Methods for generating double-stranded DNA

Abstract

The present invention provides a method for generating a double-stranded DNA (dsDNA) molecule. In particular, the present invention provides a method for utilizing a plurality of single-stranded DNA (ssDNA) molecules to form a dsDNA complex, in which the ssDNA molecules are ligated to generate a dsDNA molecule.

Description

[Technical field]

[0001] The present invention is in the field of synthetic biology and relates to a method for generating double-stranded DNA (dsDNA) molecules, e.g., long dsDNA molecules, such as those comprising at least about 1000 bp. In particular, the present invention provides a method utilizing a plurality of single-stranded DNA (ssDNA) molecules to form a dsDNA complex, in which the ssDNA molecules are ligated to generate a dsDNA molecule. Some of the ssDNA molecules may be extended prior to ligation. The ssDNA molecules used in the present invention may be the product of an enzymatic synthesis, such as an enzyme-involved rolling circle amplification (RCA) reaction. The present invention also provides the use of a plurality of ssDNA molecules in a ligase-involved reaction to generate a dsDNA molecule. Double-stranded DNA molecules obtained by the method are also provided. A library of dsDNA molecules comprising a plurality of different (e.g., functionalized or modified) dsDNA molecules obtained by the method is also provided. [Background technology]

[0002] Synthetic biology is widely recognized as an important and rapidly developing scientific field. It is an interdisciplinary area of ​​research, and a key aspect of it is related to genetic engineering, including the synthesis of new genes and the modification of existing genes. Significant developments over the last two decades have made gene synthesis more accessible, which in turn has enabled advances in many fields and the development of new applications.

[0003] Currently, gene synthesis (i.e., DNA synthesis) is based on the assembly of short, contiguous oligonucleotides that are typically synthesized by solid-phase synthesis, specifically phosphoramidite chemistry. Generally, these ssDNA segments are 60-80 nucleotides (nt) long and contain both strands of the resulting DNA duplex. To reduce the amount of oligonucleotides required to form a desired sequence (e.g., a gene sequence), each ssDNA segment is designed to have an annealing sequence of 15-25 nucleotides to the ssDNA segment present on the opposite strand. A thermostable DNA polymerase then randomly extends the assembled oligonucleotides to form products of various lengths, which are then used as templates for a PCR reaction driven by the excess outermost ends of the assembled oligonucleotides. This is known as polymerase cycling assembly (PCA) or assembly PCR.

[0004] Importantly, both the synthetic and assembly aspects of the above methods pose distinct limitations to the precise production of dsDNA, eg, gene synthesis.

[0005] First and foremost, 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 at the same time decreasing the product yield. The error rate in the production of polynucleotides by solid-phase synthesis is significantly higher than that of polymerase enzymes observed in nature and increases dramatically with the length of the polynucleotide, so that commercially available polynucleotides of approximately 50 nucleotides in length are typically only 70% pure. This error rate makes the solid-phase synthesis method unsuitable for the production of long polynucleotides. For example, solid-phase synthesis typically has an error rate of >0.5%, which means that the production of ssDNA molecules containing 100 nucleotides will result in a mixture of ssDNA molecules in which more than 40% of the ssDNA molecules contain at least one error, i.e., the purity of the synthesis product is less than 60%. Finally, the assembly of ssDNA segments (oligos) to form target genes is error-prone, especially when multiple oligonucleotides with similar sequences are present, at least in part because the typical overlap between opposite strands is short.

[0006] Thus, errors in the starting products further increase errors during the assembly process, resulting in errors in the final assembly products that need to be removed. Therefore, efficient and accurate synthesis of long DNA sequences (e.g., genes) using current methods is extremely difficult, time-consuming, labor-intensive, and expensive. This is due to the extremely labor-intensive post-synthesis quality control procedures that require identifying and selecting products with the correct sequence, or more commonly, performing sequencing / error correction / recloning cycles. Furthermore, even with these post-assembly processes, the generation of molecules with the correct sequence cannot always be achieved, especially for molecules with long and / or complex or repetitive sequences.

[0007] In view of the problems with existing methods, there is a need for alternative methods for generating dsDNA molecules, particularly methods that are effective in forming long dsDNA molecules (eg, greater than about 1 kb) in high yield and purity. Summary of the Invention [Means for solving the problem]

[0008] In the research leading to the present invention, the inventors unexpectedly determined that ligase-related assembly of ssDNA molecules can be used to accurately and efficiently generate long dsDNA molecules. In particular, the methods and uses provided herein do not involve PCR methods, which typically result in side products, especially when long complex sequences are desired. In particular, the inventors have discovered that long, pure ssDNA building blocks (e.g., each containing 150-10,000 nucleotides) can be used to accurately assemble stable dsDNA complexes that can be efficiently combined to generate the desired dsDNA molecules.

[0009] Without wishing to be bound by theory, it is believed that the purity of the starting material (e.g., enzymatically synthesized ssDNA molecules) (drastically reducing the cases of errors due to misannealing events) in combination with the length of the annealing sequence, e.g., at least 50 bp, results in the accurate assembly of ultra-pure dsDNA molecules (e.g., genes or gene fragments). Advantageously, this minimizes (e.g., eliminates) the need for post-assembly processes, allowing the assembly of any sequence, regardless of length and complexity. Unexpectedly, the examples show that dsDNA molecules can be generated in a one-pot reaction that includes both the synthesis of ssDNA molecules and their assembly into the desired dsDNA molecule. This is particularly advantageous, as it further simplifies the method.

[0010] Representative strategies for assembling dsDNA complexes that can be combined to form the desired dsDNA molecule are shown in Figure 1. These can be characterized as "full overlap" (Figure 1A), "partial overlap" (Figure 1B) and "minimal overlap" (Figure 1C) strategies and are presented below.

[0011] Complete overlap: the plus strand of a target dsDNA (e.g., a gene) is provided as four segments A, B, C and D (e.g., each containing 1000 nucleotides) and the minus strand is provided as five segments E, F, G, H and I (e.g., three segments (F, G and H) each containing 1000 nucleotides and two segments (E and I) each containing 500 nt). The smaller segments (E and I) are positioned to form the ends (3' and 5' ends, respectively) of the minus strand of the target dsDNA, such that the longer segments (F, G and H) contain regions of large complementarity (overlap) with the segments in the plus strand. There are no gaps between adjacent segments (i.e., their 5' and 3' ends are directly adjacent) so that the segments can be joined in a ligation reaction involving a ligase using overlapping segments of opposite strands to serve as templates for a ligation reaction, i.e., segment A serves as a template for the ligation of segments E and F, segment F serves as a template for the ligation of segments A and B, etc.

[0012] Partial overlap: The plus strand of the target dsDNA (e.g., a gene) is provided as three segments A, J and D (e.g., each containing 1000 nucleotides), where segments A and D form the ends of the plus strand of the target dsDNA (5' and 3' ends, respectively), and segment J comprises the central portion of the plus strand of the target dsDNA (e.g., it comprises nucleotides 1501 to 2500 of the plus strand). The minus strand is provided as four segments E, K, L and I (e.g., two segments (K and L) each containing 1000 nucleotides, and two segments (E and I) each containing 500 nt). Similar to the complete overlap method described above, the small segments E and I are positioned to form the ends of the minus strand of the target dsDNA (3' and 5' ends, respectively). The segments are arranged such that there are gaps between adjacent segments (i.e., the 5' and 3' ends are indirectly adjacent), and the longer segments overlap only partially, e.g., containing a region of complementarity of about 250 bp. Upon formation of a DNA complex by hybridization of complementary sequences, the inner 3' ends of the segments become templates for extension reactions, i.e., segment A becomes a template for the extension of segment K, segment K becomes a template for the extension of segment A, etc., and can be extended in a polymerase extension reaction involving a template using overlapping segments of the opposite strand. The extended segments can then be ligated in a ligation reaction involving a ligase using overlapping segments of the opposite strand, similar to the complete overlap method, i.e., segment A becomes a template for the ligation of segments E and K, segment K becomes a template for the ligation of segments A and J, etc.

[0013] Minimal overlap: the plus strand is identical to the complete overlap strategy, with segments A, B, C and D used. The minus strand is provided by four smaller segments M, N, O and P (e.g., each containing 200 nucleotides). Only one of the segments provides the end of the minus strand, i.e., segment P provides the 5' end of the minus strand. The other minus strand segments (M, N and O) partially overlap with the plus strand segments, e.g., containing a region of complementarity of about 100 bp. Similar to the partial overlap strategy described above, the formation of a DNA complex by hybridization of complementary sequences allows the inner 3' ends of the minus strand segments to become templates for the extension reaction, i.e., segment A becomes a template for the extension of segment M, segment B becomes a template for the extension of segment N, etc., and become extendable in a polymerase extension reaction involving a template using the overlapping segments of the plus strand. The extended segments can then be joined in a ligation reaction involving a ligase using overlapping segments of opposite strands, similar to the perfect overlap strategy, i.e., segment B is a template for ligation of segments M and N, segment M is a template for ligation of segments A and B, etc.

[0014] Those skilled in the art will understand that the above outlined strategy can be modified to increase the number of segments, for example to generate longer dsDNA molecules. Similarly, the length of the segments may also be modified. For example, not all of the segments of the plus and / or minus strands need to be the same length. In particular, the segments can be designed to ensure that the junction locations (i.e., the positions in the dsDNA complex where the 5' and 3' ends of the segments directly meet) do not coincide with repeat sequences and / or sequences that can form secondary structures, in order to minimize misalignment within the DNA complex.

[0015] It will also be apparent that multiple types (e.g., sequence variants) of one or more ssDNA segments may be provided to form a mixture of dsDNA molecules, i.e., to generate a gene library, such as for use in phage display methods. By way of example, and with reference to the above and FIG. 1 strategies, several types (e.g., sequence variants) of segment K could be provided as a reaction mixture with a common region of complementarity and different sequences in opposite regions of the gap (i.e., the gap between segments A and J) on opposite strands. As shown in Example 4, a library of ssDNA variants may be generated by including manganese ions in the DNA synthesis reaction. The product of the extension and ligation reaction will be a mixture of dsDNA molecules that contain different sequences in the regions contributed (and encoded) by multiple types of segment K. In particular, the different types of ssDNA segments would not need to be sequence variants. For example, multiple ssDNA molecules, each of which contains modified nucleotides (e.g., functionalized nucleotides such as methylated nucleotides), could be provided, as shown in Example 5. The different types may contain modifications in different positions and / or different modifications.

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

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

[0018] Similarly, the segment providing the positive strand 3' end (segment D) may be truncated so as not to include the positive strand 3' end of the target dsDNA, since segment D can be extended using segment I as a template to obtain the positive strand 3' end. Thus, in the partial overlap strategy, a positive strand segment and / or a negative strand segment can provide the 3' end of the target dsDNA molecule by a templated extension reaction, i.e., a polymerase-associated extension reaction.

[0019] Thus, there is provided herein the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-involving ligation reaction to generate a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, (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 a 5'-terminus of the first strand of the dsDNA molecule, and the second ssDNA molecule (a) comprises a 3'-terminus of the first strand of the dsDNA molecule, or (b) is capable of providing a 3'-terminus of the first strand of the dsDNA molecule by a polymerase-involved extension reaction using an ssDNA molecule comprising the 5'-terminus of the second strand as a template; (ii) each of the second plurality of ssDNA molecules comprises (e.g., consists of) a non-overlapping portion 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, and optionally, a second ssDNA molecule comprises a 3'-end of the second strand of the dsDNA molecule; each of the at least two plurality of ssDNA molecules from (1)(ii) comprises a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms a contiguous portion of a first strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts; When the first and second pluralities of ssDNA molecules are contacted under conditions suitable for hybridization of the DNA molecules, they form a dsDNA complex that contains all the ssDNA molecules that can be ligated in a reaction involving a ligase to produce a dsDNA molecule.

[0020] Thus, there is provided herein the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-involving ligation reaction to generate a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, (i) each of the first plurality of single-stranded DNA (ssDNA) molecules comprises (e.g., consists of) a non-overlapping portion of a first strand of a dsDNA molecule, the first ssDNA molecule comprises a 5' end of the first strand of the dsDNA molecule and the second ssDNA molecule comprises a 3' end of the first strand of the dsDNA molecule; (ii) each of the second plurality of ssDNA molecules comprises (e.g., consists of) a non-overlapping portion 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, and optionally, a second ssDNA molecule comprises a 3'-end of the second strand of the dsDNA molecule; each of the at least two plurality of ssDNA molecules from (1)(ii) comprises a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms a contiguous portion of a first strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts; When the first and second pluralities of ssDNA molecules are contacted under conditions suitable for hybridization of the DNA molecules, they form a dsDNA complex that contains all the ssDNA molecules that can be ligated in a reaction involving a ligase to produce a dsDNA molecule.

[0021] Also provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-involving ligation reaction to generate a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, (i) the first plurality of ssDNA molecules comprises at least three ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3'-end of the first strand of the dsDNA molecule; (ii) the second plurality of ssDNA molecules comprises at least four ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion of the second strand of the dsDNA molecule, a first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule and is fully complementary to a region of the second ssDNA molecule of (i), and a second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule of (i); at least two of the at least four ssDNA molecules from (1)(ii), which do not include the 5' and 3' ends of the second strand of the dsDNA molecule, each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms an immediately adjacent portion of a first strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts; When the first and second pluralities of ssDNA molecules are contacted under conditions suitable for hybridization of the DNA molecules, they form a dsDNA complex that contains all the ssDNA molecules that can be joined in a ligation reaction involving a ligase to generate a dsDNA molecule.

[0022] Further provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-involving ligation reaction to generate a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, (i) the first plurality of ssDNA molecules comprises at least three ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein the first ssDNA molecule comprises a 5'-terminus of the first strand of the dsDNA molecule, and the second ssDNA molecule (a) comprises a 3'-terminus of the first strand of the dsDNA molecule, or (b) is capable of providing a 3'-terminus of the first strand of the dsDNA molecule by a polymerase-involved extension reaction using an ssDNA molecule comprising the 5'-terminus of the second strand as a template; (ii) the second plurality of ssDNA molecules comprises at least three (e.g., at least four) ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of the second strand of the dsDNA molecule, a first ssDNA molecule comprising the 5' end of the second strand of the dsDNA molecule and fully complementary to a region of the second ssDNA molecule of (i), and optionally, a second ssDNA molecule comprising the 3' end of the second strand of the dsDNA molecule and fully complementary to a region of the first ssDNA molecule of (i); at least two of the at least three ssDNA molecules from (1)(ii) (e.g., at least two of at least four), which do not include the 5' and 3' ends of the second strand of the dsDNA molecule, each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms an indirectly contiguous portion of a first strand of a dsDNA molecule, and the ssDNA molecule of (ii) to which the ssDNA molecule of (i) is complementary forms an indirectly contiguous portion of a second strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts; When the first and second plurality of ssDNA molecules are contacted under conditions suitable for hybridization of the DNA molecules, they form a partially double-stranded DNA complex containing all of the ssDNA molecules, and the inner 3' ends of the ssDNA molecules in the partially double-stranded DNA complex can be extended in an extension reaction involving a polymerase to generate a fully double-stranded DNA complex that can be joined in a ligation reaction involving a ligase to generate a dsDNA molecule.

[0023] Further provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-involving ligation reaction to generate a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, (i) the first plurality of ssDNA molecules comprises at least three ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-terminus of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3'-terminus of the first strand of the dsDNA molecule; (ii) the second plurality of ssDNA molecules comprises at least four ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion of the second strand of the dsDNA molecule, a first ssDNA molecule comprises the 5' end of the second strand of the dsDNA molecule and is fully complementary to a region of the second ssDNA molecule of (i), and a second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule of (i); at least two of the at least four ssDNA molecules from (1)(ii), which do not include the 5' and 3' ends of the second strand of the dsDNA molecule, each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms an indirectly contiguous portion of a first strand of a dsDNA molecule, and the ssDNA molecule of (ii) to which the ssDNA molecule of (i) is complementary forms an indirectly contiguous portion of a second strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts; When the first and second plurality of ssDNA molecules are contacted under conditions suitable for hybridization of the DNA molecules, they form a partially double-stranded DNA complex containing all of the ssDNA molecules, and the inner 3' ends of the ssDNA molecules in the partially double-stranded DNA complex can be extended in an extension reaction involving a polymerase to generate a fully double-stranded DNA complex that can be joined in a ligation reaction involving a ligase to generate a dsDNA molecule.

[0024] Also provided herein is the use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-involving ligation reaction to generate a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, (i) the first plurality of ssDNA molecules comprises at least three ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-terminus of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3'-terminus of the first strand of the dsDNA molecule; (ii) the second plurality of ssDNA molecules comprises at least three ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion 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 and is fully complementary to a region of the second ssDNA molecule of (i); at least two of the at least three ssDNA molecules from (1)(ii), which do not include the 5' end of the second strand of the dsDNA molecule, each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms a directly adjacent portion of a first strand of a dsDNA molecule, and the ssDNA molecule (ii) to which the ssDNA molecule (i) is complementary forms an indirectly adjacent portion of a second strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts; When the first and second plurality of ssDNA molecules are contacted under conditions suitable for hybridization of the DNA molecules, they form a partially double-stranded DNA complex containing all of the ssDNA molecules, and the inner 3' ends of the ssDNA molecules in the partially double-stranded DNA complex (the inner 3' ends of the second plurality of ssDNA molecules) can be extended in an extension reaction involving a polymerase to generate a fully double-stranded DNA complex that can be joined in a ligation reaction involving a ligase to generate a dsDNA molecule.

[0025] Viewed another way, there is provided herein a method for generating a double stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising: (a) (i) a plurality of single-stranded DNA (ssDNA) molecules each comprising (e.g., consisting of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the first strand of the dsDNA molecule, and a second ssDNA molecule (a) comprises a 3'-end of the first strand of the dsDNA molecule, or (b) is capable of providing (providing) the 3'-end of the first strand of the dsDNA molecule by a polymerase-involved extension reaction using an ssDNA molecule comprising the 5'-end of the second strand as a template; and (ii) a plurality of ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion of a second strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5' end of the second strand of the dsDNA molecule, and optionally a second ssDNA molecule comprises a 3' end of the second strand of the dsDNA molecule. and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce a dsDNA complex containing all of the ssDNA molecules from (a); (c) joining adjacent ssDNA molecules to generate a dsDNA molecule; Including, each of the at least two plurality of ssDNA molecules from (1)(ii) comprises a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms a contiguous portion of a first strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts.

[0026] Also provided herein is a method for generating a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising: (a) (i) a plurality of single-stranded DNA (ssDNA) molecules, each of which comprises (e.g., consists of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5' end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3' end of the first strand of the dsDNA molecule; and (ii) a plurality of ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion of a second strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5' end of the second strand of the dsDNA molecule, and optionally a second ssDNA molecule comprises a 3' end of the second strand of the dsDNA molecule. and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce a dsDNA complex containing all of the ssDNA molecules from (a); (c) joining adjacent ssDNA molecules to generate a dsDNA molecule; Including, each of the at least two plurality of ssDNA molecules from (1)(ii) comprises a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms a contiguous portion of a first strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts.

[0027] Also provided herein is a method for generating a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising: (a) (i) at least three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5' end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3' end of the first strand of the dsDNA molecule; and (ii) at least four ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a second strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the second ssDNA molecule of (i), and a second ssDNA molecule comprises a 3'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule of (i). and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce an entirely double-stranded DNA complex containing all ssDNA molecules from (a); (c) ligating directly adjacent ssDNA molecules to generate a dsDNA molecule; Including, at least two of the at least four ssDNA molecules from (1)(ii), which do not include the 5' and 3' ends of the second strand of the dsDNA molecule, each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms an immediately adjacent portion of a first strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts.

[0028] Further provided herein is a method for generating a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising: (a) (i) at least three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a first strand of a dsDNA molecule, where a first ssDNA molecule comprises a 5'-end of the first strand of the dsDNA molecule and a second ssDNA molecule either (a) comprises a 3'-end of the first strand of the dsDNA molecule or (b) is capable of providing (providing) a 3'-end of the first strand of the dsDNA molecule by a polymerase-involved extension reaction using an 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 comprising (e.g., consisting of) a non-overlapping portion of a second strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the second ssDNA molecule in (i), and optionally, a second ssDNA molecule comprises a 3'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule in (i). and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce a partially double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) extending the 3' ends of the ssDNA molecules to generate fully double-stranded DNA complexes; (d) ligating directly adjacent ssDNA molecules to generate a dsDNA molecule; Including, at least two of the at least three ssDNA molecules from (1)(ii) (e.g., at least two of at least four), which do not include the 5' and 3' ends of the second strand of the dsDNA molecule, each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms an indirectly contiguous portion of a first strand of a dsDNA molecule, and the ssDNA molecule of (ii) to which the ssDNA molecule of (i) is complementary forms an indirectly contiguous portion of a second strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts.

[0029] Further provided herein is a method for generating a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising: (a) (i) at least three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3'-end of the first strand of the dsDNA molecule; and (ii) at least four ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a second strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the second ssDNA molecule of (i), and a second ssDNA molecule comprises a 3'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule of (i). and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce a partially double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) extending the 3' ends of the ssDNA molecules to generate fully double-stranded DNA complexes; (d) ligating directly adjacent ssDNA molecules to generate a dsDNA molecule; Including, at least two of the at least four ssDNA molecules from (1)(ii), which do not include the 5' and 3' ends of the second strand of the dsDNA molecule, each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms an indirectly contiguous portion of a first strand of a dsDNA molecule, and the ssDNA molecule of (ii) to which the ssDNA molecule of (i) is complementary forms an indirectly contiguous portion of a second strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts.

[0030] Further provided herein is a method for generating a double-stranded DNA (dsDNA) molecule comprising at least 1500 bp, the method comprising: (a) (i) at least three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3'-end of the first strand of the dsDNA molecule; and (ii) at least three ssDNA molecules, each of which comprises (e.g., consists of) a non-overlapping portion 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 completely complementary to a region of the second ssDNA molecule of (i). and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce a partially double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) extending the 3' ends of the ssDNA molecules of (ii) to generate fully double-stranded DNA complexes; (d) ligating directly adjacent ssDNA molecules to generate a dsDNA molecule; Including, at least two of the at least three ssDNA molecules from (1)(ii), which do not include the 5' end of the second strand of the dsDNA molecule, each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms a directly adjacent portion of a first strand of a dsDNA molecule, and the ssDNA molecule (ii) to which the ssDNA molecule (i) is complementary forms an indirectly adjacent portion of a second strand of a dsDNA molecule; (3) the complementary region comprises at least 50 nucleotides; (4) all ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts.

[0031] Although the methods and uses provided herein prove to be particularly useful for the production (i.e., synthesis) of long dsDNA molecules (e.g., comprising at least 1500 bp), those skilled in the art will understand that the methods and uses provided herein may also be particularly advantageous for the production of dsDNA molecules that contain repeat sequences and / or sequences that can form secondary structures. In view of the problems associated with the production of small dsDNA molecules that contain repeat sequences and / or sequences that can form secondary structures, when the methods and uses provided herein are utilized for the production of dsDNA molecules that contain repeat sequences and / or sequences that can form secondary structures, the above size requirements do not need to be applied. Thus, for example, the methods and uses provided herein can be used to produce dsDNA molecules that contain 500 bp or more (e.g., 750 bp or 1000 bp or more) when the dsDNA molecules contain repeat sequences and / or sequences that can form secondary structures.

[0032] Also provided herein are dsDNA molecules produced by the methods and uses provided herein.

[0033] Further provided herein is a library of dsDNA molecules comprising a plurality of different (eg, functionalized or modified) dsDNA molecules obtained by the methods and uses provided herein.

[0034] The invention will now be described in further detail in the following non-limiting examples, with reference to the following figures. [Brief description of the drawings]

[0035] [Figure 1] FIG. 1 shows a schematic diagram of the assembly strategies: (A) complete overlap, (B) partial overlap, and (C) minimal overlap, where the ssDNA molecules labeled A-P refer to SEQ ID NOs: 1-16, respectively. [Diagram 2]Figure 2 shows a photograph of an agarose gel stained with ethidium bromide and visualized with Bio-Rad Chemidoc MP: (A) shows in lane A the products of the full overlap reaction described in Example 1, N the negative control reaction, and (B) shows in lane B the products of the partial overlap reaction described in Example 1. [Diagram 3] 3 shows a photograph of an agarose gel stained with ethidium bromide and visualized with Bio-Rad Chemidoc MP. Lanes 1-6 show digestion product plasmids containing products from the partial overlap reaction described in Example 2, and lanes 7-12 show digestion product plasmids containing products from the minimal overlap reaction described in Example 1. [Figure 4] Figure 4 shows a comparative assembly strategy using shorter ssDNA molecules to generate dsDNA. (A) shows a schematic of ssDNA oligonucleotides and their annealing patterns, and (B) shows a photograph of an agarose gel stained with ethidium bromide and visualized with Bio-Rad Chemidoc MP to compare the final gene assembly products, lane 1 shows 100 ng of the assembly mixture loaded, lane 2 shows 500 ng of the assembly mixture loaded (ladder: Gene ruler 1 kb plus). [Diagram 5] Figure 5 shows the minimal overlap approach using oligonucleotides containing short regions of complementarity. (A) shows a schematic of the assembly using parts A, B, C and D and ssDNA connectors (each 50 nt long), (B) shows a photograph of an agarose gel stained with ethidium bromide to observe the final gene assembly product and visualized with Bio-Rad Chemidoc MP, lane 1 shows 50 ng of assembly mixture loaded, lane 2 shows 100 ng of assembly mixture loaded (ladder: Gene ruler 1 kb plus). [Figure 6]Figure 6 shows a photograph of an agarose gel stained with ethidium bromide to visualize the RCA digestion products obtained using different concentrations of magnesium and manganese and visualized with Bio-Rad Chemidoc MP (samples 1–3 visualized in lanes 1–3, respectively; see Table 1 for sample components). [Figure 7] FIG. 7 shows a photograph of an agarose gel stained with ethidium bromide to observe the assembly of segments C, D and H containing 50% or 100% 5-methyl-dCTP and visualized with Bio-Rad Chemidoc MP. [Figure 8] Figure 8 shows the results of the experiment described in Example 6. (A) shows a photograph of an agarose gel stained with ethidium bromide and visualized with Bio-Rad Chemidoc MP (lane 1: 50 ng total DNA loaded, lane 2: 100 ng total DNA loaded) to show assembly from ssDNA molecules (plus and minus ssDNA molecules) generated in two separate one-pot synthesis reactions, (B) shows E. coli colonies (kanamycin selection) transformed with plasmids formed from ligation reactions with the assembly mixture, and (C) shows a photograph of an agarose gel stained with ethidium bromide and visualized with Bio-Rad Chemidoc MP showing the correct digestion pattern for plasmids from eight colonies digested with Ndel (there are two bands in each lane corresponding to 4.8 kbp and 0.45 kbp). [Figure 9]Figure 9 shows one-pot synthesis and assembly using the minimal overlap approach described in Example 7. (A) shows a photograph of an agarose gel stained with ethidium bromide and visualized with Bio-Rad Chemidoc MP to show the digestion products of the one-pot RCA containing all segments required for the minimal overlap strategy, (B) is a photograph of a plate containing kanamycin showing E. coli colonies transfected with the plasmid formed from ligation of the dsDNA complex generated from hybridization of the linearized plasmid and the digestion products of the one-pot RCA, and (C) shows a photograph of an agarose gel stained with ethidium bromide and visualized with Bio-Rad Chemidoc MP to show digestion of the plasmid with Ndel after extraction from two single colonies. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The terms "double-stranded DNA (dsDNA)" and "dsDNA molecule" are used interchangeably herein to refer to a nucleic acid molecule composed of two antiparallel polynucleotides containing deoxyribonucleotides (dNTPs) held together by hydrogen bonds through Watson-Crick or similar base pairing interactions. The two antiparallel polynucleotides of a dsDNA molecule are sometimes referred to as DNA "strands" and may be distinguished from the "first and second strands" or "plus and minus strands."

[0037] In view of the fact that synthetic and / or functionalized nucleotides (i.e., modified nucleotides) can participate in Watson-Crick or similar base pairing interactions, it will be apparent that dsDNA need not be entirely composed of standard or conventional dNTPs, i.e., dsDNA may include synthetic and / or functionalized nucleotides. For example, the 5' and / or 3' nucleotides in a dsDNA molecule may be modified, e.g., post-synthetically. Additionally, synthetic and / or functionalized nucleotides may be incorporated into a DNA molecule during its synthesis, e.g., during the synthesis of a single-stranded DNA (ssDNA) molecule used to generate a double-stranded molecule described herein. Thus, it is envisioned that dsDNA generated according to the methods and uses provided herein may include one or more functionalized, modified, or non-standard nucleotides, e.g., methylated nucleotides, such as methylated cytosine. Thus, the dsDNA molecules provided by the methods and uses described herein may be modified (e.g., functionalized) dsDNA molecules, e.g., methylated dsDNA molecules. In particular, the dsDNA molecule provided by the methods and uses described herein may comprise one or more (e.g., multiple) site-specific modifications. For example, the dsDNA molecule may comprise one or more site-specific modifications compared 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. Thus, the dsDNA molecule produced according to the methods and uses provided herein may be composed of conventional dNTPs.

[0038] Viewed another way, one or more of the plurality of ssDNA molecules provided for use in the present methods and uses may comprise one or more modified nucleotides (e.g. functionalized nucleotides such as methylated nucleotides), e.g., site-specific modifications, i.e., such that the modified nucleotides are incorporated into the dsDNA molecule that is generated.

[0039] The terms "conventional nucleotide" and "standard nucleotide" are used interchangeably herein and refer to a deoxynucleotide that contains one of the four bases found in DNA: adenine, guanine, cytosine, and thymine. Thus, the term "conventional nucleotide" encompasses, for example, dATP, dGTP, dCTP, and dTTP. Although uracil is not generally found in DNA naturally, dUTP can readily be used in place of or in addition to dTTP. Thus, in the context of the present invention, dUTP may also be viewed as a "conventional" nucleotide.

[0040] The term "functionalized nucleotide" or "functionalized dNTP" refers to a nucleotide that includes a modification relative to an unmodified conventional nucleotide, i.e., the modification results in the functionalized nucleotide and / or polynucleotide including at least one functionalized nucleotide having additional or alternative properties or characteristics compared to the corresponding conventional nucleotide. Thus, functionalized nucleotides may also be referred to as synthetic, modified or non-standard nucleotides. For example, the modification may make the nucleotide detectable, e.g., by incorporation of a label, or may make it capable of interacting and / or reacting with another component, i.e., a component with which the corresponding conventional nucleotide does not interact or react. The modification may render the polynucleotide that includes the nucleotide resistant to degradation, e.g., chemical and / or enzymatic degradation (e.g., nuclease degradation), or may alter the metabolism of the nucleotide. The modification may be a naturally occurring modification in vivo, e.g., methylation (e.g., the modified nucleotide may be 5-methylcytosine). Examples of functionalized nucleotides are provided in WO2020 / 161187, which is incorporated herein by reference.

[0041] Two antiparallel polynucleotides hybridize to form a dsDNA molecule because they contain sufficiently complementary nucleotide sequences to form a double strand by Watson-Crick base pairing or similar base pairing interactions. Two nucleotide sequences are "complementary" to one another if the molecules share base pairing homology. "Complementary" nucleotide sequences will combine with specificity under appropriate hybridization conditions to form a stable duplex. For example, two sequences are complementary if a portion of the first sequence can bind (hybridize) with a portion of the second sequence in an antiparallel orientation, such that the 3' end of each sequence binds with the 5' end of the other sequence, and then each A, T(U), G, and C of one sequence lines up with a T(U), A, C, and G of the other sequence, respectively. Thus, two sequences do not need to have perfect homology to be "complementary." Polynucleotides that contain sequences that do not have complete homology may also be referred to as having partial complementarity (i.e., being partially complementary) and will hybridize to form a partially double-stranded DNA molecule, i.e., a dsDNA molecule that contains regions of mismatched base pairs that result in regions of the molecule that are single stranded. Thus, polynucleotides that contain sequences that have complete homology may also be referred to as having complete complementarity (i.e., being fully complementary) and will hybridize to form a complete dsDNA molecule, i.e., a dsDNA molecule that does not contain regions of mismatched base pairs.

[0042] Of course, a partially double-stranded DNA molecule may also refer to a DNA molecule that does not have mismatched base pairs, i.e., a molecule that includes a region of dsDNA and a region of ssDNA that does not have an opposite strand (so that a gap exists in one of the DNA strands). Such a molecule may also be referred to as a partially double-stranded DNA complex herein. As described above, the strategy of resulting in a partially double-stranded DNA complex may be used to form a mixture of dsDNA molecules (e.g., a library of dsDNA molecules, such as a gene library) by using multiple ssDNA molecules that have a common region of complementarity adjacent (i.e., adjacent) to an intervening variant sequence.

[0043] The dsDNA molecule produced according to the methods and uses provided herein may be fully complementary, i.e., fully double-stranded, and this is preferred.However, it is envisaged that in some circumstances it may be desirable to produce partially complementary dsDNA molecules, for example, where at least about 90% (most preferably at least about 95%, 96%, 97%, 98% or 99%) of nucleotides share base pairing throughout the full length of the molecule.Thus, for example, a dsDNA molecule may have fully complementary regions separated by one or more regions of partial or no complementarity, so that at least about 90% of nucleotides in total form base pairs with complementary nucleotides on opposite strands.

[0044] The method or use provided herein proves to be particularly useful in generating long dsDNA molecules. Long dsDNA refers to any length of dsDNA molecule that cannot be efficiently and / or accurately synthesized using the above-mentioned conventional methods such as PCA. Since the efficiency and accuracy of the synthesis reaction varies based on the complexity of the dsDNA to be generated, long dsDNA molecules may also include dsDNA molecules that are less than 1 kbp in length, e.g., 500-1000 bp, which cannot be efficiently and / or accurately synthesized using conventional methods such as PCA. For example, the method or use provided herein proves to be useful in synthesizing any dsDNA molecule (regardless of length) that cannot be generated by conventional methods such as PCR assembly methods with an accuracy of more than 70%, i.e., 30% or more of the synthesis products contain incorrect sequences.

[0045] Preferably, the dsDNA molecules generated by the methods or uses provided herein may contain at least about 1500 bp (base pairs), e.g., at least about 1750 bp, 2000 bp, 2250 bp, 2500 bp, 3000 bp, 3500 bp, or 4000 bp. The dsDNA molecules generated by the methods or uses provided herein may be very long, e.g., 5 kbp, 6 kbp, 7 kbp, 8 kbp, 9 kbp, 10 kbp or more, e.g., 15 kbp, 20 kbp, 25 kbp, 30 kbp, 35 kbp, 40 kbp or more. For example, the dsDNA may contain about 1 to 50 kbp, e.g., about 1.5 to 50 kbp, 2 to 45 kbp, 3 to 40 kbp, 4 to 35 kbp, or 5 to 30 kbp.

[0046] The methods and uses provided herein can be advantageously used to generate dsDNA molecules comprising any sequence. The dsDNA molecule may comprise a coding sequence (i.e., an RNA coding sequence) and / or a non-coding sequence (e.g., a promoter). The coding sequence may code for any cellular or viral RNA. It may therefore be an mRNA, a tRNA, an rRNA, a viral RNA, a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a microRNA (miRNA), a small interfering RNA (siRNA), a piwi-binding RNA (piRNA), a ribozyme RNA, an antisense RNA or a non-coding RNA (e.g., a long non-coding RNA). Thus, the dsDNA may code for an mRNA and therefore may also be viewed as comprising a polypeptide coding sequence. The non-coding sequence may be any non-coding DNA, for example, an expression control sequence (e.g., a promoter, an enhancer, a terminator or a functional part thereof) or a recombinant DNA cloning vehicle or vector. For example, dsDNA may include a non-coding sequence (e.g., a promoter) operably linked to a coding sequence (e.g., a polypeptide coding sequence). Thus, dsDNA may include a gene. Of course, dsDNA may include any combination of coding and non-coding sequences, including combinations that do not occur in nature, such as a promoter sequence operably linked to a heterologous polypeptide coding sequence. The dsDNA generated by the methods and uses provided herein may be viewed as a "target dsDNA" or a "desired dsDNA", and these terms are used interchangeably herein.

[0047] The methods and uses provided herein may be particularly useful for generating dsDNA molecules that contain repeat sequences and / or sequences that can form secondary structures. In this regard, the formation of dsDNA complexes using single-stranded polynucleotides (e.g., ssDNA) that contain repeat sequences and / or sequences that can form secondary structures generally results in a mixture of various complexes (both intermolecular and intramolecular complexes) because each polynucleotide may have more complementarity than the polynucleotides in the mixture. Thus, the formation of dsDNA molecules from these dsDNA complexes using conventional methods (e.g., using PCA) results in a mixture of various dsDNA molecules, only some of which may contain the desired sequence. However, it is hypothesized that the use of single-stranded polynucleotides that contain a long, e.g., at least 50 bp, region of complementarity will result in the formation of dsDNA complexes that contain the desired (i.e., correct) sequence, resulting in a greater number of these complexes. Furthermore, formation of dsDNA molecules from these complexes using a ligation reaction involving a ligase (optionally preceded by an extension reaction involving a polymerase) is believed to improve the yield of dsDNA molecules having desired sequences, rather than PCR-based methods (e.g., PCA).Thus, the methods or uses provided herein can be used to generate dsDNA molecules that include repeat sequences and / or sequences that can form secondary structures, and optionally the dsDNA includes at least about 500bp, e.g., at least about 750bp, 1000bp, 1250bp or 1500bp.

[0048] Therefore, the method or use provided herein does not involve polymerase chain reaction (PCR), such as PCA or assembly PCR, to generate dsDNA. In another view, the step of forming or generating dsDNA molecules does not use polymerase chain reaction (PCR), such as PCA or assembly PCR. As mentioned above, the step of generating dsDNA molecules from dsDNA complexes involves a ligation reaction involving ligase, optionally preceded by an extension reaction involving polymerase. In particular, the ssDNA molecules used in the method provided herein may be generated using a method involving PCR, such as asymmetric PCR. Therefore, it will be understood that the step of forming dsDNA molecules does not involve PCR.

[0049] A repeat sequence or a repeat sequence refers to a nucleotide sequence that occurs more than once in a polynucleotide. For example, a region of a polynucleotide (e.g., two or more nucleotides, typically 3-10 or more nucleotides, e.g., 10-60 nucleotides) that contains one type of nucleotide (e.g., dATP) can be viewed as a repeat. Similarly, more complex sequences (i.e., containing more than one type of nucleotide) that are repeated more than once in a polynucleotide can also be viewed as repeats. Repeat sequences can be tandem (i.e., directly adjacent to each other) or interspersed (i.e., with non-repeat sequences separating the repeats). Thus, a repeat sequence includes a sequence of at least 3 nucleotides (e.g., at least 4, 5 or 6 nucleotides, e.g., about 10, 15 or 20 nucleotides) that occurs more than once in a polynucleotide, e.g., at least 2, 3, 4 or more times, e.g., 5, 6, 7, 8, 9, 10 or more times.

[0050] A sequence capable of forming a secondary structure refers to a nucleotide sequence that has a region of self-complementarity (complete or partial complementarity), such that a polynucleotide comprising the sequence, when subjected to suitable conditions to allow hybridization of complementary sequences, forms one or more secondary structures, for example, one or more intramolecular double-stranded regions. The intramolecular double-stranded regions may be separated by single-stranded regions, such that the polynucleotide comprises one or more so-called stem-loop regions.

[0051] The methods and uses provided herein use a plurality of single-stranded DNA (ssDNA) molecules (polynucleotides) to form each strand of a dsDNA molecule. Thus, each ssDNA molecule provides a "non-overlapping portion" of the strand of the dsDNA molecule. Thus, the term "non-overlapping portion" means that each ssDNA molecule provides a separate (separate or different) portion (fragment, segment, etc.) of one strand of the dsDNA molecule, which portion is not included in any of the other ssDNA molecules. Two or more ssDNA molecules providing a portion of one strand of a dsDNA molecule may contain the same sequence, but these sequences are found in different parts of the strand of the dsDNA molecule, for example, due to sequence repetition in the strand of the dsDNA molecule. Thus, when compared over their full length, each ssDNA molecule of the plurality of ssDNA molecules has a different nucleotide sequence.

[0052] Thus, when the multiple ssDNA molecules provided by the present methods and uses are aligned end-to-end in the correct order, they form at least a portion of each strand of a dsDNA molecule and include at least the 5'-ends of the first and second strands, preferably the 5'- and 3'-ends of the first strand and at least the 5'-end of the second strand.

[0053] As mentioned above, each ssDNA molecule used in the methods and uses provided herein may be provided as multiple types of strands of dsDNA molecules, i.e., corresponding non-overlapping portions of different types (e.g., sequence variants), such that the product of the ligation reaction involving ligase is a mixture of dsDNA molecules. The dsDNA molecules in the mixture are different based on which type of ssDNA molecule is incorporated into the dsDNA complex. When the different types of ssDNA molecules differ by the functional groups on the nucleotides (e.g., the position and / or type of the functional group), the sequences of the different types may be identical.

[0054] 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 method, a sequence variant is a ssDNA molecule that contains identical sequences (i.e., sequences that form regions of complementarity) adjacent to non-identical intervening sequences. The intervening sequences may be similar to each other (e.g., all of the intervening sequences may have at least 60%, 70%, 80%, 90% or more sequence identity with each other, e.g., 95%, 96%, 97%, 98% or 99% sequence identity with each other), or may be different from each other (e.g., all of the intervening sequences may have less than 60%, less than 50% or less than 40% sequence identity, e.g., 30%, 25%, 20% or less sequence identity). Of course, the above sequence identity values ​​may be applied to the entire sequence, taking into account identical end sequences. Sequence identity can be determined by any suitable method known in the art, for example, using the BLAST alignment algorithm.

[0055] The term "different" in relation to different non-overlapping portions of the strands of a dsDNA molecule refers to ssDNA molecules that contain nucleotide sequences with one or more different nucleotides. Thus, different ssDNA molecules may contain nucleotide sequences that differ by one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides, e.g., 20, 30, 40, 50, 60, 70, 80, 90 or more nucleotides. The difference may be the length and / or sequence of the ssDNA molecule. In another view, different ssDNA molecules have less than 100% sequence identity with each other, such as less than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 70%, 60%, 50%.

[0056] For example, in the "complete overlap" strategy described above, end-to-end alignment of non-overlapping ssDNA molecules results in the complete sequence of both strands of the dsDNA, i.e., there are no "gaps" in the sequence. Thus, the ssDNA molecules used in the "complete overlap" strategy can be viewed as contiguous ssDNA molecules.

[0057] In the above "partial overlap" strategy, the alignment of non-overlapping ssDNA molecules end-to-end results in incomplete alignment of both strands of dsDNA, i.e., there is a "gap" in the sequence of both strands. Thus, the ssDNA molecules provided in the "partial overlap" strategy can be viewed as non-contiguous ssDNA molecules.

[0058] The term "gap" as used herein refers to the portion of the nucleotide sequence of the strand of a dsDNA molecule that is not directly provided by multiple ssDNA molecules.As described in more detail below, the gap is "filled" by using the sequence of the opposite strand as a template for the extension reaction involving polymerase.Therefore, these portions of the nucleotide sequence of the strand of a dsDNA molecule can also be considered to be indirectly provided.For example, in the above-mentioned "partial overlap" strategy, the 3' end of the first and / or second strand of a dsDNA molecule can be indirectly provided.

[0059] In the above "minimal overlap" strategy, the alignment of non-overlapping ssDNA molecules end-to-end results in a complete sequence of the first strand of dsDNA and an incomplete sequence of the second strand. Thus, the multiple ssDNA molecules that contribute to the first strand in the "minimal overlap" strategy can be viewed as continuous ssDNA molecules, and the multiple ssDNA molecules that contribute to the second strand in the "minimal overlap" strategy can be viewed as non-contiguous ssDNA molecules.

[0060] Of course, the methods outlined above may be combined such that one or both strands may be provided by a mixture of contiguous and non-contiguous ssDNA molecules, such that only a portion of the ssDNA molecules need to be extended in a polymerase-involved extension reaction.Thus, in some embodiments, the plurality of ssDNA molecules that provide one strand of the dsDNA is a mixture of contiguous and non-contiguous ssDNA molecules, and / or the plurality of ssDNA molecules that provide the other strand of the dsDNA is a mixture of contiguous and non-contiguous ssDNA molecules.

[0061] However, in some embodiments, the plurality of ssDNA molecules provided by the method and use are all continuous ssDNA molecules.In some embodiments, the plurality of ssDNA molecules provided by the method and use are all non-contiguous ssDNA molecules.In some embodiments, the plurality of ssDNA molecules that provide one strand of dsDNA molecule are all continuous ssDNA molecules, and the plurality of ssDNA molecules that provide the other strand of dsDNA molecule are all non-contiguous ssDNA molecules.

[0062] The ssDNA molecule (polynucleotide) may be of any size suitable for generating a dsDNA molecule of the above size, as further defined below, and further suitable for generating a region of complementarity with its opposite strand of at least 50 base pairs (bp). It will be apparent that the size of each ssDNA molecule of each of the plurality will depend on the number of molecules and the strategy (e.g., complete overlap, partial overlap, etc.) used to generate each strand of the dsDNA molecule. In general, contiguous ssDNA molecules will be longer than non-contiguous ssDNA molecules. Thus, each ssDNA of the plurality of ssDNA molecules may be of different sizes. However, in some embodiments, each ssDNA molecule of the plurality of ssDNA molecules used to generate a strand of the dsDNA molecule may be substantially the same size, for example, within about 20% (e.g., within about 15% or within 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 of the plurality of ssDNA molecules used to provide the strands of dsDNA may consist of about 450-550 nucleotides (i.e., 500 nucleotides ± 50 nucleotides). However, it is clear that the ssDNA molecule providing the 5' and 3' ends of one strand of the dsDNA molecule (e.g., the second strand as defined above) may be smaller than the other plurality of ssDNA molecules. For example, in a complete overlap strategy, the ssDNA molecule providing the 5' and 3' ends of the second strand of the dsDNA molecule is smaller than the other plurality of ssDNA molecules.

[0063] Each ssDNA molecule of the plurality may be about 150 to 10,000 nucleotides in length, e.g., about 200 to about 9,000 nucleotides in length, about 250 to about 8,000 nucleotides in length, about 350 to about 7,000 nucleotides in length, about 400 to about 6,000 nucleotides in length, about 450 to about 5,000 nucleotides in length, about 500 to about 4,000 nucleotides in length, about 600 to about 3,500 nucleotides in length, about 700 to about 3,000 nucleotides in length, about 800 to about 2,500 nucleotides in length, about 900 to about 2,000 nucleotides in length, about 1,000 to about 1,500 nucleotides in length, etc.

[0064] Viewed alternatively, each ssDNA molecule of the plurality may comprise at least about 200, 300, 400, 500 nucleotides, e.g., at least about 600, 700, 800, 900, 1000 nucleotides, e.g., 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more nucleotides.

[0065] As mentioned above, continuous ssDNA molecule is generally longer than non-contiguous ssDNA molecule.Therefore, for example, continuous ssDNA molecule can comprise at least about 500 nucleotides, for example, at least about 600, 700, 800, 900, 1000 nucleotides, for example, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000 or more nucleotides.In the method using a mixture of continuous ssDNA and non-contiguous ssDNA, non-contiguous ssDNA molecule is generally shorter than continuous ssDNA molecule, for example, at least about 150, 200, 300, 400 or 500 nucleotides.

[0066] Further, even in methods and uses in which all of the ssDNA molecules are contiguous, the ssDNA molecule providing the 5' and 3' ends of one strand of the dsDNA may be shorter than the other ssDNA molecule, e.g., by at least the length of the region of complementarity, e.g., by at least 50 nucleotides, e.g., by 75, 100, 125, 150 nucleotides or more.

[0067] Thus, in connection with the methods and uses provided above (i.e., the complete overlap and partial overlap strategies), the (a)(i) ssDNA molecule may comprise at least about 500 nucleotides, e.g., at least about 600, 700, 800, 900 or 1000 nucleotides, and at least two of the (a)(ii) ssDNA molecules may comprise at least about 500 nucleotides, e.g., at least about 600, 700, 800, 900 or 1000 nucleotides.

[0068] In connection with the other methods and uses provided above (i.e., the minimal overlap strategy), the (a)(i) ssDNA molecule may comprise at least about 500 nucleotides, e.g., at least about 600, 700, 800, 900 or 1000 nucleotides, and the (a)(ii) ssDNA molecule may be shorter than the (a)(i) ssDNA molecule, e.g., at least about 150, 200, 300, 400 or 500 nucleotides.

[0069] In addition to providing a plurality of ssDNA molecules of sufficient size to generate a dsDNA complex and subsequently a dsDNA molecule, it will be appreciated that each ssDNA molecule having a non-terminal 5' end (i.e., ssDNA molecules that do not provide a 5' end of their respective strand) must be phosphorylated to allow ligation to take place. It is advantageous for each ssDNA molecule having a non-terminal 5' end to be provided with a phosphorylated 5' end, although this is not essential, as the 5' end may be phosphorylated after assembly of the dsDNA complex. Thus, the method may include a further step of phosphorylating the 5' end(s) of the ssDNA molecule, for example using a kinase enzyme such as T4 polynucleotide kinase.

[0070] Similarly, it will be understood that each ssDNA molecule having a non-terminal 3' end (i.e., ssDNA molecules that do not directly provide the 3' end of their respective strand) must be extendable in an extension reaction involving a polymerase and / or ligatable in a ligation reaction involving a ligase, e.g., comprise an extendable and / or ligatable 3' end (i.e., comprise a functional hydroxyl group).

[0071] In some embodiments, it may be useful to provide one or both ssDNA molecules that provide the 5' end of the dsDNA molecule without phosphorylated 5' end, for example, to minimize inter- and intramolecular ligation events between dsDNA complexes.For example, the 5' end of one or both 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.

[0072] Similarly, in some embodiments, it may be beneficial to provide one or both ssDNA molecules that provide the 3' end of the dsDNA molecule without an extendable and / or ligatable 3' end (i.e., directly), for example, to minimize intermolecular ligation events between dsDNA complexes and / or to minimize the generation of undesired extension products.For example, the 3' end of one or both ssDNA molecules that provide the 3' end of the dsDNA molecule may be modified.As described further below, the dsDNA molecule may be subjected to further steps, for example, post-synthetic modifications, such as phosphorylation of the 5' end to improve the functionality of the dsDNA molecule, for example, to facilitate ligation with other DNA molecules, such as vectors, e.g., plasmids.

[0073] It should be understood that the term "single strand" in relation to a single stranded DNA molecule 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 (single strand). As mentioned above, this does not prevent single stranded DNA molecules from forming secondary or tertiary structures. For example, single stranded DNA molecules may contain regions of self-complementarity, and may therefore be able to form hairpin or stem loop structures that involve one region of the single stranded DNA molecule hybridizing 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.

[0074] As used herein, the term "multiple" means three or more, e.g., 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 present 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 comprise at least three ssDNA molecules, e.g., 4, 5, 6, 7, 8, 9, 10, 12, 20, 25, 30 or more ssDNA molecules, e.g., 3-50, 4-45, 5-40, 6-35, 7-30, 8-25, 9-20, or 10-15 ssDNA molecules. Of course, in some approaches (i.e., full overlap and some partial overlap approaches), one strand of dsDNA is contributed by more ssDNA molecules than the other strand, i.e., at least one more ssDNA molecule. In a representative example, when the first strand is provided by four ssDNA molecules, the second strand is provided by five ssDNA molecules.Therefore, the number of ssDNA molecules provided as each of the multiple ssDNA molecules may be different.However, in some methods (i.e., the minimum overlap method or some partial overlap methods), the number of ssDNA molecules provided as each of the multiple ssDNA molecules is the same.

[0075] When multiple copies (i.e., a plurality) of each of the multiple ssDNA molecules are provided in the methods and uses described herein, this represents a multiple dsDNA complex and subsequently a multiple dsDNA molecule, e.g., 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 It can be seen that each dsDNA molecule will generate more than one copy. Therefore, the methods and uses provided herein can be seen as generating a plurality of dsDNA molecules. In this regard, each copy of the ssDNA molecule that provides the non-overlapping portion of the strand of the dsDNA molecule can be the same, i.e., contain the same sequence. When the ssDNA molecule contains modified nucleotides, the modified nucleotides of each copy can be the same, i.e., the number, type and position of functional groups on each copy of the ssDNA molecule can be the same.

[0076] Similarly, when different types (e.g., sequence variants) of ssDNA molecules are provided, the term multiple can refer to at least three different types (e.g., three sequence variants), e.g., 4, 5, 6, 7, 8, 9, 10, 12, 20, 25, 30 or more different types, including multiple copies of each type (e.g., 10 copies of each type). 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 In cases where different types are used to generate a library of dsDNA molecules (a gene library, e.g., for phage display), the term plurality refers to at least 100 (e.g., 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 109 , 10 10 or more types), and multiple copies of each type (e.g., 10 of each type). 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 Copies of the above may be provided.

[0077] As mentioned above, it is assumed that the use of extremely 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 the polynucleotides produced using solid-phase chemical synthesis (e.g., phosphoramidite synthesis) generally contain impurities (e.g., by-products of chemical synthesis reaction) and incorrect sequences, especially for polynucleotides that contain more than 50 nucleotides.In contrast, the polynucleotides produced using enzymatic synthesis, especially using so-called high fidelity DNA polymerase, can be easily highly purified and contain only minimal sequence errors.

[0078] Thus, the ssDNA molecules provided in the methods and uses described herein are not produced using chemical synthesis methods, for example solid phase chemical synthesis methods such as phosphoramidite synthesis.

[0079] In another view, each ssDNA molecule provided in the methods and uses described herein is generated by enzymatic synthesis, i.e., an enzymatically generated ssDNA molecule. Numerous methods for enzymatic generation of ssDNA molecules are known in the art, such as asymmetric PCR (e.g., LATE-PCR), monoclonal stoichiometric (MOSIC) polynucleotide generation (Ducani et al., 2013, Nature Methods, p. 647-652, incorporated herein by reference), terminal deoxynucleotidyl transferase (TdT) synthesis, etc. Of course, any suitable enzymatic synthesis method can be used to generate ssDNA molecules for use in the methods and uses provided herein. Thus, ssDNA molecules for use in the methods and uses provided herein may be generated using asymmetric PCR.

[0080] Although it is known that PCR-based synthesis methods can result in polynucleotides containing errors, polynucleotides containing about 1000 or fewer nucleotides are less likely to produce errors. Thus, when ssDNA molecules are generated using asymmetric PCR, the ssDNA molecules may contain 1000 or fewer nucleotides. However, it will be appreciated 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 by Perler et al., Proceedings of The National Academy of Sciences of The United States of America (1992) 89:5577-5581; Pyrococcus species GB-D (Deep Vent); Pyrococcus furiosus DNA polymerase (Pfu), described by Lundberg et al., Gene (1991) 108:1-6; Pyrococcus woesei (Pwo); and the like.

[0081] Therefore, enzymatic synthesis (e.g., asymmetric PCR) methods require the use of high-fidelity polymerases, i.e., 2.0 × 10 -5 For example, 1.0×10 -5 Below, 9.0 x 10 -6 Below, 8.0 x 10 -6 or less than 7.0 x 10 -6 Polymerases with the following error rates may be used:

[0082] The "error rate" (f) is calculated as f=n / S(target size x d), where n is the number of mutations observed for all polynucleotides sequenced, (target size x d) is for each polynucleotide cloned, and (d) is the average doubling value for each PCR reaction, where doubling is calculated from the formula d=(ng DNA after PCR / ng DNA input).

[0083] Any method of enzymatic synthesis can be used to generate ssDNA molecules for use in the methods provided herein, although ssDNA molecules generated using methods that do not involve PCR may be preferred to avoid the introduction of sequence errors, especially with respect to the generation of long ssDNA molecules (e.g., containing at least about 500 nucleotides, e.g., 1000 or more nucleotides). For example, ssDNA molecules may be generated using rolling circle amplification (RCA)-based methods, in particular the MOSIC method. Methods for generating ssDNA molecules using the MOSIC method are described in Ducany et al. (supra), WO 2020 / 161187 and WO 2021 / 170656 (all of which are incorporated herein by reference).

[0084] Thus, the ssDNA molecules for use in the methods described herein include (a) providing a circular DNA molecule comprising a nucleotide sequence encoding a cleavage domain and a flanking ssDNA molecule; (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule of (a) as a template; (c) cleaving (e.g., enzymatically cleaving) the product of the RCA reaction at a cleavage domain to release multiple copies of the ssDNA molecule; and optionally (d) isolating or purifying the ssDNA molecules produced in (c); The composition may be produced by a method including the steps of:

[0085] The step of preparing a circular DNA molecule may be accomplished by any suitable means and may depend on the structure of the circular DNA molecule. For example, the circular DNA molecule may be provided by a minicircle plasmid as described in WO2021 / 170656 (herein incorporated by reference). In this regard, the circular DNA molecule may be a single-stranded DNA molecule or a double-stranded DNA molecule.

[0086] It will be apparent that in embodiments where the circular DNA molecule is a double-stranded molecule, the circular DNA molecule must be treated to prepare the RCA template. Thus, in some embodiments, the method includes an additional step of cleaving one strand of the circular DNA molecule to prepare the RCA template before the RCA reaction is performed.

[0087] Thus, step (a) of the method provided herein may include the above-mentioned method for generating ssDNA molecules of a plurality of ssDNA molecules. Step (a) of the method provided herein may include the above-mentioned method for generating all of the plurality of ssDNA molecules used in the method provided herein. Of course, each of the plurality of ssDNA molecules may be generated in a separate reaction and then pooled or combined to form a plurality of ssDNA molecules for one or both strands. Alternatively, the plurality of ssDNA molecules for one or both strands may be generated as a single reaction mixture in a so-called multiplex reaction. For example, as shown in the examples, each ssDNA molecule may be encoded by a separate circular DNA molecule, which may be combined into a single reaction mixture so that the ssDNA molecules are generated simultaneously.

[0088] It will be appreciated that the circular DNA molecule may comprise multiple nucleotide sequences encoding the ssDNA molecules provided in (a), each flanked by a cleavage domain, such that more than one of the multiple ssDNA molecules, e.g., all of the multiple ssDNA molecules of a strand of a dsDNA molecule, can be provided in a single step.

[0089] The ssDNA molecules for one or both strands can be generated by combining the multiplex reaction method described above with a circular DNA molecule that contains multiple nucleotide sequences, each encoding a ssDNA molecule.

[0090] Of course, each ssDNA molecule used in the methods and uses provided herein can be prepared separately and thus can be generated using different methods.Thus, for example, some ssDNA molecules can be generated by asymmetric PCR and / or TdT synthesis and some by MOSIC method, and then combined (i.e., mixed) to prepare multiple ssDNA molecules.However, in order to avoid additional steps, it may be preferable to generate all ssDNA molecules by the same method, for example, the MOSIC method described above.

[0091] As shown in the examples, it has been unexpectedly shown that all ssDNA molecules for both strands of dsDNA molecules can be generated and assembled as a single reaction mixture, i.e., 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.Therefore, all steps of the method, including the step of generating ssDNA molecules, can be carried out in a single reaction mixture and / or vessel.In other words, the method provided herein can be a one-pot reaction.

[0092] The term "cleavage domain" as used herein generally refers to a domain in a circular DNA molecule that results in a domain in an RCA product that can be specifically cleaved to release ssDNA molecules.Thus, the cleavage domain in a circular DNA molecule may be capable of direct cleavage, or may simply code for a cleavage domain that is functional only in the RCA product, or that is functional under certain conditions, such as when contacted with a cofactor.

[0093] In some embodiments, the cleavage domain comprises or consists of a sequence that can form a hairpin structure. A hairpin structure may also be known as a hairpin loop or stem loop, and these terms are used interchangeably herein. A hairpin is an intramolecular base-pairing pattern that can occur in single-stranded DNA or RNA molecules. A hairpin occurs when two regions of the same strand, which are usually complementary in nucleotide sequence when read in opposite directions, base-pair (hybridize) to form a double-stranded stem (duplex) and an unpaired, i.e., single-stranded loop. The resulting structure can be shown as a carrot-and-lollipop shape.

[0094] Thus, in some embodiments, the cleavage domain comprises or consists of a sequence that can form a hairpin structure, the cleavage domain comprises a self-complementary sequence.As the RCA product extends, the hybridization of these self-complementary regions results in a hairpin structure, and the double-stranded part of the hairpin structure comprises the sequence that is recognized by the cleavage enzyme.Therefore, the cleavage of the double-stranded part of the hairpin structure in the RCA product releases the ssDNA molecule and the hairpin structure (i.e., the oligonucleotide that forms the hairpin structure).

[0095] "Cleavage" includes any means of breaking a covalent bond. Thus, in the context of the present invention, cleavage includes cleavage of a covalent bond in a nucleotide chain (i.e., strand scission or strand splitting), for example, by cleavage of a phosphodiester bond.

[0096] Thus, in some embodiments, the cleavage domain may comprise a sequence recognized by one or more enzymes capable of cleaving a nucleic acid molecule, i.e., breaking a phosphodiester bond between two or more nucleotides. For example, the cleavage domain may comprise a restriction endonuclease (restriction enzyme) recognition sequence. Restriction enzymes cleave double-stranded or single-stranded DNA at specific recognition nucleotide sequences known as restriction sites, and suitable enzymes are well known in the art. For example, to facilitate the design of the polynucleotide sequence(s) in the circular DNA molecule, e.g., to avoid including cleavage recognition sites that occur within the sequence of a ssDNA molecule, it may be particularly advantageous to use a low-frequency-cutting restriction enzyme, i.e., an enzyme with a long recognition site (at least 8 base pairs in length).

[0097] The cleavage domain may comprise a sequence recognized by a type II restriction endonuclease, more preferably a type IIs restriction endonuclease. While any suitable cleavage domain and cleavage enzyme can 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 can be used include BsrDI, BtsI, BtsIMutI, MlyI or an isoschizomer thereof.

[0098] As mentioned above, the methods provided herein may utilize long ssDNA molecules, for example, ssDNA molecules at least about 500, 750 or 1000 nucleotides in length. Of course, the longer the sequence, the more likely it is to contain a sequence recognized by an endonuclease, particularly a type IIs restriction endonuclease. Thus, the cleavage domain adjacent to the polynucleotide may be a homing endonuclease cleavage domain, i.e., the cleavage enzyme used in the cleavage step may be a homing endonuclease. For example, the cleavage domain adjacent to the polynucleotide may be a meganuclease cleavage domain, i.e., the cleavage enzyme used in the cleavage step may be a meganuclease.

[0099] The term "meganuclease" refers to endonucleases characterized by large recognition sites, e.g., double-stranded DNA sequences of 12 to 40 base pairs. Thus, many homing endonucleases, such as I-SceI, can be viewed as meganucleases. Chimeric meganucleases may be generated by fusing a nucleic acid binding domain and an endonuclease cleavage domain from different proteins. For example, any protein domain capable of site-specific recognition (binding) of a DNA sequence as described above may be fused with a cleavage domain from an endonuclease that cleaves outside the sequence recognized by the endonuclease, e.g., at a specific distance from the recognition sequence. Any suitable meganuclease known in the art may be used in the methods described herein, e.g., in the step of cleaving the RCA product.

[0100] Thus, cleaving the product of the RCA reaction may comprise contacting the RCA product with a cleavage enzyme under suitable conditions to selectively cleave the RCA product at the cleavage domain.

[0101] Upon cleavage of the RCA product, a ssDNA polynucleotide is released. The released polynucleotide may consist of the polynucleotide sequence alone (i.e., without additional nucleotides) or may contain one or more additional nucleotides from the cleavage domain adjacent to the polynucleotide sequence at one or both ends. Thus, the ssDNA polynucleotide may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleotides from the cleavage domain at one or both ends. Preferably, the sequence of the circular DNA molecule is designed such that when the RCA product is cleaved, the released polynucleotide does not contain any additional nucleotides from the cleavage domain. In another view, the cleavage domain adjacent to the polynucleotide sequence(s) may be positioned in the circular DNA molecule such that its cleavage in the RCA product releases a ssDNA polynucleotide that does not contain any additional nucleotides from the cleavage domain.

[0102] A cleavage enzyme may cleave a nucleic acid molecule at a position outside the cleavage enzyme recognition sequence, so that there may be one or more nucleotides between the cleavage domain and the polynucleotide sequence. Alternatively, the cleavage domain may contain nucleotide sequences in addition to the cleavage enzyme recognition sequence to ensure that cleavage preferably releases an intact ssDNA polynucleotide that does not contain any additional nucleotides (e.g., nucleotides that form part of the cleavage domain).

[0103] Thus, the term "adjacent" in relation to a polynucleotide sequence adjacent to a cleavage domain refers to a cleavage domain that is directly or indirectly adjacent to the polynucleotide sequence. In another view, the cleavage domain is located at either end of the polynucleotide sequence. That is, the cleavage domain is upstream and downstream (5' and 3' ends) of the polynucleotide sequence. The cleavage site of the cleavage domain (e.g., the site at which the cleavage enzyme cleaves the cleavage domain) may be directly adjacent to the end of the polynucleotide sequence to which it is adjacent. However, the polynucleotide sequence and the cleavage domain sequence may overlap. That is, for example, if the cleavage site is an internal site within the cleavage domain, the end of the polynucleotide sequence may form part of the cleavage domain, i.e., the cleavage domain may form the end or part of the end of the polynucleotide sequence. Thus, there may be one or more nucleotides between the cleavage domain and the polynucleotide sequence (i.e., between the ends of the sequence), e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more. However, the cleavage domain and the polynucleotide sequence may overlap by one or more, for example, by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides.

[0104] The size of the cleavage domain in the circular DNA molecule is not particularly limited, and will depend on the type of cleavage domain as described above. The relative length of the polynucleotide sequence and the cleavage domain are designed or selected to be different from each other, so that the ssDNA polynucleotide can be easily purified after the RCA product is cleaved by the cleavage domain. Therefore, the cleavage domain(s) can be selected to be shorter than the polynucleotide sequence(s) in the circular DNA molecule. When the circular DNA molecule comprises multiple polynucleotide sequences of different lengths, the cleavage domain(s) can be selected to be shorter than the shortest polynucleotide sequence in the circular DNA molecule.

[0105] The cleavage domain may be about 4-50 nucleotides in length, e.g., about 5-45, 6-40, 7-35, or 8-30 nucleotides in length. In some embodiments, the cleavage domain may range from about 10 to 25 nucleotides in length, including about 12 to 22 or about 14 to 20. However, it will be apparent that a cleavage domain of any suitable length may be used in the present invention so long as it meets the above functional requirements. Thus, longer cleavage domains are envisioned, e.g., about 22 to 70 nucleotides in length, including about 25 to 60 or about 30 to 55 nucleotides in length. Alternatively, the cleavage domain may range from about 35 to 80 nucleotides in length, including about 40 to 70 or about 45 to 60 nucleotides in length.

[0106] The polynucleotide sequence and the adjacent cleavage domains may be the same or different. It is advantageous that the polynucleotide sequence and the adjacent cleavage domains are the same so that a single cleavage step is sufficient to release all single-stranded DNA polynucleotides. However, as mentioned above, some cleavage enzymes may cleave nucleic acid molecules at positions outside the cleavage enzyme recognition sequence or may recognize more than one sequence (e.g., when changes in the enzyme recognition sequence are allowed). Therefore, the entire sequence of the cleavage domains does not need to be the same in order for the cleavage domains to be cleaved by the same enzyme. For example, the step of cleaving the RCA product may include a step of contacting the RCA product with a single cleavage enzyme under conditions suitable for cleaving the cleavage domain in the RCA product.

[0107] The suitable conditions for cleaving the cleavage domain in RCA product will depend on the means used to achieve cleavage.For example, when cleavage is achieved using a cleavage enzyme such as restriction endonuclease or homing endonuclease, the conditions will vary depending on the enzyme selected, and suitable conditions are well known in the art, for example, cleavage step can follow the manufacturer's instructions.

[0108] For example, a cleavage enzyme, e.g., a restriction or 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 buffered saline (HBS), and Tris buffered saline (TBS), with and without EDTA. Cleavage may occur over a wide range of temperatures, e.g., 0-70° C., and at a pH range of about 3.0-10.0, e.g., 4.0-9.0, 5.0-8.0. Those of skill in the art will be able to readily determine other suitable conditions.

[0109] Thus, the method for generating ssDNA molecules may include performing rolling circle amplification using circular DNA molecules as templates. Rolling circle amplification (RCA) is well known in the art and described in Dean et al., 2001 (Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification, Genome Research, 11, p.1095-1099), the disclosure of which is incorporated herein by reference. Briefly, RCA involves the synthesis of nucleic acid molecules using a circular single-stranded nucleic acid molecule, e.g., a circular or circular oligonucleotide, as a rolling circle template (RCA template) and a strand-displacing polymerase to extend the primer hybridized to the template. The addition of polymerase and nucleotides initiates the synthesis reaction, i.e., polymerization. Because the rolling circle template is endless, the resulting product is a long single-stranded nucleic acid molecule composed of tandem repeats complementary to the rolling circle template.

[0110] A typical RCA reaction mixture includes a circular DNA molecule serving as a template and one or more primers utilized in a primer extension reaction; for example, RCA may be templated by a single primer to form a single concatemer product, or by multiple primers, each of which anneals to a different region of the circular template to generate multiple concatemer products per round. The oligonucleotide primers that the circular DNA molecule may be contacted with will be of sufficient length to provide hybridization with the circular DNA molecule under annealing conditions. Of course, the primers can be obtained by cutting (e.g., nicking) one strand of the double-stranded circular DNA molecule prepared in step (a).

[0111] The RCA reaction mixture may further comprise an aqueous buffer medium comprising a source of monovalent ions, a source of divalent cations and a buffering agent. 4 , K glutamate, NH 4 Any convenient source of monovalent ions may be utilized, such as Cl, ammonium sulfate, etc. The divalent cation may be magnesium, manganese, zinc, etc., and the cation is typically magnesium. MgCl 2 Any convenient source of magnesium cations may be utilized, including Mg acetate, etc. 2+ The amount of may range from 0.5 to 10 mM, but will preferably be in the range of about 3 to 6 mM, and ideally about 5 mM. Representative buffering agents or salts that may be present in the buffer include Tris, Tricine, HEPES, MOPS, and the like, and the amount of buffering agent will generally range from about 5 to 150 mM, usually about 10 to 100 mM, more usually about 20 to 50 mM, and the buffering agent may be present in an amount sufficient to provide a pH in the range of about 6.0 to 9.5. Other agents that may be present in the buffer medium include chelating agents such as EDTA, EGTA, and the like.

[0112] In addition to the components described above, the reaction mixture generally includes a polymerase (defined further below, e.g., phi29 DNA polymerase), one or more nucleotides, and other components required for a DNA polymerase reaction as set forth below. The desired polymerase activity may be provided by one or more different polymerase enzymes.

[0113] The RCA reaction ultimately produces a polynucleotide product that contains adjacent (tandem) repeats of the complementary sequence of the circular DNA molecule. This product may also be known as a concatemer, RCA product, or "RCP." Thus, the RCA product contains a linear sequence that consists of a polynucleotide sequence (or specifically the reverse complement of the polynucleotide sequence of the circular DNA molecule template) flanked by a cleavage domain.

[0114] Any DNA polymerase with at least some strand displacement activity may be used in the RCA reaction used to generate ssDNA molecules for use in the methods provided herein. Strand displacement activity ensures that once the polymerase has extended around the circular DNA molecule, it can remove the primer sequence and extension product and continue "wrapped" around the template. In embodiments where the nicked strand of the DNA minicircle provides the primer for RCA extension, strand displacement activity ensures that the polymerase can remove the nicked strand. Suitable DNA polymerase enzymes with at least some strand displacement activity include phi29 DNA polymerase, E. 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 all such modified derivatives, including derivatives of naturally occurring DNA polymerase enzymes. For example, in some embodiments, the DNA polymerase may be modified to remove 5'-3' exonuclease activity.

[0115] Particularly preferred DNA polymerase enzymes for use in RCA reactions include phi29 DNA polymerase, Bst DNA polymerase and derivatives thereof, such as sequence modified derivatives or mutants.

[0116] Sequence modified derivatives or mutants of DNA polymerase enzymes include mutants that retain at least some functional activity of the wild-type sequence, such as DNA polymerase activity and at least some strand displacement activity. Mutations may affect the activity profile of the enzyme under various reaction conditions, such as temperature, template concentration, primer concentration, etc., e.g., increase or decrease the rate of polymerization. Mutations or sequence modifications may also affect the exonuclease activity and / or thermostability of the enzyme.

[0117] As mentioned above, the RCA reaction may be carried out using conventional nucleotides, functionalized nucleotides, or a mixture of conventional and functionalized nucleotides.

[0118] The term "releasing" is used in this context to refer to cleaving the RCA product at a cleavage domain adjacent to the polynucleotide sequence to sever or separate the polynucleotide from the cleavage domain. It is desirable for release of a given polynucleotide to involve cleavage at both cleavage domains adjacent to the polynucleotide sequence.

[0119] It is not necessary that all cleavage domains in the RCA product are cleaved to form a single-stranded DNA polynucleotide. Cleavage of a portion of a cleavage domain will also release a portion of a single-stranded DNA polynucleotide. Thus, the step of cleaving the RCA product may 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 may result in the cleavage of at least about 90%, for example, 95% or more of the cleavage domains in the RCA product.

[0120] Alternatively, the step of cleaving the RCA product may result in the release of at least about 30%, e.g., at least about 35%, 40%, 45%, 50%, 60%, 70% or 80% of the single-stranded DNA polynucleotides contained in the RCA product. Further, the step of cleaving the RCA product may result in the release of at least about 90%, e.g., 95% or more of the single-stranded DNA polynucleotides contained in the RCA product.

[0121] Once the single-stranded DNA polynucleotides are released, it may be desirable to isolate, separate or purify the single-stranded DNA polynucleotides from the cleavage reaction mixture (e.g., reaction components and / or degradation products, e.g., cleavage domains, uncleaved RCA products, etc.) to prepare ssDNA molecules for use in the methods and uses provided herein.

[0122] Thus, the method of producing a ssDNA molecule may further comprise the step of isolating, separating or purifying the single-stranded DNA polynucleotides, which may be carried out by any suitable method known in the art.

[0123] After the isolation, separation or purification step, the single-stranded DNA polynucleotide is preferably substantially free of any contaminating components (e.g., reaction components and / or degradation products, e.g., cleavage domains, uncleaved RCA products, etc.) from the materials or components used in the isolation procedure or its preparation. The single-stranded DNA polynucleotide is purified to a degree of purity greater than about 70, 80 or 90%, e.g., greater than about 95 or 99%, when assessed by w / w (dry weight). Such purity levels may include degradation products of the single-stranded DNA polynucleotide.

[0124] For example, the products of the cleavage reaction may be separated by size using gel electrophoresis using agarose or polyacrylamide gels. The desired polynucleotides may then be isolated from the gel and further purified, if necessary, by methods known in the art. Other methods for purifying, isolating or separating the polynucleotides of the present invention utilize chromatography (e.g., HPLC, size exclusion, ion exchange, affinity, hydrophobic interaction, reverse phase) or capillary electrophoresis.

[0125] However, it will be appreciated that in some embodiments, e.g., "one-pot" embodiments, isolation, separation or purification of the ssDNA molecules is not required or desirable. Thus, in some embodiments, the ssDNA molecules are not isolated, separated or purified prior to the step of hybridizing the ssDNA molecules.

[0126] The methods and uses provided herein utilize the complementary regions between ssDNA molecules in each of the plurality of ssDNA molecules to form a DNA complex.Each ssDNA molecule of the plurality of ssDNA molecules (i.e., the plurality of ssDNA molecules that provide all parts of the strand of a dsDNA molecule) comprises a region of complementarity with at least one ssDNA molecule of the other plurality of ssDNA molecules.The region of complementarity generally comprises the "terminal region" of the ssDNA molecule, i.e., the region of the ssDNA molecule that comprises the 5' or 3' end.

[0127] Of course, the terminal ssDNA molecule of one strand (i.e., the ssDNA molecule that includes the 5' or 3' of a strand of the dsDNA molecule) will have a sequence that is complementary to only one of the ssDNA molecules of the other strand. However, the inner ssDNA molecule (i.e., the ssDNA molecule that does not include the 5' or 3' of a strand of the dsDNA molecule), and the terminal ssDNA molecule of the other strand, will have two regions of complementarity, i.e., a first region that is completely complementary to a terminal region of the ssDNA molecule of the opposite strand and a second region that is completely complementary to a terminal region of a different ssDNA molecule of the opposite strand.

[0128] The two regions of complementarity of an ssDNA molecule (e.g., an inner ssDNA molecule) may be directly adjacent to each other, such that there is no intervening nucleotide sequence between the regions (e.g., as in a complete overlap strategy). This means that the 5' and 3' ends of the ssDNA molecules of opposite strands that hybridize to the ssDNA molecule are directly adjacent to each other, i.e., there is no gap between the 5' and 3' ends.

[0129] However, in other approaches, the two regions of complementarity of the ssDNA molecules (e.g., inner ssDNA molecules) may be indirectly adjacent to each other, such that there is an intervening nucleotide sequence between the regions (e.g., as in the partial overlap approach). This means that the 5' and 3' ends of the ssDNA molecules of opposite strands that hybridize 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 are ligated.

[0130] The ssDNA molecules of each of the plurality of ssDNA molecules are designed such that the region of complementarity hybridizes with the adjacent ssDNA molecules of the opposite strand, so that when the ssDNA molecules are ligated, the portions of each strand are joined in the correct order to generate a dsDNA molecule. As described above, the ssDNA molecules may hybridize with the ssDNA molecules of the opposite strand in a direct abutment (without a gap) or indirect abutment (i.e., with a gap). The directly abutting ssDNA molecules may be considered to form a junction (e.g., a nick site) between the two molecules, so that the molecules can be joined in a ligation reaction involving a ligase.

[0131] The term "region" refers to a portion of a ssDNA molecule.

[0132] As stated above, it is preferred that the region of a ssDNA molecule that hybridizes to a ssDNA molecule of the opposite strand is perfectly complementary as defined above.

[0133] Without wishing to be bound by theory, it is hypothesized that the size of the region of complementarity 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, e.g., 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 50-750, 60-700, 70-650, 80-600, 90-550 or 100-500 nucleotides, e.g., 150-500, 200-500 or 250-500 nucleotides.

[0134] The ssDNA molecules are provided in equimolar (i.e., stoichiometric 1:1:1, etc.) amounts, so that there are approximately the same number of each ssDNA molecule. This is believed to reduce the formation of undesirable dsDNA complexes. Thus, when different types of ssDNA molecules (e.g., sequence variants of ssDNA molecules) are provided, the total concentration of the different types is equimolar to the concentration of the other ssDNA molecules, i.e., each type of ssDNA molecule is provided at a lower concentration than ssDNA molecules where only one type is provided.

[0135] The ssDNA molecules may be prepared by any suitable method or means. For example, each of the multiple ssDNA molecules may be combined separately to obtain two separate mixtures of ssDNA molecules (i.e., one for each strand), which are then combined, i.e., combined to allow hybridization to occur. Alternatively, all ssDNA molecules may be combined as a single mixture in any order and under any suitable conditions, e.g., conditions suitable for maintaining the integrity of the ssDNA molecules, i.e., conditions that avoid degradation of the ssDNA molecules, e.g., in a buffer, e.g., 20-100 mM Tris-HCl, pH 7-9, optionally containing a chelating agent such as EDTA or EGTA.

[0136] Thus, the methods and uses provided herein may include a step of combining or mixing ssDNA molecules, i.e., to obtain a mixture of ssDNA molecules. However, as described above, the ssDNA molecules may be produced as a single reaction mixture, and in such an embodiment, a step of combining or mixing ssDNA molecules is not required.

[0137] The method and use involves hybridizing ssDNA molecules that contain regions of complementarity to generate a dsDNA complex that includes all the ssDNA molecules. This step can be seen as assembling the dsDNA complex. The hybridizing step involves subjecting the mixture of ssDNA molecules to annealing conditions. It will be apparent that the method and use provided herein involves a single hybridization step followed by a single ligation step, i.e., all ssDNA molecules that form the dsDNA molecule are combined or mixed (present in a mixture) prior to hybridization and ligation. Thus, the method and use does not involve sequential hybridization and / or sequential ligation steps or multiple hybridization and / or ligation steps, e.g., a first subset of ssDNA molecules are hybridized and ligated to form a partially double-stranded DNA molecule that is used for the next hybridization and ligation step to form a complete dsDNA molecule.

[0138] The term "annealing conditions" refers to the conditions under which two nucleic acid molecules that contain complementary nucleotide sequences specifically hybridize with each other.Various parameters affect hybridization, including temperature, salt concentration, nucleic acid concentration, composition and length, and buffer composition.Those skilled in the art can easily determine the annealing conditions that are suitable for a particular combination of ssDNA molecules as a matter of routine.

[0139] In a representative example, the step of hybridizing the ssDNA molecules to generate a dsDNA complex includes subjecting the mixture of ssDNA molecules (i.e., all ssDNA molecules) to a temperature suitable for denaturing the DNA molecules, e.g., heating the mixture to at least 94°C, e.g., 94-100°C (e.g., 94, 95, 96, 97, 98 or 99°C), and then cooling the mixture to a temperature that allows the ssDNA molecules to specifically hybridize to each other, e.g., cooling the mixture to less than 40°C, e.g., 4-40°C (e.g., 10-35, 15-30, 20-25°C, e.g., room temperature). As mentioned above, the methods and uses provided herein advantageously use long regions of complementarity. Thus, it will be appreciated that ssDNA molecules can hybridize to each other at relatively high temperatures, e.g., as high as about 85°C. Thus, cooling the mixture to a temperature that allows the ssDNA molecules to specifically hybridize to one another may include cooling the mixture to less than about 90°C, e.g., about 40-90°C (e.g., about 50-89°C, 60-88°C, 65-87°C, 70-86°C, or 75-85°C). For subsequent extension and / or ligation steps, a further cooling step may be required, i.e., to a temperature at which the polymerase and / or ligation enzyme is active (e.g., to avoid denaturing the enzyme(s)). However, if the extension and / or ligation steps use a thermostable polymerase and / or ligase, e.g., Taq polymerase and / or Taq ligase (or a functional variant thereof), a further cooling step may not be necessary.

[0140] Thus, the methods and uses provided herein may involve heating and cooling ssDNA molecules (i.e. a mixture of ssDNA molecules) to allow hybridization of ssDNA molecules containing regions of complementarity, thereby generating a dsDNA complex.

[0141] The terms "hybridization" or "hybridize," as used herein, refer to the formation of a duplex between nucleotide sequences that are sufficiently complementary to form a duplex by Watson-Crick base pairing.

[0142] The term "dsDNA complex" refers to a dsDNA molecule in which each strand is composed of two or more ssDNAs. Alternatively, it refers to multiple ssDNA molecules that are non-covalently linked (i.e., hybridized) to form a dsDNA molecule in which the phosphodiester backbone of one or both strands is not fully bound, i.e., one or both strands contain two or more 5' and 3' ends available for reaction.

[0143] A dsDNA complex can be viewed as "fully double-stranded" or "fully double-stranded" when all ssDNA molecules in each strand are in direct contact with one another, i.e., the ssDNA molecules of each strand form ligatable junctions. Thus, a fully double-stranded DNA complex can be viewed as a "nicked dsDNA," i.e., containing one or more breaks in the phosphodiester backbone.

[0144] A dsDNA complex can be viewed as a "partially double-stranded" DNA complex when two or more ssDNA molecules of the strands are indirectly adjacent to each other, i.e., the ssDNA molecules of at least one strand form a gap.

[0145] Thus, the step of hybridizing ssDNA molecules may result in a fully double-stranded DNA complex (e.g., a completely overlapping strategy) or a partially double-stranded DNA complex (e.g., a partially or minimally overlapping strategy). The step of hybridizing ssDNA molecules may be viewed as a step of assembling a dsDNA complex.

[0146] It will be apparent that when ssDNA molecules are non-covalently associated (i.e., hybridized) with their complementary regions in a dsDNA complex, they are no longer entirely single-stranded (they may be partially single-stranded). However, in the context of the methods and uses provided herein, it is convenient to refer to them as ssDNA molecules on the basis that they will form separate ssDNA portions of the dsDNA molecule under denaturing conditions.

[0147] If the step of hybridizing the ssDNA molecules results in the formation (i.e., assembly) of a partially double-stranded DNA complex, the gaps between the ssDNA molecules must be "filled" (to generate a fully double-stranded DNA complex) before the ssDNA molecules are ligated, i.e., joined in a ligation reaction involving a ligase.

[0148] The gap between the ssDNA molecules in the partially double-stranded DNA complex is "filled" by a polymerase enzyme using the ssDNA molecule of the opposite strand as a template. Thus, the method provided herein may include a step of extending the 3' end of the ssDNA molecule to generate a fully double-stranded DNA complex. Of course, the extension step involves the extension of the inner 3' end of the ssDNA molecule, i.e., 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. The nucleotide sequences of the ssDNA molecules opposite the gap may be different, i.e., by providing multiple types of ssDNA molecules in the reaction mixture, so that the extension reaction involving a polymerase can facilitate the generation of a mixture of dsDNA molecules (i.e., containing different sequences).

[0149] Any suitable DNA polymerase may be used for the extension step and, as a matter of routine, can be readily selected by the skilled artisan. In this regard, it will be clear that the polymerase enzyme should not have strand displacement or 5' exonuclease activity in order to avoid displacement or degradation of the ssDNA molecule of the partially double-stranded DNA complex downstream of (3' to) the ssDNA molecule providing the 5' end of the dsDNA molecule. For example, the DNA polymerase may be T4 DNA polymerase, Klenow fragment of DNA polymerase I, Taq polymerase or sequence modified derivatives, or variants thereof. The polymerase may be a thermostable polymerase and / or a high fidelity polymerase, for example Q5® High Fidelity DNA Polymerase (NEB). Thus, the extension step involving the polymerase may be carried out at a relatively high temperature, such as between 55-80°C, for example 60-75°C or 65-75°C. This may involve increasing the temperature of the reaction mixture containing the dsDNA complex, i.e., a heating step after the hybridization step (i.e., after assembly of the dsDNA complex). The extension step may be performed simultaneously with the hybridization step.

[0150] Sequence modified derivatives or mutants of DNA polymerase enzymes include mutants that retain at least some of the functional activity of the wild-type sequence, e.g., DNA polymerase activity. Mutations may affect the activity profile of the enzyme under various reaction conditions, e.g., temperature, template concentration, primer concentration, etc., e.g., increase or decrease the rate of polymerization. Mutations or sequence modifications may also affect the exonuclease activity and / or thermostability of the enzyme.

[0151] After formation of the fully double-stranded DNA complex, i.e., after the hybridizing and (if applicable) extending steps, the ssDNA molecules are ligated (synthesized, formed, etc.) to produce a dsDNA molecule. Viewed another way, the hybridizing and (if applicable) extending steps form a ligatable junction, where the ssDNA molecules are in direct contact with each other and are held in place by regions of complementarity on the ssDNA molecules of the opposite strand. The step of ligating adjacent ssDNA molecules may be accomplished by any suitable enzymatic means, i.e., by the addition of a ligase enzyme under suitable conditions. As described further below, the ligase activity (e.g., ligase enzyme) may be obtained in a host cell containing the dsDNA complex, i.e., when the dsDNA complex is introduced into a host cell (e.g., a bacterial cell) along with a linearized nucleic acid vector (e.g., a plasmid).

[0152] Thus, the methods and uses provided herein do not involve chemical ligation, in particular click chemistry, the coupling of adjacent nucleotides by CuAAC reaction between adjacent terminal cytidine and thymidine residues resulting in 1,4-linked 1,2,3-triazoles.However, as described elsewhere herein, the dsDNA molecules generated by the present methods may be subjected to click chemistry reactions in a subsequent step.

[0153] As known in the art, ligase catalyzes the formation of a phosphodiester bond between the juxtaposed 3'-hydroxyl and 5'-phosphate ends of two directly adjacent nucleic acids. Thus, a ligase can be viewed as any enzyme capable of catalyzing this reaction. Any convenient ligase may be utilized, and representative ligases of interest include, but are not limited to, temperature-sensitive 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 may be obtained from thermophilic or hyperthermophilic microorganisms, including, but not limited to, prokaryotes, eukaryotes, or archaea. Enzymes suitable for the ligation step are known in the art and include, for example, T4 DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermococcus species (strain 9°N) DNA ligase (9°N™ DNA Ligase, New England Biolabs), and Ampligase™ (Epicentre Biotechnologies).

[0154] 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 for ligation of the junctions in the resulting dsDNA complex. As further described below, the ligation reaction may be carried out in a host cell, for example, a bacterial cell. Ligation reaction conditions are well known to those skilled in the art. During ligation, the reaction mixture may be maintained at a temperature ranging from about 4°C to about 50°C, for example, from about 20°C to about 37°C, for a time ranging from about 5 seconds to about 16 hours, for example, from about 1 minute to about 1 hour, in certain embodiments. In yet another embodiment, the reaction mixture may be maintained at a temperature ranging from about 35°C to about 75°C, for example, from about 37°C to about 75°C, for example, or about 40°C, 50°C, 60°C, 70°C or 75°C, for a time ranging from about 5 seconds to about 16 hours, for example, from about 1 minute to about 1 hour, including about 2 minutes to about 8 hours. In a representative example, the ligation reaction mixture is 50 mM Tris, pH 7.5, 10 mM MgCl 2 , 10 mM DTT, 1 mM ATP, 25 mg / ml BSA, and 0.125 units / ml T4 DNA ligase. Yet another representative example utilizes 2.125 mM magnesium ion, and 0.125 units / ml DNA ligase.

[0155] As mentioned above, the ligation step generally synthesizes a linear dsDNA molecule, although the ligation step may also synthesize a circular dsDNA molecule containing the desired dsDNA or target dsDNA molecule if the dsDNA complex is contacted with a suitable linearized nucleic acid vector (e.g., a plasmid) prior to ligation.

[0156] The dsDNA molecules resulting from the methods and uses provided herein provide a further aspect of the invention.

[0157] Of course, the dsDNA molecules may be subjected to further processing steps after synthesis, for example to isolate, modify and / or amplify the dsDNA molecules.

[0158] The ligation step of the methods and uses provided herein may not be completely efficient, i.e., there may be some unreacted (unligated) ssDNA molecules and / or undesired by-products.Therefore, it may be desirable to separate dsDNA molecules from unreacted components and / or other components in the reaction mixture, such as ligase.Any suitable means may be used to separate dsDNA molecules from other components in the reaction mixture.

[0159] Therefore, the method may include the further step of separating or purifying dsDNA molecules.For example, the product of the ligation reaction may be separated by size using gel electrophoresis using agarose gel or polyacrylamide gel.The desired dsDNA molecules may then be isolated from the gel and, if necessary, further purified according to methods known in the art.Other methods for purifying, isolating or separating dsDNA molecules utilize chromatography (e.g., HPLC, size exclusion, ion exchange, affinity, hydrophobic interaction, reverse phase) or capillary electrophoresis.

[0160] As mentioned above, the dsDNA molecules produced by the above methods may contain another chemical group, e.g., a reactive group that can react with a chemical group on a molecule or component that is attached to the dsDNA molecule, e.g., by click chemistry. For example, attaching an additional molecule or component (which may itself contain a functional group or be viewed as 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 method if present in the ssDNA molecule.

[0161] Thus, the method may include the step of attaching a molecule or moiety to the dsDNA molecule via a functional (eg reactive) group in the dsDNA molecule, such as by click chemistry.

[0162] Viewed alternatively, the method may include the step of modifying the dsDNA molecule, for example to include a functional group, such as a label.

[0163] Thus, the methods and uses provided herein can result in a mixture of dsDNA molecules, i.e., a library of dsDNA molecules comprising a plurality of different (e.g., functionalized or modified) dsDNA molecules.

[0164] As mentioned above, a mixture of dsDNA molecules may be achieved by providing one or more ssDNA molecules of multiple types. The types of ssDNA molecules may have the same sequence, but may differ with respect to the nucleotides that contain functional groups (e.g., the position and / or number of functional groups). Additionally or alternatively, the types of ssDNA molecules may have the same sequence, but may differ with respect to the type of functional group on the modified nucleotide. For example, each type of ssDNA molecule may contain multiple functional groups.

[0165] It will also be apparent to one skilled in the art that a mixture of dsDNA molecules may be achieved by providing one or more ssDNA molecules of multiple types, where the types of ssDNA molecules may have different sequences (i.e., the types may be sequence variants). Such sequence variants may be achieved by using any suitable means known in the art. For example, site-specific variants may be generated separately and mixed to obtain multiple types of ssDNA molecules. In a further representative example, sequence changes can be randomly incorporated when enzymatically synthesizing ssDNA molecules, for example, by performing the synthesis reaction in the presence of high salt concentration or manganese ions to induce mutations. The product of the synthesis reaction will be a mixture of ssDNA molecules that includes multiple types of ssDNA molecules (i.e., sequence variants of ssDNA molecules). As described above, when the multiple types of ssDNA molecules are sequence variants, each ssDNA molecule of the multiple will include a common region of complementarity.

[0166] Alternatively, a mixture of dsDNA molecules may be achieved by carrying out an extension reaction in the presence of high salt or manganese ions to introduce mutations into the synthesized strand, which may then be incorporated into the opposite strand in a subsequent processing step.

[0167] The method may also include amplifying the dsDNA molecule. Methods for amplifying dsDNA molecules are well known in the art, and any suitable method may be utilized. However, as described above, PCR-based methods may incorporate errors (mutations) into the dsDNA product. Therefore, it may be preferable to use a method other than a PCR-based method to amplify the dsDNA molecule. Alternatively, when using a PCR-based method to amplify the dsDNA molecule, the method preferably uses a high-fidelity polymerase as defined above.

[0168] The method may involve amplifying dsDNA using a host cell. For example, the method may comprise: (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) for example, isolating or purifying the dsDNA molecule from the vector nucleic acid. Step (ii) may include transfecting the vector (e.g., a DNA plasmid) into bacteria and propagating the bacteria.

[0169] Suitable plasmid sequences are well known in the art. In particular, inserting dsDNA into a nucleic acid vector and amplifying the vector allows the sequence of the dsDNA to be checked (e.g., by sequencing), for example by repeated sequencing and mutagenesis using any suitable method, and allows any errors in the sequence to be corrected. Thus, the method may include, for example, after the above steps (i), (ii) or (iii), checking the sequence of the dsDNA molecule, for example, sequencing the dsDNA molecule. The method may also include a step of correcting one or more errors in the sequence of the dsDNA molecule, and then the corrected dsDNA may be amplified, for example, by the above methods, i.e., the above steps (ii) to (iv).

[0170] Insertion of a dsDNA molecule into a plasmid also facilitates the formation of a significant number of copies of the dsDNA molecule. For example, a single bacterial colony containing a plasmid containing a sequence-verified dsDNA molecule may be grown to amplify the plasmid, which may then be purified from the bacteria using any suitable means known in the art.

[0171] dsDNA molecule can be excised from plasmid, for example, by using restriction enzyme as described above.Excised linear dsDNA molecule can be purified by PAGE and gel extraction, or other suitable method.Purified dsDNA molecule can then be used as desired.

[0172] In addition to amplification, the process of transfecting dsDNA containing plasmids into bacteria also allows for the generation of glycerol stocks of bacteria. Bacteria containing the desired dsDNA molecule plasmid can be prepared in glycerol, frozen, and stably stored for long periods of time.

[0173] The inventors have found that the dsDNA complex formed in the hybridization step is very stable. Therefore, when it is desired to insert the DNA molecule into a nucleic acid vector (e.g., a plasmid), the ligation step and the insertion step can advantageously be carried out in a single reaction. Thus, the ligation step may involve contacting (mixing) the dsDNA complex with a nucleic acid vector (e.g., a linearized plasmid) and a means for linking polynucleotides (e.g., a ligase) under conditions suitable for forming a dsDNA molecule and linking it to the nucleic acid vector. This step may also have an additional advantage because any single-strand breaks (i.e., nicks) in the vector are repaired when the vector is introduced into a host cell.

[0174] One of skill in the art will appreciate that the ligation step can be accomplished or performed in a host cell (see, e.g., Examples 1, 6, and 7). In this regard, the ligation step may 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) under conditions suitable for the host cell to take up the assembled dsDNA complex:nucleic acid vector, and ligase activity in the host cell will join the molecules to form a complete nucleic acid vector containing the target dsDNA molecule. In another view, when the assembled dsDNA complex:nucleic acid vector is introduced into a host cell, the host cell will repair the single-strand breaks (i.e., nicks) in the dsDNA complex and the vector.

[0175] It will be apparent that any suitable host cell may be used, e.g., a host cell that contains an enzyme having ligase activity, e.g., a ligase enzyme. In a typical example, the host cell is a bacterial host cell, e.g., an E. coli cell.

[0176] The stability of the dsDNA complex also facilitates the use of a thermostable polymerase to perform the extension step after assembly of the dsDNA complex; i.e., a 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 optimal for the polymerase activity of the enzyme, e.g., a temperature higher than the temperature to which the reaction mixture was cooled during the hybridization step.

[0177] Thus, in a representative example, a method is provided herein for generating a double-stranded DNA (dsDNA) molecule comprising at least 3000 bp (e.g., at least 4000 bp or 5000 bp), the method comprising: (a) (i) at least three (e.g., at least four or five) ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5' end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3' end of the first strand of the dsDNA molecule; and (ii) at least four (e.g., at least five or six) ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a second strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the second ssDNA molecule in (i), and a second ssDNA molecule comprises a 3'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule in (i). and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce an entirely double-stranded DNA complex containing all ssDNA molecules from (a); (c) ligating immediately adjacent ssDNA molecules in the fully double-stranded DNA complex to generate a dsDNA molecule; Including, at least two (e.g., at least three or four) of the at least four (e.g., at least five or six) ssDNA molecules from (1)(ii) that do not include the 5' and 3' ends of the second strand of the dsDNA molecule 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 a different ssDNA molecule in (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms an immediately adjacent portion of a first strand of a dsDNA molecule; (3) the complementary region comprises at least 150 (e.g., at least 200 or 250) nucleotides; (4) all ssDNA molecules contain at least 400 (e.g., at least 500) nucleotides, and optionally all ssDNA molecules from (i) and ssDNA molecules from (ii) not including the 5' and 3' ends of the second strand of the dsDNA molecule contain at least 800 (e.g., at least 900 or 1000) nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts.

[0178] In a further representative example, provided herein is a method for generating a double-stranded DNA (dsDNA) molecule comprising at least 3000 bp (e.g., at least 4000 bp or 5000 bp), the method comprising: (a) (i) at least three (e.g., at least four or five) ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5' end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3' end of the first strand of the dsDNA molecule; and (ii) at least four (e.g., at least five or six) ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a second strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the second ssDNA molecule in (i), and a second ssDNA molecule comprises a 3'-end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule in (i). and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce a partially double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) extending the 3' ends of the ssDNA molecules in the partially double-stranded DNA complex to generate a fully double-stranded DNA complex; (d) ligating immediately adjacent ssDNA molecules in the fully double-stranded DNA complex to form a dsDNA molecule; Including, at least two (e.g., at least three or four) of the at least four (e.g., at least five or six) ssDNA molecules from (1)(ii) that do not include the 5' and 3' ends of the second strand of the dsDNA molecule 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 a different ssDNA molecule in (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms an indirectly contiguous portion of a first strand of a dsDNA molecule, and the ssDNA molecule of (ii) to which the ssDNA molecule of (i) is complementary forms an indirectly contiguous portion of a second strand of a dsDNA molecule; (3) the complementary region comprises at least 150 (e.g., at least 200 or 250) nucleotides; (4) all ssDNA molecules contain at least 400 (e.g., at least 500) nucleotides, and optionally all ssDNA molecules from (i) and ssDNA molecules from (ii) not including the 5' and 3' ends of the second strand of the dsDNA molecule contain at least 800 (e.g., at least 900 or 1000) nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts.

[0179] In yet another representative example, provided herein is a method for generating a double-stranded DNA (dsDNA) molecule comprising at least 3000 bp (e.g., at least 4000 bp or 5000 bp), the method comprising: (a) (i) at least three (e.g., at least four or five) ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion of a first strand of a dsDNA molecule, wherein a first ssDNA molecule comprises a 5' end of the first strand of the dsDNA molecule and a second ssDNA molecule comprises a 3' end of the first strand of the dsDNA molecule; and (ii) at least three (e.g., at least four or five) ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion 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 and is completely complementary to a region of the second ssDNA molecule of (i). and preparing (b) hybridizing ssDNA molecules containing regions of complementarity to produce a partially double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) extending the 3' ends of the ssDNA molecules of (ii) in the partially double-stranded DNA complex to produce a fully double-stranded DNA complex; (d) ligating immediately adjacent ssDNA molecules in the fully double-stranded DNA complex to generate a dsDNA molecule; Including, at least two (e.g., at least three or four) of the at least three (e.g., at least four or five) ssDNA molecules from (1)(ii) that do not include the 5' end of the second strand of the dsDNA molecule 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 a different ssDNA molecule in (i); (2) the ssDNA molecule (i) to which the ssDNA molecule (ii) is complementary forms a directly adjacent portion of a first strand of a dsDNA molecule, and the ssDNA molecule (ii) to which the ssDNA molecule (i) is complementary forms an indirectly adjacent portion of a second strand of a dsDNA molecule; (3) the complementary region comprises at least 50 (e.g., at least 75 or 100) nucleotides; (4) all of the ssDNA molecules of (i) contain at least 800 (e.g., at least 900 or 1000) nucleotides, and all of the ssDNA molecules of (ii) contain at least 150 (e.g., at least 175 or 200) nucleotides; (5) The ssDNA molecules are prepared in equimolar amounts. EXAMPLES

[0180] Example 1: Generation of dsDNA using various overlapping methods The synthetic gene (4000 bp) contains 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). Single-stranded polynucleotides containing non-overlapping portions (fragments) of the gene were designed by various strategies (see FIG. 1) and enzymatically synthesized using the RCA-based MOSIC method. Solution: 1) Complete overlap: The positive strand of the gene was divided into four parts (A, B, C, D) of 1000 nucleotides (nt) each, and the negative strand was divided into three parts of 1000 nt and two parts of 500 nt (E, F, G, H, I). The two parts of 500 nt (E, I) were placed at both ends, so that the 1000 nt long parts overlap each other by 500 nucleotides. 2) Partial overlap: The segments were designed to have an overlap of 250 nucleotides. The plus strand has three segments (A, J, D) of 1000 nucleotides, where A and D are the same as in the complete overlap strategy, and J extends from 1501 to 2500 nucleotides. The minus strand has segments E and I at both ends with two 1000 nt long segments (K, L) between them, so that the overlap between the 1000 segments is 250 bases long. 3) Minimal overlap: The plus strand is identical to the complete overlap strategy and uses segments A, B, C and D. The minus strand consists of four 200 nt long segments, three of which connect the 1000 nt segment with an overlap of 100 bases (M, N, O) and the fourth anneals 5' to the minus strand (P).

[0181] The ssDNA molecules (polynucleotides) were purified, mixed in stoichiometric amounts, and heated to 95°C for 10 minutes. The mixture was cooled from 95°C to room temperature. In the case of the partial and minimal overlap strategies, the gaps between the assembled fragments were sealed by polymerase-mediated extension using a high-fidelity thermostable polymerase (Q5® High-Fidelity DNA Polymerase (NEB)). The assembled fragments (see Figure 2) were ligated and inserted into bacterial plasmids either in separate ligation steps (using T4 DNA ligase) or in a single step (using Taq DNA ligase). E. coli DH5 alpha competent cells were transformed with the final ligated plasmid. Cells were selected with kanamycin, and plasmids were extracted from single colonies and tested first by restriction endonuclease digestion (Figure 3) and then by Sanger sequencing, which confirmed that the synthesized and amplified dsDNA molecules contained the correct nucleotide sequence.

[0182] Example 2 (Comparative): Attempted generation of dsDNA using one-pot assembly with short ssDNA molecules To prove that long ssDNA molecules with long regions of complementarity are essential for the overlap method of the present disclosure to work, comparative experiments were performed using a one-pot assembly method with short oligonucleotides (SEQ ID NOs: 18-117). Each oligonucleotide is 60 nt long, except for SEQ ID NO: 68, which is only 40 nt long like Segment I in FIG. 1B. In addition, the oligonucleotide containing the 3' end of the second strand (corresponding to Segment E in FIG. 1B) was omitted, on the basis that the penultimate oligonucleotide (corresponding to Segment K in FIG. 1B) can be extended using the 5'-most oligonucleotide of the first strand (corresponding to Segment A in FIG. 1B) to obtain the 3' end of the second strand of the desired dsDNA molecule. Similarly, the 3'-most oligonucleotide of the first strand (corresponding to Segment D in FIG. 1B) does not contain the 3' end of the desired dsDNA molecule, on the basis that the 3' end of the first strand of the desired dsDNA molecule can be obtained by extension of the 3'-most oligonucleotide of the first strand using as a template an oligonucleotide providing the 5' end of the second strand of the desired dsDNA.

[0183] Each oligonucleotide has at least one region of complementarity (overlap) of 20 bases to the oligonucleotide that forms the opposite strand. The arrangement of partially overlapping oligonucleotides is repeated along the entire length of the generated dsDNA (see FIG. 4A), i.e., using the partial overlap strategy as defined herein.

[0184] Two different concentrations of oligonucleotides were tested: 200 ng and 1000 ng. The oligonucleotides were mixed in PCR buffer and incubated using a temperature gradient (95° C. for 5 min, followed by a 1-h temperature ramp to 22° C. at a rate of 0.1° C. / s). Gaps between assembled fragments were sealed by polymerase-mediated extension using a high-fidelity thermostable polymerase (Q5® High-Fidelity DNA Polymerase (NEB)) with 1 mM nucleotides during a 15-min incubation at 72° C. as recommended by the manufacturer.

[0185] Half of the reaction samples were then loaded onto an agarose gel, followed by gel electrophoresis and ethidium bromide staining. No bands corresponding to assembly products (i.e., dsDNA complexes) were observed on the gel (see FIG. 4B). Combining the assembly mixture with linearized plasmids for ligase-mediated generation of plasmids containing the desired dsDNA molecules and subsequent transformation of E. coli strains did not yield any colonies.

[0186] These results indicate that short oligonucleotides, i.e., oligonucleotides containing less than 150 nt, with short overlaps, i.e., overlaps of less than 50 nt, cannot be used in the methods disclosed herein to generate long dsDNA molecules, i.e., dsDNA molecules containing at least 1500 bp.

[0187] Example 3 (Comparative): Use of the minimal overlap strategy with oligonucleotides having short regions of complementarity (overlap) For this comparative experiment, we attempted 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: 118-121) to form the second strand. With the exception of the oligonucleotide that is fully complementary to segment D and forms the 5' end of the second strand (corresponding to segment P in Figure 1C), the short oligonucleotides have a 25 nt complementary region with adjacent ssDNA molecules in the first strand (see Figure 5A).

[0188] Assembly was attempted with two different concentrations of DNA (sample 1 = 100 ng and sample 2 = 200 ng). Segments were mixed in PCR buffer and incubated using a temperature gradient (95°C for 5 min, followed by a 1 h temperature decrease to 22°C at a rate of 0.1°C / s). When using the minimal overlap strategy, gaps between assembled fragments were sealed by polymerase-mediated extension using a high-fidelity thermostable polymerase (Q5® High-Fidelity DNA Polymerase (NEB)) with 1 mM nucleotides during a 15 min incubation at 72°C.

[0189] Half of the reaction sample was loaded onto an agarose gel, followed by gel electrophoresis and ethidium bromide staining, after which no band corresponding to the assembly product (dsDNA complex) was observed on the gel (Figure 5B).

[0190] Example 4: Use of a single long oligonucleotide mutation for the assembly of a degenerate DNA library As described elsewhere herein, the methods disclosed herein allow for the assembly of denatured DNA libraries, where the denatured regions may be site-specific to long DNA sequences. To demonstrate this, denatured ssDNA (1 kb long) was synthesized using the MOSIC method described herein for assembly in a desired dsDNA molecule.

[0191] Three RCA reactions using phi29 DNA polymerase, 1 mM dNTPs, and different concentrations of magnesium and manganese (see Table 1 for concentrations used in each reaction) were performed at 30° C. for 16 hours using the nicked circular dsDNA template encoding segment B described in Example 1. After inactivation at 70° C., the RCA products were digested using BtsCI restriction enzyme (to remove the hairpin and release the ssDNA product). The digestion products were run on an agarose gel (see FIG. 6) after heat inactivation at 80° C., and analyzed by Illumina sequencing.

[0192] Sequencing showed that the error rate increased as the concentration of manganese increased.

[0193] Denatured ssDNA molecules generated using this method can be used in the methods disclosed herein to generate libraries of denatured dsDNA molecules.

[0194] [Table 1]

[0195] Example 5: Assembly of ssDNA molecules containing modified nucleotides The methods disclosed herein allow for the assembly of dsDNA molecules containing site-specific modifications. To demonstrate this, a single modified segment (ssDNA molecule) was generated as previously described (WO 2020 / 161187). The modification selected was 5-methyl-2'-deoxycytidine-5'-triphosphate (5-methyl-dCTP).

[0196] Segments C, D and H were synthesized in a single tube using the RCA and BtsCl digestion procedures described in Example 4. Each segment was generated using two different RCA conditions with native dCTP substituted 50% or 100% with 5-methyl-dCTP.

[0197] After desalting the generated ssDNA molecules using Nucleospin, the segments were assembled in different combinations using 50 ng of each segment in each reaction as follows: 1) segments C, D and H containing 50% 5-methyl-dCTP; 2) segments D and H containing 50% 5-methyl-dCTP; 3) segments C, D and H containing 100% 5-methyl-dCTP; 4) Segments D and H containing 100% 5-methyl-dCTP.

[0198] The segments were then mixed in PCR buffer and incubated using a temperature gradient (95°C for 5 min, followed by a temperature decrease at a rate of 0.1°C / s to 22°C for 1 h) to form dsDNA complexes.

[0199] The samples were then loaded onto an agarose gel followed by gel electrophoresis and ethidium bromide staining. A negative control (part H only) was also loaded onto the gel.

[0200] Bands corresponding to assemblies 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).

[0201] This demonstrates that ssDNA molecules containing modified nucleotides can be used in the methods disclosed herein to generate dsDNA molecules that contain site-specific modifications.

[0202] Example 6: Separate synthesis of plus- and minus-strand forming ssDNA molecules and one-pot assembly of the desired dsDNA molecule The ssDNA molecules that form the plus and minus strands for the assembly of dsDNA molecules using the methods disclosed herein may be synthesized separately and then assembled into dsDNA molecules by one-pot assembly. It is advantageous to minimize the number of synthesis reactions required to generate a ssDNA molecule, since this will simplify the assembly process.

[0203] To see if it was possible to reduce the number of synthesis steps, the desired dsDNA molecules were synthesized using a minimal overlap strategy, where ssDNA molecules for each strand were synthesized together, i.e., in a synthesis pool. The ssDNA molecules were synthesized using the MOSIC method described herein.

[0204] Four nicked circular templates, each encoding a single segment, were mixed to generate segments A, B, C, and D in a single tube (i.e., segments A-D were generated from separate circular templates). One circular template encoding M, N, O, and P was used to generate segments M, N, O, and P in a second one-pot RCA reaction.

[0205] In both cases, RCA was performed with phi29 DNA polymerase at 30°C for 16 h, and the RCA products were digested with BtsCl at 50°C for 4 h to release the ssDNA segments from the cutter hairpins. After incubation at 70°C for 15 min to inactivate the polymerase and desalting the digestion mixture with Nucleospin, the ssDNA pool was used in two different amounts (sample 1 = 100 ng and sample 2 = 200 ng).

[0206] Segments for both strands were resuspended in PCR buffer in a single tube and incubated using a temperature gradient (95° C. for 5 min, followed by a 1-h temperature ramp to 22° C. at a rate of 0.1° C. / s) to form dsDNA complexes. When using the minimal overlap strategy, gaps between assembled segments were sealed by polymerase-mediated extension using a high-fidelity thermostable polymerase (Q5® High-Fidelity DNA Polymerase (NEB)) with 1 mM nucleotides during a 15-min incubation at 72° C. as recommended by the manufacturer.

[0207] Half of the sample reactions were loaded onto an agarose gel, followed by gel electrophoresis and ethidium bromide staining. The bands on the gel indicate that different states of assembly were recognizable, i.e., various dsDNA complexes were formed (see Figure 8A). Both assembly mixtures (samples 1 and 2) were mixed with linearized plasmid and transformed into E. coli, and many colonies grew overnight (see Figure 8B), indicating that the ligation step had occurred within the bacterial cells. Eight random colonies were selected, from which plasmids were extracted and digested with Ndel to confirm the presence of the complete insert.

[0208] All digestion mixtures yielded digestion patterns on agarose gels corresponding to fully assembled dsDNA molecules (Figure 8C). Sanger sequencing showed that 100% correct sequences for each insert were derived from the selected colonies.

[0209] Example 7: One-pot synthesis and assembly using a minimal overlap strategy A one-pot method for the synthesis and assembly of long ssDNA molecules is particularly advantageous, as it would further simplify the method compared to Example 6, which involves separate synthesis of ssDNA molecules for each strand. The possibility of using one-pot assembly and synthesis was investigated using a minimal overlap strategy.

[0210] All ssDNA molecules (segments A, B, C, D, M, N, O and P) were synthesized in a single tube as described in Example 6. RCA was performed by incubating four nicked circular dsDNAs encoding segments A, B, C and D, and one nicked circular dsDNA template encoding segments M, N, O and P with phi29 DNA polymerase and dNTPs at 30° C. for 16 hours.

[0211] After heat inactivation, the RCA products were digested using BtsCl as previously described to obtain single ssDNA molecules. Even after digestion, partial assembly of the sequences could be observed using an agarose gel followed by gel electrophoresis and ethidium bromide staining (Figure 9A).

[0212] After desalting with Nucleospin, the digestion mixture was resuspended in PCR buffer and incubated using a temperature gradient (95°C for 5 min, followed by a 1-h temperature decrease to 22°C at a rate of 0.1°C / s) for assembly to form dsDNA complexes. Since a minimal overlap strategy was used, the gaps between the assembled fragments were sealed by polymerase-mediated extension using a high-fidelity thermostable polymerase (Q5® High-Fidelity DNA Polymerase (NEB)) with 1 mM nucleotides during a 15-min incubation at 72°C as recommended by the manufacturer. Other polymerases such as Phusion DNA polymerase or Taq polymerase were tested with the same results.

[0213] The resulting dsDNA complex was mixed with the linearized plasmid and transformed into E. coli, and many colonies grew on the selection plates (Figure 9B). This indicates that the ligation step occurred inside the bacterial cells. Two colonies were selected, from which the plasmid was extracted and digested with Ndel. The digested plasmid samples were applied to an agarose gel, followed by gel electrophoresis and ethidium bromide staining, which showed that the plasmids formed the correct bands (0.45 kbp and 4.8 kbp, upper and lower arrows, respectively, in Figure 9C). This confirmed that the assembly strategy worked correctly. Sanger sequencing also confirmed that both inserts were 100% correct.

[0214] This experiment demonstrates that the methods disclosed herein can advantageously and surprisingly be carried out in a one-pot reaction involving both the synthesis of ssDNA molecules and their subsequent assembly into the desired dsDNA molecules.

Claims

1. 1. A method for generating double-stranded DNA (dsDNA) molecules comprising at least 1500 bp, said method comprising: (a) (i) a plurality of single-stranded DNA (ssDNA) molecules, each comprising (e.g., consisting of) a non-overlapping portion of a first strand of the dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the first strand of the dsDNA molecule, and a second ssDNA molecule (a) comprises a 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 an ssDNA molecule comprising the 5'-end of a second strand as a template; and (ii) a plurality of ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion 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 molecule comprises the 3' end of the second strand of the dsDNA molecule. and preparing a (b) hybridizing the ssDNA molecules containing regions of complementarity to produce a dsDNA complex containing all of the ssDNA molecules from (a); (c) joining adjacent ssDNA molecules using a ligase enzyme to generate said dsDNA molecule; Including, (i) at least two of the plurality of ssDNA molecules from (ii) each comprising a first region that is completely complementary to a terminal region of an ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms a contiguous portion of the first strand of the dsDNA molecule; (3) the region of complementarity comprises at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; (5) The method, wherein the ssDNA molecules are provided in equimolar amounts.

2. The method comprises: (a) (i) at least three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion 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 comprising (e.g., consisting of) a non-overlapping portion 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 a region of the second ssDNA molecule in (i), and a second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule in (i). and preparing a (b) hybridizing the ssDNA molecules containing regions of complementarity to form an entirely double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) joining directly adjacent ssDNA molecules using a ligase enzyme to generate said dsDNA molecule; Including, (1) At least two of the at least four ssDNA molecules from (ii) that do not include the 5' and 3' ends of the second strand of the dsDNA molecule each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms an immediately adjacent portion of the first strand of the dsDNA molecule; (3) the region of complementarity comprises at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are provided in equimolar amounts; 10. A method for producing a double-stranded DNA (dsDNA) molecule according to claim 1.

3. The method comprises: (a) (i) at least three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion 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 comprising (e.g., consisting of) a non-overlapping portion 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 a region of the second ssDNA molecule in (i), and a second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule in (i). and preparing a (b) hybridizing the ssDNA molecules containing regions of complementarity to produce a partially double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) extending the 3' ends of the ssDNA molecules to generate fully double-stranded DNA complexes; (d) joining directly adjacent ssDNA molecules using a ligase enzyme to generate said dsDNA molecule; Including, (1) At least two of the at least four ssDNA molecules from (ii) that do not include the 5' and 3' ends of the second strand of the dsDNA molecule each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms an indirectly adjacent portion of the first strand of the dsDNA molecule, and the ssDNA molecule of (ii) to which the ssDNA molecule of (i) is complementary forms an indirectly adjacent portion of the second strand of the dsDNA molecule; (3) the region of complementarity comprises at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are provided in equimolar amounts; 10. A method for producing a double-stranded DNA (dsDNA) molecule according to claim 1.

4. The method comprises: (a) (i) at least three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion 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 three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion 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 a region of the second ssDNA molecule of (i). and preparing a (b) hybridizing the ssDNA molecules containing regions of complementarity to produce a partially double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) extending the 3' ends of the ssDNA molecules of (ii) to generate fully double-stranded DNA complexes; (d) joining directly adjacent ssDNA molecules using a ligase enzyme to generate said dsDNA molecule; Including, (i) at least two of the at least three ssDNA molecules from (ii) that do not include the 5' end of the second strand of the dsDNA molecule each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms a directly adjacent portion of the first strand of the dsDNA molecule, and the ssDNA molecule of (ii) to which the ssDNA molecule of (i) is complementary forms an indirectly adjacent portion of the second strand of the dsDNA molecule; (3) the region of complementarity comprises at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are provided in equimolar amounts; 10. A method for producing a double-stranded DNA (dsDNA) molecule according to claim 1.

5. The method comprises: (a) (i) at least three ssDNA molecules, each comprising (e.g., consisting of) a non-overlapping portion 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 either (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 an 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 comprising (e.g., consisting of) a non-overlapping portion 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 a region of the second ssDNA molecule in (i), and optionally a second ssDNA molecule comprises the 3' end of the second strand of the dsDNA molecule and is fully complementary to a region of the first ssDNA molecule in (i). and preparing a (b) hybridizing the ssDNA molecules containing regions of complementarity to produce a partially double-stranded DNA complex containing all of the ssDNA molecules from (a); (c) extending the 3' ends of the ssDNA molecules to generate fully double-stranded DNA complexes; (d) joining directly adjacent ssDNA molecules using a ligase enzyme to generate said dsDNA molecule; Including, (1) at least two of the at least three ssDNA molecules from (ii) (e.g., at least two of the at least four) that do not include the 5' and 3' ends of the second strand of the dsDNA molecule each comprise a first region that is completely complementary to a terminal region of the ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms an indirectly adjacent portion of the first strand of the dsDNA molecule, and the ssDNA molecule of (ii) to which the ssDNA molecule of (i) is complementary forms an indirectly adjacent portion of the second strand of the dsDNA molecule; (3) the region of complementarity comprises at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; (5) The ssDNA molecules are provided in equimolar amounts; 10. A method for producing a double-stranded DNA (dsDNA) molecule according to claim 1.

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

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

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

9. 6. The method of any one of claims 1 to 5, wherein the region of complementarity comprises at least 100 nucleotides, optionally at least 150, 200, 250, 300, 400 or 500 nucleotides.

10. 6. The method of any one of claims 1 to 5, wherein the dsDNA molecule comprises at least 2000 bp, 2500 bp, 3000 bp, 3500 bp or 4000 bp.

11. 6. The method of any one of claims 1 to 5, wherein the dsDNA molecule comprises 1500 to 50000 bp.

12. 6. The method of claim 1, wherein the ssDNA molecule is produced using enzymatic synthesis.

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

14. One or more (preferably all) of the ssDNA molecules provided in (a) (a) providing a circular DNA molecule comprising a nucleotide sequence encoding said ssDNA molecule flanked by a cleavage domain; (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule of (a) as a template; (c) cleaving the product of the RCA reaction with the cleavage domain to release the ssDNA; The method of any one of claims 1 to 5, wherein the method is produced using a method comprising:

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

16. 15. The method of claim 14, wherein the circular DNA molecule comprises a plurality of nucleotide sequences encoding the ssDNA molecules provided in (a), each flanked by a cleavage domain.

17. 6. The method of claim 1, wherein the dsDNA molecule is linear.

18. 6. The method of claim 1, further comprising amplifying the dsDNA molecule.

19. amplifying the dsDNA molecules; (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 said dsDNA molecule from said vector; and optionally (iv) isolating or purifying the dsDNA molecule, e.g., from the vector nucleic acid; 20. The method of claim 18, comprising:

20. 20. The method of claim 19, wherein step (ii) comprises transfecting the vector (e.g., a DNA plasmid) into bacteria and propagating the bacteria.

21. 6. The method of any one of claims 1 to 5, further comprising isolating the dsDNA molecule.

22. 6. The method of claim 1, further comprising modifying the dsDNA molecule.

23. The method of claim 1 , wherein multiple types of one or more of the multiple ssDNA molecules are provided.

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

25. 24. The method of claim 23, wherein the multiple types of one or more of the multiple ssDNA molecules comprise one or more modified (e.g., functionalized) nucleotides, and the types differ with respect to (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. 6. The method of any one of claims 1 to 5, wherein one or more of the plurality of ssDNA molecules comprises one or more modified (e.g., functionalized) nucleotides.

27. 6. The method of any one of claims 1 to 5, wherein each copy of the ssDNA molecule providing the non-overlapping portions of the strands of the dsDNA molecule is identical.

28. 24. A library of dsDNA molecules comprising a plurality of different dsDNA molecules obtained from the method of claim 23.

29. 1. Use of a plurality of single-stranded DNA (ssDNA) molecules in a ligase-involving ligation reaction to generate double-stranded DNA (dsDNA) molecules comprising at least 1500 bp, comprising: (i) each of a first plurality of single-stranded DNA (ssDNA) molecules comprises (e.g., consists of) a non-overlapping portion of a first strand of the dsDNA molecule, wherein a first ssDNA molecule comprises a 5'-end of the first strand of the dsDNA molecule, and a second ssDNA molecule (a) comprises a 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 an ssDNA molecule comprising the 5'-end of a second strand as a template; (ii) each of the second plurality of ssDNA molecules comprises (e.g., consists of) a non-overlapping portion of the second strand of said dsDNA molecule, wherein a first ssDNA molecule comprises the 5' end of said second strand of said dsDNA molecule, and optionally, a second ssDNA molecule comprises the 3' end of said second strand of said dsDNA molecule; (i) at least two of the plurality of ssDNA molecules from (ii) each comprising a first region that is completely complementary to a terminal region of an ssDNA molecule of (i) and a second region that is completely complementary to a terminal region of a different ssDNA molecule of (i); (2) the ssDNA molecule of (i) to which the ssDNA molecule of (ii) is complementary forms a contiguous portion of the first strand of the dsDNA molecule; (3) the region of complementarity comprises at least 50 nucleotides; (4) all of the ssDNA molecules contain at least 150 nucleotides; (5) the ssDNA molecules are provided in equimolar amounts; When the first and second plurality of ssDNA molecules are contacted under conditions suitable for hybridization of the DNA molecules, they form a dsDNA complex containing all the ssDNA molecules that can be joined in a reaction involving a ligase to produce the dsDNA molecule.

30. (i) (1) the ssDNA molecule of (a)(i) comprises at least 500 nucleotides; (2) the ssDNA molecules of (a)(i) comprise at least 500 nucleotides, and at least two of the ssDNA molecules of (a)(ii) comprise at least 500 nucleotides; (3) the ssDNA molecule of (a)(i) comprises at least 500 nucleotides, and the ssDNA molecule of (a)(ii) is shorter than the ssDNA molecule of (a)(i); (4) the ssDNA molecule is produced using enzymatic synthesis; or (5) The ssDNA molecule is (a) providing a circular DNA molecule comprising a nucleotide sequence encoding said ssDNA molecule flanked by a cleavage domain; (b) performing a rolling circle amplification (RCA) reaction using the circular DNA molecule of (a) as a template; (c) cleaving the product of the RCA reaction with the cleavage domain to release the ssDNA; generated using a method comprising: (ii) (1) one or more of the types of the plurality of ssDNA molecules are provided; (2) one or more of the plurality of ssDNA molecules comprises one or more modified (e.g., functionalized) nucleotides; or (3) each copy of the ssDNA molecule providing the non-overlapping portions of the strands of the dsDNA molecule is identical; (iii) the region of complementarity comprises at least 100 nucleotides; and / or (iv) the dsDNA molecule is (1) At least 2000 bp, 2500 bp, 3000 bp, 3500 bp, or 4000 bp; (2) containing 1,500 to 50,000 bp, or (3) It is linear.

30. The use according to claim 29.

Images