shown, wherein: lanes 1 to 6 show the enzyme cleavage products of the plasmid containing the partially overlapping reaction products described in Example 2; lanes 7 to 12 show the enzyme cleavage products of the plasmid containing the minimal overlapping reaction products described in Example 1.

[0349] Figure 4 A comparative assembly strategy for generating dsDNA using shorter ssDNA molecules is shown, wherein: (A) a schematic diagram of ssDNA oligonucleotides and their annealing patterns is shown; (B) a photograph of an agarose gel stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP is shown to measure the final gene assembly products, wherein lane 1 shows 100 ng of the assembly mix loaded and lane 2 shows 500 ng of the assembly mix loaded (ladder: Gene Ruler 1 kb Plus).

[0350] Figure 5 A minimal overlap method using oligonucleotides containing short complementary regions is shown, wherein: (A) a schematic diagram of assembly using parts A, B, C and D and ssDNA linkers (each 50 nucleotides long) is shown; (B) a photograph of an agarose gel stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP is shown to observe the final gene assembly products, wherein lane 1 shows 50 ng of the assembly mixture loaded and lane 2 shows 100 ng of the assembly mixture loaded (ladder: Gene Ruler 1 kb Plus).

[0351] Figure 6 Shown are photographs of agarose gels stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP to image RCA cleavage products obtained at different concentrations of magnesium and manganese (samples 1 to 3 are imaged in lanes 1 to 3, respectively; see Table 1 for sample compositions).

[0352] Figure 7 Shown are photographs of agarose gels stained with ethidium bromide and imaged by Bio-Rad Chemidoc Mp to visualize the assembly products of segments C, D, and H containing 50% or 100% 5-methyl-dCTP.

[0353] Figure 8 The results of the experiment described in Example 6 are shown, wherein: (A) shows a photograph of an agarose gel stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP to show the assembly of ssDNA molecules produced in two separate one-pot synthesis reactions (positive and negative ssDNA molecules) (lane 1: 50 ng total DNA loaded, lane 2: 100 ng total DNA loaded); (B) shows E. coli colonies transformed with plasmids produced by ligation reactions (kanamycin selection) using the assembly mixture; (C) shows a photograph of an agarose gel stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP, which shows the correct enzyme digestion pattern after plasmids extracted from eight colonies were digested with Ndel (two bands in each lane corresponding to 4.8 kbp and 0.45 kbp, respectively).

[0354] Fig. 9 One-pot synthesis and assembly using the minimal overlap method described in Example 7 are shown, wherein: (A) shows a photograph of an agarose gel stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP to show the one-pot RCA digestion products containing all the necessary segments of the minimal overlap strategy; (B) is a photograph of a plate containing kanamycin, which shows E. coli colonies transfected with a plasmid, which is generated by ligating a linearized plasmid and a dsDNA complex, which is generated by hybridization of the one-pot RCA digestion products; (C) shows a photograph of an agarose gel stained with ethidium bromide and imaged by a Bio-Rad Chemidoc MP to show the results of Ndel digestion of plasmids extracted from two single colonies.

[0355] Example

[0356] Example 1: Production of dsDNA using different overlapping strategies

[0357] A synthetic gene (4000 bp) comprising a nucleotide sequence encoding GFP operably linked to a promoter for expression in mammalian cells and a kanamycin / neomycin resistance cassette (SEQ ID NO: 17). Figure 1 ) Single-stranded polynucleotides containing non-overlapping portions (fragments) of genes were designed and enzymatically synthesized using the RCA-based MOSIC method.

[0358] Strategy:

[0359] 1) Complete overlap: The positive strand of the gene is divided into four 1000 nucleotide (nt) long segments (A, B, C, D); the negative strand is divided into three 1000 nucleotide long segments and two 500 nucleotide long segments (E, F, G, H, I). The two 500 nucleotide long segments (E, I) are located at both ends, so that the 1000 nucleotide long segments overlap each other by 500 nucleotides.

[0360] 2) Partial overlap: These segments are designed to overlap by 250 nucleotides. The positive strand has three 1000-nucleotide segments (A, J, D), where A and D are the same as the complete overlap strategy, and J extends from 1501 nucleotides to 2500 nucleotides. The negative strand has segments E and I at both ends, with two 1000-nucleotide segments (K, L) between the two segments, so that the overlap length between the 1000-nucleotide segments is 250 bases.

[0361] 3) Minimum overlap: The positive strand is the same as the full overlap strategy, using segments A, B, C, and D. The negative strand consists of four 200nt segments: three of the segments (M, N, O) are connected to a 1000nt segment with a 100 base overlap, and the fourth segment (P) is annealed at the 5' end of the negative strand.

[0362] The ssDNA molecules (polynucleotides) were purified, mixed in stoichiometric amounts and heated to 95°C for 10 minutes. The mixture underwent a temperature ramp from 95°C to room temperature. In the case of the partial and minimal overlap strategies, a high-fidelity thermostable polymerase ( High-fidelity DNA polymerase (NEB)) fills in gaps between assembled fragments by polymerase-mediated extension. The assembled fragments (see Figure 2 ) and inserted into the bacterial plasmid. The final ligated plasmid was used to transform E. coli DH5α competent cells. The cells were selected by kanamycin, and the plasmid was extracted from a single colony and first tested by restriction endonuclease digestion ( Figure 3 ), and then tested by Sanger sequencing to confirm that the synthesized and amplified dsDNA molecules contained the correct nucleotide sequence.

[0363] Example 2 (Comparative Example): Attempt to produce dsDNA by one-pot assembly using short ssDNA molecules

[0364] To demonstrate that long ssDNA molecules with long complementary regions are essential for the disclosed overlapping strategy, a comparative experiment was performed with short oligonucleotides (SEQ ID NOs: 18 to 117) using a one-pot assembly method. Each oligonucleotide was 60 nt long, except for SEQ ID NO: 68, which was only 40 nt long, similar to Figure 1 In addition, based on the penultimate oligonucleotide (corresponding to Figure 1 The segment K in B of the first strand can be formed using the 5' end oligonucleotide (corresponding to Figure 1 The segment A in the B of the dsDNA is extended to provide the 3' end of the second strand of the desired dsDNA molecule, and thus the oligonucleotide comprising the 3' end of the second strand (corresponding to Figure 1 Similarly, based on the oligonucleotide providing the 5' end of the second strand of the desired dsDNA molecule, the 3' end oligonucleotide of the first strand can be used as an extension template to provide the 3' end of the first strand of the desired dsDNA molecule. Figure 1 Segment D) in B does not contain the 3' end of the desired dsDNA molecule.

[0365] Each oligonucleotide has at least one region of complementarity (overlap) of 20 bases with the oligonucleotide forming the opposite strand. The arrangement of partially overlapping oligonucleotides is repeated along the entire length of the dsDNA to be generated (see Figure 4 A), i.e., the partial overlapping strategy as defined in this article is used.

[0366] Two different concentrations of oligonucleotides were tested: 200 ng and 1000 ng. The oligonucleotides were mixed in PCR buffer and incubated using a temperature ramp (95°C for 5 min, then ramped to 22°C at a rate of 0.1°C per second for 1 h). The incubation was performed at 72°C for 15 min using a high-fidelity thermostable polymerase ( High-fidelity DNA polymerase (NEB) and 1 mM nucleotides were used to fill the gaps between assembled fragments by polymerase-mediated extension.

[0367] Subsequently, half of the reaction sample was loaded onto an agarose gel, followed by gel electrophoresis and ethidium bromide staining. No bands corresponding to the assembly product (i.e., dsDNA complex) were observed on the gel (see Figure 4 B) When the assembly mixture was combined with the linearized plasmid to produce a plasmid containing the desired dsDNA molecule via ligase-mediated transformation into an E. coli strain, no colonies were obtained.

[0368] These results indicate that short oligonucleotides (ie containing less than 150 nt) with short overlaps (ie less than 50 nt) cannot be used in the methods disclosed herein to generate long dsDNA molecules, ie dsDNA molecules comprising at least 1500 bp.

[0369] Example 3 (Comparative Example): Minimum overlap strategy using oligonucleotides with short complementary regions (overlap)

[0370] For this comparative experiment, attempts were made to generate dsDNA molecules by the minimal overlap strategy as defined herein, using ssDNA molecules A, B, C and D (as described in Example 1) to form the first strand and short oligonucleotides (50 nt long, SEQ ID NOs: 118 to 121) to form the second strand. The short oligonucleotides had a complementary region of 25 nucleotides to the adjacent ssDNA molecule in the first strand, but the oligonucleotide forming the 5' end of the second strand (corresponding to Figure 1 C), which is completely complementary to segment D (see Figure 5 A).

[0371] Two different concentrations of DNA were attempted for assembly (sample 1 = 100 ng, sample 2 = 200 ng). The segments were mixed in PCR buffer and incubated using a temperature ramp (95°C for 5 minutes, then ramped to 22°C at a rate of 0.1°C per second for 1 hour). Given the minimal overlap strategy used, the DNA was incubated at 72°C for 15 minutes using a high-fidelity thermostable polymerase ( High-fidelity DNA polymerase (NEB) and 1 mM nucleotides were used to fill the gaps between assembled fragments by polymerase-mediated extension.

[0372] Half of the reaction sample was loaded onto an agarose gel, followed by gel electrophoresis and ethidium bromide staining. Subsequently, no bands corresponding to the assembly product (dsDNA complex) were observed on the gel ( Figure 5 B).

[0373] Example 4: Assembly of a degenerate DNA library using a single long oligonucleotide mutagenesis

[0374] As described elsewhere herein, the methods disclosed herein enable the assembly of degenerate DNA libraries, wherein degenerate regions can be site-specific for long DNA sequences. To verify this, degenerate ssDNA (1 kb long) was synthesized using the MOSIC method described herein for assembly into desired dsDNA molecules.

[0375] Three RCA reactions were performed using phi29 DNA polymerase, 1 mM dNTPs, and varying concentrations of magnesium and manganese (see Table 1 for the concentrations used in each reaction) at 30°C for 16 hours using a circularly nicked dsDNA template encoding segment B described in Example 1. After inactivation at 70°C, the RCA products were cleaved using the BtsCI restriction enzyme (to remove the hairpin structure and release the ssDNA product). After heat inactivation at 80°C, the cleavage products were separated by agarose gel electrophoresis (see Figure 6 ) and analyzed by Illumina sequencing.

[0376] The sequencing results showed that the error rate increased with the increase of manganese concentration.

[0377] The degenerate ssDNA molecules generated using this method can be used in the methods disclosed herein to generate libraries of degenerate dsDNA molecules.

[0378] Table 1: Error rates measured according to the concentrations of magnesium and manganese used in each reaction

[0379] sample 1 2 3 <![CDATA[MgCl 2 ]]> 5mM 5mM / <![CDATA[MnCl 2 ]]> 1.5mM 3mM 10mM Error rate (Illumina) <![CDATA[1.59×10 -3 ]]> <![CDATA[2.61×10 -3 ]]> <![CDATA[2.68×10 -3 ]]>

[0380] Example 5: Assembly of ssDNA molecules containing modified nucleotides

[0381] The method disclosed herein is capable of assembling dsDNA molecules containing site-specific modifications. To verify this, a single modified segment (ssDNA molecule) was generated using the method described above (WO2020 / 161187); the selected modification was 5-methyl-2'-deoxycytidine-5'-triphosphate (5-methyl-dCTP).

[0382] Segments C, D and H were synthesized in a single tube using the RCA and BtscI digestion protocol described in Example 4. Each segment was generated using two different RCA conditions, in which either 50% or 100% of the native dCTP was replaced with 5-methyl-dCTP.

[0383] After desalting the generated ssDNA molecules with Nucleospin, the segments were assembled in different combinations using 50 ng of each segment in each reaction as follows:

[0384] 1) Segments C, D and H containing 50% 5-methyl-dCTP;

[0385] 2) segments D and H containing 50% 5-methyl-dCTP;

[0386] 3) Segments C, D, and H containing 100% 5-methyl-dCTP;

[0387] 4) Segments D and H contain 100% 5-methyl-dCTP.

[0388] The segments were then mixed in PCR buffer and incubated using a temperature ramp (95°C for 5 minutes, then ramped to 22°C at a rate of 0.1°C per second for 1 hour) to form dsDNA complexes.

[0389] The samples were then loaded onto an agarose gel followed by gel electrophoresis and ethidium bromide staining. A negative control (segment H only) was also loaded onto the gel.

[0390] Bands corresponding to the assembly products of segments C, D and H containing 50% or 100% 5-methyl-dCTP (i.e., dsDNA complexes containing segments C, D and H) were observed on the gel (see Figure 7 ).

[0391] This demonstrates that ssDNA molecules comprising modified nucleotides can be used in the methods disclosed herein to generate dsDNA molecules containing site-specific modifications.

[0392] Example 6: Separate synthesis of ssDNA molecules forming positive and negative strands and one-pot assembly of desired dsDNA molecules

[0393] The ssDNA molecules that form the positive and negative strands for dsDNA molecule assembly used in the methods disclosed herein can be synthesized separately and subsequently assembled into dsDNA molecules by one-pot assembly. It would be advantageous to minimize the number of synthetic reactions required to generate ssDNA molecules because this would simplify the assembly process.

[0394] To investigate whether the number of synthesis steps could be reduced, the desired dsDNA molecules were synthesized using a minimal overlap strategy, where the ssDNA molecules of each strand were synthesized together, ie in a synthesis pool. The ssDNA molecules were synthesized using the MOSIC method described herein.

[0395] Segments A, B, C, and D were generated in a single tube, mixing four circular nicking templates, each encoding a single segment (i.e., segments A to D were generated from separate circular templates). Segments M, N, O, and P were generated in a second one-pot RCA reaction, with one circular template encoding M, N, O, and P.

[0396] In both cases, RCA reactions were performed using phi29 DNA polymerase at 30°C for 16 hours, and RCA products were digested with BtscI at 50°C for 4 hours to release ssDNA segments from the cleaved hairpin structures. After incubation at 70°C for 15 minutes to inactivate the polymerase and desalting the digestion mixture with Nucleospin, the ssDNA pool was used in two different amounts (sample 1 = 100 ng, sample 2 = 200 ng).

[0397] The segments of both strands were resuspended in PCR buffer in a single tube and incubated using a temperature ramp (95°C for 5 minutes, then ramped to 22°C at a rate of 0.1°C per second for 1 hour) to form dsDNA complexes. Given the minimal overlap strategy used, the dsDNA complexes were incubated at 72°C for 15 minutes using a high-fidelity thermostable polymerase ( High-fidelity DNA polymerase (NEB) and 1 mM nucleotides were used to fill the gaps between assembled fragments by polymerase-mediated extension.

[0398] Half of the reaction sample was loaded onto an agarose gel, followed by gel electrophoresis and ethidium bromide staining. The bands on the gel showed different assembly states, i.e., a variety of dsDNA complexes were formed (see Figure 8 A). The two assembly mixtures (samples 1 and 2) were mixed with the linearized plasmid and transformed into E. coli, and many colonies grew in overnight culture (see Figure 8 B), demonstrating that the ligation step occurred inside the bacterial cell. Eight colonies were randomly selected, and plasmids were extracted from them and digested with Ndel to verify the presence of the intact insert.

[0399] All digestion mixtures produced digestion patterns corresponding to fully assembled dsDNA molecules on agarose gels ( Figure 8 C). Sanger sequencing results showed that 100% correct sequence of each insert fragment originated from the selected colony.

[0400] Example 7: One-pot synthesis and assembly using a minimal overlap strategy

[0401] A one-pot method for the synthesis and assembly of long ssDNA molecules would be particularly advantageous as this would further simplify the process compared to the ssDNA molecules of Example 6 which involved the separate synthesis of each strand. The possibility of one-pot assembly and synthesis using a minimal overlap strategy was investigated.

[0402] All ssDNA molecules (segments A, B, C, D, M, N, O and P) were synthesized as described in Example 6, but in a single tube. The four circular nicked dsDNAs encoding segments A, B, C and D and the circular nicked dsDNA template encoding segments M, N, O and P were incubated with phi29 DNA polymerase and dNTPs at 30°C for 16 hours to perform the RCA reaction.

[0403] After heat inactivation, the RCA products were digested with BtscI as described above to obtain single ssDNA molecules. Even after digestion, partial assembly of the sequences could be visualized using agarose gels followed by gel electrophoresis and ethidium bromide staining ( Fig. 9 A).

[0404] After desalting with Nucleospin, the digestion mixture was resuspended in PCR buffer and assembled using a temperature ramp (incubated at 95°C for 5 minutes, then cooled to 22°C at a rate of 0.1°C per second for 1 hour) to form dsDNA complexes. Since the minimum overlap strategy was used, the dsDNA complex was incubated at 72°C for 15 minutes as recommended by the manufacturer, using a high-fidelity thermostable polymerase ( High-fidelity DNA polymerase (NEB) and 1 mM nucleotides were used to fill the gaps between assembled fragments by polymerase-mediated extension. Other polymerases, such as Phusion DNA polymerase or Taq polymerase, were tested and the same results were obtained.

[0405] The resulting dsDNA complex was mixed with the linearized plasmid and transformed into E. coli, and many colonies grew on the selection plate ( Fig. 9 B), proving that the ligation step occurred inside the bacterial cell. Two colonies were selected, and plasmids were extracted from them and digested with Ndel. The digested plasmid samples were loaded onto an agarose gel, followed by gel electrophoresis and ethidium bromide staining. The results showed that the plasmid produced the correct bands (0.45 kbp and 4.8 kbp, respectively). Fig. 9The assembly strategy was confirmed to be correct and effective as shown in the top and bottom arrows in C. Sanger sequencing also confirmed that both inserts were 100% correct.

[0406] This experiment demonstrates that the methods disclosed herein can be advantageously and surprisingly performed in a one-pot reaction, involving the synthesis of ssDNA molecules and their subsequent assembly into desired dsDNA molecules.

Claims

1. A method for producing a double-stranded DNA (dsDNA) molecule, the dsDNA molecule comprising at least 1500 bp; the method comprising: (a) Provide: (i) a plurality of single-stranded DNA (ssDNA) molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of the dsDNA molecule, wherein a first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule, and a second ssDNA molecule: (a) comprises the 3' end of the first strand of the dsDNA molecule, or (b) is capable of providing the 3' end of the first strand of the dsDNA molecule by a polymerase-mediated extension reaction using the ssDNA molecule comprising the 5' end of the second strand as a template; and (ii) a plurality of ssDNA molecules, each ssDNA molecule comprising (e.g. consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein a first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule, and optionally, a second ssDNA comprises the 3' end of the second strand of the dsDNA molecule, in: (1) At least two of the plurality of ssDNA molecules in (ii) each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i); (2) the ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms an adjacent portion of the first strand of the dsDNA molecule; (3) the complementary region contains at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; and (5) the ssDNA molecules are provided in equimolar amounts; (b) hybridizing the ssDNA molecules containing complementary regions to produce a dsDNA complex comprising all of the ssDNA molecules in (a); and (c) ligating adjacent ssDNA molecules using a ligase to produce the dsDNA molecule.

2. The method for producing a double-stranded DNA (dsDNA) molecule according to claim 1, comprising: (a) Provide: (i) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of the dsDNA molecule, wherein a first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and (ii) at least four ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: a first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule and is fully complementary to the region of the second ssDNA in (i); a second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule and is fully complementary to the region of the first ssDNA molecule in (i), in: (1) At least two of the at least four ssDNA molecules in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i); (2) the ssDNA molecule described in (i) is complementary to the ssDNA molecule described in (ii) and forms a directly adjacent portion of the first strand of the dsDNA molecule; (3) the complementary region contains at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; and (5) the ssDNA molecules are provided in equimolar amounts; (b) hybridizing said ssDNA molecules containing complementary regions to produce a complete dsDNA complex comprising all of said ssDNA molecules in (a); and (c) ligating directly adjacent ssDNA molecules using a ligase to produce said dsDNA molecule.

3. The method for producing a double-stranded DNA (dsDNA) molecule according to claim 1, comprising: (a) Provide: (i) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of the dsDNA molecule, wherein: a first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; a second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and (ii) at least four ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: a first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule, which is completely complementary to the region of the second ssDNA in (i); a second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule, which is completely complementary to the region of the first ssDNA molecule in (i), in: (1) At least two of the at least four ssDNA molecules in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i); (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming an indirectly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule; (3) the complementary region contains at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; and (5) the ssDNA molecules are provided in equimolar amounts; (b) hybridizing the ssDNA molecules containing complementary regions to produce a partial dsDNA complex comprising all of the ssDNA molecules in (a); (c) extending the 3' end of the ssDNA molecule to produce a complete dsDNA complex; and (d) ligating directly adjacent ssDNA molecules using a ligase to produce said dsDNA molecule.

4. The method for producing a double-stranded DNA (dsDNA) molecule according to claim 1, comprising: (a) Provide: (i) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of the dsDNA molecule, wherein: a first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; a second ssDNA molecule comprises the 3' end of the first strand of the dsDNA molecule; and (ii) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: a first ssDNA molecule comprises a 5' end of the second strand of the dsDNA molecule that is fully complementary to a region of the second ssDNA molecule in (i), in: (1) At least two of the at least three ssDNA molecules in (ii) do not include the 5' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of one of the ssDNA molecules in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i); (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming a directly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule; (3) the complementary region contains at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; and (5) the ssDNA molecules are provided in equimolar amounts; (b) hybridizing the ssDNA molecules containing complementary regions to produce a partial dsDNA complex comprising all of the ssDNA molecules in (a); (c) extending the 3' end of the ssDNA molecule described in (ii) to produce a complete dsDNA complex; and (d) ligating directly adjacent ssDNA molecules using a ligase to produce said dsDNA molecule.

5. The method for producing a double-stranded DNA (dsDNA) molecule according to claim 1, comprising: (a) Provide: (i) at least three ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of a first strand of the dsDNA molecule, wherein: a first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule; and a second ssDNA molecule: (a) comprises the 3' end of the first strand of the dsDNA molecule, or (b) is capable of providing the 3' end of the first strand of the dsDNA molecule by a polymerase-mediated extension reaction using the ssDNA molecule comprising the 5' end of the second strand as a template; and (ii) at least three (e.g., at least four) ssDNA molecules, each ssDNA molecule comprising (e.g., consisting of) non-overlapping portions of the second strand of the dsDNA molecule, wherein: a first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule, which is fully complementary to a region of the second ssDNA in (i); and optionally, a second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule, which is fully complementary to a region of the first ssDNA molecule in (i), in: (1) The at least three ssDNA molecules (e.g., at least two of the at least four ssDNA molecules) in (ii) do not include the 5' end and the 3' end of the second strand of the dsDNA molecule, and each ssDNA molecule includes a first region that is completely complementary to the terminal region of a certain ssDNA molecule in (i) and a second region that is completely complementary to the terminal region of another ssDNA molecule in (i); (2) The ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii), forming an indirectly adjacent portion of the first strand of the dsDNA molecule; the ssDNA molecule in (ii) is complementary to the ssDNA molecule in (i), forming an indirectly adjacent portion of the second strand of the dsDNA molecule; (3) the complementary region contains at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; and (5) the ssDNA molecules are provided in equimolar amounts; (b) hybridizing the ssDNA molecules containing complementary regions to produce a partial dsDNA complex comprising all of the ssDNA molecules in (a); (c) extending the 3' end of the ssDNA molecule to produce a complete dsDNA complex; and (d) ligating directly adjacent ssDNA molecules using a ligase to produce said dsDNA molecule.

6. The method of any one of claims 1 to 5, wherein the ssDNA molecule in (a)(i) comprises at least 500 nucleotides, optionally at least 600, 700, 800, 900 or 1000 nucleotides.

7. The method according to any one of claims 1 to 3 or 5, wherein: The ssDNA molecules in (A)(a)(i) comprise at least 500 nucleotides, optionally at least 600, 700, 800, 900 or 1000 nucleotides; at least two of the ssDNA molecules in (B)(a)(ii) comprise at least 500 nucleotides, optionally at least 600, 700, 800, 900 or 1000 nucleotides.

8. The method according to claim 1 or 4, wherein: The ssDNA molecule in (A)(a)(i) comprises at least 500 nucleotides, optionally at least 600, 700, 800, 900 or 1000 nucleotides; the ssDNA molecule in (B)(a)(ii) is shorter than the ssDNA molecule in (a)(i).

9. The method according to any one of claims 1 to 8, wherein the region of complementarity comprises at least 100 nucleotides, optionally at least 150, 200, 250, 300, 400 or 500 nucleotides.

10. The method of any one of claims 1 to 9, wherein the dsDNA molecule comprises at least 2000 bp, 2500 bp, 3000 bp, 3500 bp or 4000 bp.

11. The method according to any one of claims 1 to 9, wherein the dsDNA molecule comprises 1500 to 50000 bp.

12. The method of any one of claims 1 to 11, wherein the dsDNA molecule is produced by enzymatic synthesis.

13. The method of claim 12, wherein the enzymatic synthesis is asymmetric PCR, TdT synthesis, RCA-based synthesis method or a combination thereof.

14. The method according to any one of claims 1 to 13, wherein one or more (preferably all) of the ssDNA molecules provided in (a) are produced by a method comprising: (a) providing a circular DNA molecule, the circular DNA molecule comprising a nucleotide sequence encoding the ssDNA molecule adjacent to a cleavage domain; (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule in (a) as a template; and (c) cleaving the product of the RCA reaction at the cleavage domain to release the ssDNA.

15. The method of claim 14, wherein all of the ssDNA molecules provided in (a) are produced in a single reaction mixture.

16. The method according to claim 14 or 15, wherein the circular DNA molecule comprises a plurality of nucleotide sequences adjacent to the cleavage domain, each nucleotide sequence encoding the ssDNA molecule provided in (a).

17. The method of any one of claims 1 to 16, wherein the dsDNA molecule is a linear dsDNA molecule.

18. The method according to any one of claims 1 to 17, further comprising the step of amplifying the dsDNA molecule.

19. The method of claim 18, wherein the step of amplifying the dsDNA molecule comprises: (i) inserting the dsDNA molecule into a nucleic acid vector (e.g., a DNA plasmid); (ii) amplifying the vector (e.g., a DNA plasmid); (iii) excising the dsDNA molecule from the vector; and optionally (iv) isolating or purifying the dsDNA molecule, for example, from a vector nucleic acid.

20. The method of claim 19, wherein step (ii) comprises transfecting the vector (eg, DNA plasmid) into bacteria and culturing the bacteria.

21. The method according to any one of claims 1 to 20, further comprising the step of isolating the dsDNA molecules.

22. The method according to any one of claims 1 to 21, further comprising the step of modifying the dsDNA molecule.

23. The method of any one of claims 1 to 22, wherein multiple versions of one or more of the plurality of ssDNA molecules are provided.

24. The method of claim 23, wherein the multiple versions of one or more of the multiple ssDNA molecules are sequence variants.

25. The method of claim 23 or 24, wherein the multiple versions of one or more of the multiple ssDNA molecules contain one or more modified (e.g., functionalized) nucleotides, wherein the versions differ in: (i) the position and / or number of modified (e.g., functionalized) nucleotides; and / or (ii) the type of functional group on the one or more modified nucleotides.

26. The method of any one of claims 1 to 22, wherein one or more of the plurality of ssDNA molecules contains one or more modified (eg, functionalized) nucleotides.

27. The method of any one of claims 1 to 22 or 26, wherein each copy of the ssDNA molecule providing the non-overlapping portions of the strands of the dsDNA molecule is identical.

28. A dsDNA molecule library comprising a plurality of different dsDNA molecules obtained by the method according to any one of claims 23 to 25.

29. Use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-mediated ligation reaction to produce a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, wherein: (i) each of the first plurality of single-stranded DNA (ssDNA) molecules comprises (e.g., consists of) non-overlapping portions of a first strand of the dsDNA molecule, wherein the first ssDNA molecule comprises the 5' end of the first strand of the dsDNA molecule, and the second ssDNA: (a) comprises the 3' end of the first strand of the dsDNA molecule, or (b) is capable of providing the 3' end of the first strand of the dsDNA molecule by a polymerase-mediated extension reaction using the ssDNA molecule comprising the 5' end of the second strand as a template; and (ii) each of the second plurality of ssDNA molecules comprises (e.g., consists of) non-overlapping portions of the second strand of the dsDNA molecule, wherein a first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule, and optionally, a second ssDNA comprises the 3' end of the second strand of the dsDNA molecule, in: (1) At least two of the plurality of ssDNA molecules in (ii) each comprise a first region that is completely complementary to a terminal region of a certain ssDNA molecule in (i) and a second region that is completely complementary to a terminal region of another ssDNA molecule in (i); (2) the ssDNA molecule in (i) is complementary to the ssDNA molecule in (ii) and forms an adjacent portion of the first strand of the dsDNA molecule; (3) the complementary region contains at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; and (5) the ssDNA molecules are provided in equimolar amounts; And wherein the first and second plurality of ssDNA molecules form a dsDNA complex comprising all of the ssDNA molecules when contacted under conditions suitable for DNA molecular hybridization, the ssDNA molecules can be ligated in a ligase-mediated reaction to produce the dsDNA molecule.

30. The use according to claim 29, wherein: (i) the ssDNA molecule is as defined in any one of claims 6 to 8, 12 to 16 or 23 to 27; (ii) the region of complementarity is as defined in claim 9; and / or (iii) The dsDNA molecule is as defined in any one of claims 10, 11 or 17.

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