Nucleic acid construct and method for producing same

Abstract

The present invention relates to novel artificially synthesized single-stranded nucleic acid molecules, as well as templates and methods for making the same, which can be used in many applications. There are many uses for single-stranded nucleic acid molecules, including, but not limited to, vectors for the delivery of sequences (e.g., gene sequences, or templates for gene editing, gene knock-in or knock-down), or in biotechnology, such as for constructing highly ordered materials from nanoparticle building blocks.

Description

[Technical field]

[0001] The present invention relates to new artificially synthesized single-stranded nucleic acid molecules, which can be used in many applications, as well as templates and methods for making them. Single-stranded nucleic acid molecules have many applications, including but not limited to vectors for the delivery of sequences (e.g., gene sequences, or templates for gene editing, gene knock-in or knock-down), or in biotechnology. For example, when building highly ordered materials from nanoparticle building blocks. Single-stranded nucleic acids can take various shapes and can provide functions, such as aptamers and nucleic acid enzymes. When single-stranded nucleic acids are used as vectors, they can be used to transfer nucleic acid sequences / fragments into target cells, either directly or encapsulated by additional components. [Background technology]

[0002] There is an increasing appreciation of the functions that nucleic acids assume within the cell, beyond and beyond coding for the production of proteins. Double-stranded structures have been studied extensively due to their nature, but it is necessary to understand that they may form rigid assemblies within the cell due to base pairing between complementary nucleotides. The most flexible regions of nucleic acids are often non-base paired and include single-stranded deoxyribose nucleic acid (ssDNA) and ribonucleic acid (ssRNA) regions involved in important processes within the cell. For example, double-stranded DNA (dsDNA) can be unwound by enzymes such as DNA polymerases, exposing ssDNA sections. These sections are available for transcription into ssRNA, such as messenger RNA (mRNA), or for interaction with other proteins that recognize ssDNA.

[0003] Single-stranded nucleic acid molecules are of particular interest to those skilled in the art of delivering nucleic acids to cells, since they are immediately available in the cell transfected with the nucleic acid and do not require "unwinding" by appropriate enzymes to expose the relevant genetic information (e.g., transcription and translation or insertion into the genome). They are considered to be the delivery vector of choice for several applications, particularly gene transfer, gene editing, and biosensing. Another potential application is the provision of DNA vaccines. Alternatively, single-stranded DNA may have functions related to its conformation, namely as aptamers. However, longer single-stranded nucleic acids, for example in the range of several thousand nucleotides in length, are currently generated inefficiently or inaccurately, limiting their usefulness, as will be further discussed below. The most common method of generating long oligonucleotides involves cloning the sequence into a plasmid for cultivation in bacteria, followed by restriction digestion and purification of the dsDNA sequence, which is then strand-stripped to generate ssDNA. Despite the problems of bacterial propagation, there are many inefficiencies and purification issues. After the entire plasmid backbone sequence is amplified, it must be separated and discarded along with the bacterial genome. Stripping of the secondary strand to reveal single-stranded nucleic acid molecules reduces efficiency by an additional 50 percent.

[0004] To harness the therapeutic potential of single-stranded nucleic acids, manufacturing methods are needed that are efficient, scalable, and generate commercial-scale quantities of material. Current technologies are limited in their ability to scale up to generate materials in a cost-effective, accurate, rapid, and safe manner. It is also desirable to accurately generate single-stranded molecules that are 200 nucleotides or more in length.

[0005] Both ssDNA and dsDNA donor sequences can serve as efficient gene editing templates, but the choice of donor construct is often dictated by the length of the sequence to be introduced. ssDNA donors have been used primarily for applications requiring small edits. This is mainly because, as discussed above, generating longer ssDNA has proven problematic. ssDNA templates have been found to offer unique advantages in terms of repair specificity when used in gene editing (Design and specificity of long ssDNA donors for CRISPR-based knock-in Han Li, Kyle A. Beckman, Veronica Pessino, Bo Huang, Jonathan S. Weissman, Manuel D. Leonetti bioRxiv 178905), and therefore their use is desirable.

[0006] By their very nature, linear single-stranded nucleic acids are rapidly degraded within cells, since the free 3' and 5' ends are available to enzymes, such as single-stranded nucleases, which "chew" the ends and destroy the nucleic acid. Therefore, there is a need to provide stabilized single-stranded nucleic acid constructs for such purposes, in which the free 3' and 5' ends are protected from immediate degradation.

[0007] Many viral vectors used to deliver genetic material into cells have a single-stranded genome, either as RNA or DNA, so there is precedent for using single-stranded nucleic acids for gene delivery.

[0008] For example, adeno-associated viruses (AAVs) are interesting gene therapy vehicles that belong to the parvovirus family and, in reality, depend on superinfection with other viruses (such as adenoviruses) to replicate. AAVs are essentially proteinaceous shells that surround a single-stranded DNA genome of about 4.7 kilobases (kb). There are hundreds of unique AAV strains. The single-stranded genome contains, among other things, the Rep (replication) and Cap (capsid) genes. These coding sequences are flanked at both ends by inverted terminal repeats (ITRs), usually 145 nucleotides long.

[0009] Recombinant AAV (rAAV), which lacks viral DNA, is essentially a transgene flanked by ITRs, protected in a protein-based nanoparticle designed for DNA cargo delivery to the nucleus of the cell. The main consideration in the design of such rAAV vectors is the package size of the transgene and the associated sequence between the two ITRs. 5 kb (including the viral ITRs) appears to be the current limit to ensure that the ITR-flanked transgene is packaged. Alternatively, the ITR-flanked transgene (or other sequence of interest) can be directly introduced into the cell without packaging, which means that the "artificial genome" can be indeed long.

[0010] Commonly used nucleic acid molecules in the art, such as gene delivery vectors derived from viral genomes, can be problematic because the immune system can recognize circulating "foreign" DNA and thus induce an immune response in the recipient of the gene delivery vector. When DNA is produced in bacterial cells, it has prokaryotic patterns of DNA methylation that can be identified as foreign in eukaryotes and rejected as well. For example, plasmids (pDNA) are naturally occurring circular dsDNA molecules, i.e. extrachromosomal DNA fragments that are stably inherited from generation to generation. Plasmids and their derivatives have been used as gene delivery vectors with varying amounts of success.

[0011] The method of generating nucleic acid vectors can also be problematic. Producing nucleic acid constructs in bacterial cells runs the risk of contaminating the final product with lipopolysaccharides (LPS), endotoxins, and other molecules specific to prokaryotes. These are effective indicators of microbial pathogens and therefore have the ability to increase the immune response in eukaryotes. In fact, producing nucleic acid vectors in cell-based systems runs the risk of contaminants from the cell culture being present in the final product, including genomic material from the host cell. The production of nucleic acids in cells is inefficient because it requires more material to be supplied to generate the nucleic acid than synthetic methods. In addition to cost issues, the use of cell cultures can often lead to difficulties in the reproducibility of the amplification process. In the complex biochemical environment of cells, it is difficult to control the quality and yield of the desired nucleic acid product. It is also difficult to deal with sequences that may be toxic to the cells in which the nucleic acid is amplified. Recombination events can also lead to problems in the faithful generation of the desired nucleic acid.

[0012] DNA can be produced synthetically without the use of cells. Oligonucleotides can be chemically synthesized by extending the chain using modified nucleotides. The preparation of these building blocks is costly. The stepwise addition of each nucleotide is an incomplete process (the probability that each chain is extended is called the "coupling efficiency"), and for longer sequences, the majority of the initiated chains do not become the full-length correct product. This makes the production of large-scale long sequences impossible, and these processes always require a sacrifice between length, precision, and scale. The main use of such oligonucleotides is still in the range of several hundred nucleotides (e.g., primers and probes), with the maximum precise length thought to be about 300 nucleotides long. Typically, synthetic oligonucleotides are single-stranded nucleic acid molecules about 15-25 bases in length.

[0013] A preferred alternative to synthetic processes is the enzymatic production of template-dependent nucleic acids. Cell-free in vitro enzymatic processes for nucleic acid synthesis are advantageous, especially when production is required according to Good Manufacturing Practice (GMP) standards, since they avoid the need for the use of host cells. As a result, enzymatically produced nucleic acids can be made much more efficiently without the risk of cell-derived contaminants.

[0014] There is therefore a need for enzymatically produced and improved constructs that are safer and more tolerated by the recipient, and ideally also resistant to rapid degradation within the cell.

[0015] Enzymatic production of single-stranded deoxyribose nucleic acid (DNA) vectors can be problematic because when a polymerase and primer are used with a double-stranded template, it essentially results in the generation of two complementary strands. These strands can be separated and the unwanted strand discarded, but this can still be considered a waste of processing resources. When scaling up production, the loss of more than 50% of the starting material in the final product is not sustainable.

[0016] The present invention particularly relates to a novel cell-free and in vitro method for efficiently and effectively producing single-stranded nucleic acid constructs, and templates that allow their generation.The templates allow the generation of single-stranded nucleic acid concatemers of any desired length for various applications, including the generation of single-stranded nucleic acid constructs.These constructs are more stable than simple linear single-stranded nucleic acids, since the ends of the nucleic acids are blocked.

[0017] The available art does not disclose processes for producing single stranded nucleic acids with blocked ends or templates for use in the processes as described herein.

[0018] Various documents describe the generation of closed linear double-stranded DNA with "capped" ends. WO2018 / 033730 from Touchlight IP relates to double-stranded closed linear DNA molecules that are not suitable for use as templates in the present invention due to the lack of adjacent processing and conformational motifs. WO2019 / 051255 and WO2019 / 143885 from Generation Bio describe linear duplex DNA molecules formed from continuous strands of complementary DNA with covalently closed ends (linear, continuous, and unencapsulated structures) including 5' inverted terminal repeat (ITR) and 3' ITR sequences. Again, this would not be suitable for use as a template molecule in the present invention.

[0019] Several RNA structures are already known, especially in the field of CRIPSR-Cas9 gene editing. Gorter de Vries, et al. (Microb Cell Fact 16, 222 (2017). https: / / doi.org / 10.1186 / s12934-017-0835-1) disclose a two ribozyme-flanked gRNA that can self-process. A similar structure is detailed in Ng et al. (Molecular Biology and Physiology, March / April 2017 Volume 2 Issue 2 e00385-16). A triple ribozyme (TRz) construct consisting of two cis-acting ribozymes flanking an internal trans-acting ribozyme is disclosed in Benedict et al., Carcinogenesis. 1998 Jul;19(7):1223-30. Such structures lack the conformational and processing motifs flanking the sequence of interest, allowing for blocking of the terminal residues of the linear single-stranded product. [Brief description of the drawings]

[0020] [Figure 1] 1 is an exemplary representation of a template of the present invention. [Diagram 2]1 is a representation of different exemplary templates, with a close-up of single-stranded nucleic acid generated by a polymerase acting on the templates. [Diagram 3] FIG. 1 is a depiction of one method of amplifying a template of the present invention, resulting in a nascent nucleic acid strand that is processed into a single-stranded nucleic acid construct with blocked ends. [Figure 4] FIG. 3 provides two depictions of sections of the nascent strand, illustrating the processing steps that result in the production of a single-stranded nucleic acid construct with blocked ends. [Diagram 5] Alternative representations of the template are shown along with the amplification and processing steps. [Figure 6] 1 is a gel photograph showing the results of an assay from Example 2, in which nucleic acid constructs were tested for their resistance to exonuclease degradation. [Figure 7] 1 is a gel photograph showing the results of an assay of the results of Example 3, in which nucleic acid constructs were tested for their resistance to degradation by cellular components. [Figure 8] 1 is a representation of the sequence of the AAV2 ITR and a representation of possible conformations of single-stranded nucleic acids of the invention having ITR-type structures. [Figure 9] This shows the template used in the first embodiment. [Figure 10] 1 is a gel photograph showing the results of Example 1. Summary of the Invention

[0021] The single-stranded nucleic acid molecules of the present invention are end-blocked. The single-stranded nucleic acid molecules of the present invention are linear single-stranded nucleic acids and therefore have a terminal nucleotide at each end. The terminal nucleic acid residue is not free, i.e., not exposed as in a purely linear single-stranded nucleic acid molecule that does not adopt any further conformation. Thus, the ends of the nucleic acid are fixed or pushed into the construct and are not readily accessible to enzymes such as single-stranded nucleases. The ends of single-stranded nucleic acids can be blocked by including the terminal nucleotide in a conformation that acts to protect the end. Thus, the terminal nucleotide at each end of the linear ssDNA is kept apart or away from agents that may act on it to initiate degradation of the nucleic acid molecule. Generally, the enzyme finds the terminal nucleotide and begins to chew the single-stranded nucleic acid from this residue.

[0022] Single-stranded nucleic acid molecules can be prepared from a template nucleic acid, the design of which is unique.

[0023] Thus, the present invention provides 1. A nucleic acid template for the cell-free in vitro production of single-stranded nucleic acid molecules having blocked ends, comprising: i) the first processing motif and adjacent ii) a first conformational motif; iii) a sequence of interest; iv) a second conformational motif adjacent to it; v) a sequence encoding a second processing motif; A nucleic acid template is provided in which the processing motif comprises a sequence capable of forming a base-pairing section that includes an endonuclease recognition site and associated cleavage site, and the conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond.

[0024] Thus, the templates of the present invention encode a single-stranded nucleic acid as described herein. A single-stranded nucleic acid is linear. A linear single-stranded nucleotide has capped ends.

[0025] Alternatively, a combination of processing and conformational motifs can be used that are adjacent to each other in either the forward (processing motif then conformational motif) or reverse (conformation motif then processing motif) orientation. These are the format elements.

[0026] Thus, the template may include the following sequences encoding the following elements, in the order listed: i) forward format elements, ii) a sequence of interest; iii) Reverse format elements.

[0027] The templates of the present invention can be amplified using any suitable polymerase enzyme to produce a single-stranded nucleic acid product.

[0028] The single-stranded nucleic acid product is linear and end-capped.

[0029] The template can be double-stranded or single-stranded. The single strand of the template is complementary to the desired linear single-stranded nucleic acid product with a blocked end, and thus directs the production of the same. The template directs the assembly of the product when contacted with a polymerase enzyme, and thus the template is replicated or amplified. The terms amplified and replicated can be used interchangeably in the art.

[0030] The template of the present invention can be contacted with a polymerase capable of rolling circle amplification (RCA). The template of the present invention can be amplified using a polymerase capable of catalyzing rolling circle amplification (RCA). RCA is an isothermal enzymatic process that uses a circular DNA template and a specific DNA or RNA polymerase to synthesize long single-stranded DNA or RNA. The RCA product is a concatemer that contains tens to hundreds or thousands of tandem repeats that are complementary to the circular template. Thus, contacting the template with the polymerase can result in "amplification" of the template, generating a complementary single strand of nucleic acid.

[0031] Thus, any template described herein can be amplified using a polymerase capable of rolling circle amplification or replication. This produces long single-stranded concatemeric nucleic acid molecules. Due to the presence of formatting elements (including processing motifs adjacent to conformational motifs, either forward or reverse), the concatemers can be easily processed by adding the required endonucleases. Cleavage within the processing motifs by the endonucleases releases the sequence of interest, which is flanked on both sides by conformational motifs. Once released, these conformational motifs act to seal the ends of single-stranded nucleic acids by forming hydrogen-bonded sections that fix the terminal nucleotides. Thus, the conformational motifs of single-stranded nucleic acid molecules adopt conformations with hydrogen bonds that block the terminal nucleotides. The terminal nucleotides can be fixed by being included or contained within the conformation adopted with or without intramolecular base pairing or hydrogen bonding. Alternatively, the terminal nucleotides may be fixed by intramolecular base pairing or hydrogen bonding, such that the conformational motif increases the stability of these intramolecular interactions.

[0032] Thus, the terminal residue of the linear single-stranded nucleic acid product is formed by the action of the endonuclease on the processing motif, and the terminal residue is the residue at the end of the molecule after the endonuclease cleaves the longer intermediate product. Thus, the format element can be described as including a processing motif adjacent to a conformational motif, and the cleavage site generates a terminal residue that is blocked by the conformational motif. The processing motif and the conformational motif can be described as adjacent, adjoining, or contiguous. Alternatively, there are no unrelated or intervening nucleic acid sequences between the processing motif and the conformational motif. The terminal residue is generated by the action of the endonuclease, which is then blocked.

[0033] Thus, the present invention provides 1. A method for producing a single stranded nucleic acid molecule having blocked ends, comprising: (a) Amplification of a circular template using a polymerase capable of rolling circle amplification, the template comprising: i) the first processing motif and adjacent ii) a first conformational motif; iii) a sequence of interest; iv) a second conformational motif adjacent to it; v) a sequence encoding a second processing motif; The processing motif comprises a sequence capable of forming a base-pairing section that includes a recognition site and associated cleavage site for an endonuclease, and the conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond; amplifying, where the amplification produces nucleic acid concatemers; and (b) processing the nucleic acid concatemer using one or more endonucleases that recognize one or more cleavage sites of the processing motif.

[0034] The single-stranded nucleic acid produced is linear and end-capped.

[0035] In other words, the present invention provides: 1. A method for producing a single stranded nucleic acid molecule having blocked ends, comprising: (a) Amplification of a circular template using a polymerase capable of rolling circle amplification, where the template is i) forward format elements, iii) a sequence of interest; iv) a reverse format element, The forward format element comprises a processing motif adjacent to a conformational motif, the reverse format element comprises a conformational motif adjacent to a processing motif, the processing motif comprises a sequence capable of forming a base-paired section that includes a recognition site and associated cleavage site for an endonuclease, the conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond, amplifying, where the amplification produces nucleic acid concatemers; and (b) processing the nucleic acid concatemer using one or more endonucleases that recognize one or more cleavage sites of the processing motif.

[0036] The single-stranded nucleic acid molecules of the present invention are linear and end-capped.

[0037] The processing step results in a single-stranded nucleic acid construct with blocked ends. In the processed form, the ends are blocked because the conformational motifs are able to form or assume their desired conformation stabilized by intramolecular hydrogen bonds. The ends of the single-stranded nucleic acid molecule are blocked by the conformation adopted by the conformational motif. The terminal nucleotide is either fixed by being included in a conformation that makes it sterically difficult for exonucleases to access, or is included in an intramolecular bond within the conformational motif, the whole of which makes the terminal nucleotide more stable to exonucleases. Since the molecule has two ends and two conformational motifs, each functions to assume a conformation that includes the associated end or terminal nucleotide. Since nucleic acids are linear, the molecule has two ends and two terminal residues.

[0038] Concatemer is an intermediate product during the production of the single-stranded nucleic acid molecule of the present invention, but due to its composition as a multimeric linked chain of target sequences, it may have some utility of its own.In applications where it may be advantageous, such as biosensing, it increases the local concentration or efficacy of the sequence.Affinity binding is one possible application.

[0039] Thus, the present invention provides A single stranded oligonucleotide concatemer having two or more repeats of a sequence unit, the sequence unit being i) the first processing motif and adjacent ii) a first conformational motif; iii) a sequence of interest; iv) a second conformational motif adjacent to it; v) a second processing motif, A single-stranded oligonucleotide concatemer is provided, in which the processing motif comprises a sequence capable of forming a base-pairing section that includes an endonuclease recognition site and associated cleavage site, and the conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond.

[0040] In other words, the present invention provides: A single stranded nucleic acid concatemer having two or more repeats of a sequence unit, the sequence unit being i) the first processing motif and adjacent ii) a first conformational motif; iii) a sequence of interest; iv) a second conformational motif adjacent to it; v) a second processing motif, A single-stranded nucleic acid concatemer is provided, wherein the processing motif comprises a sequence capable of forming a base-pairing section that includes an endonuclease recognition site and associated cleavage site, and the conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond.

[0041] The single-stranded nucleic acid molecules of the present invention are linear and end-capped.

[0042] Alternatively, when the processing motif and the conformational motif are taken together as a processing element, the present invention provides A single stranded oligonucleotide concatemer having two or more repeats of a sequence unit, the sequence unit being i) forward format elements, iii) a sequence of interest; iv) a reverse format element, A single-stranded oligonucleotide concatemer is provided, in which the forward format element comprises a processing motif adjacent to a conformational motif, and the reverse format element comprises a conformational motif adjacent to the processing motif, the processing motif comprises a sequence capable of forming a base-paired section that comprises an endonuclease recognition site and associated cleavage site, and the conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond.

[0043] The single-stranded nucleic acid molecules of the present invention are linear and end-capped.

[0044] The terminal nucleotide of a conformational motif, or indeed of a single-stranded nucleic acid construct, is usually the nucleotide adjacent to the processing motif and "released" from the concatemeric nucleic acid by the action of an endonuclease. It is this terminal nucleotide that forms the end of the single-stranded nucleic acid construct and is appropriately blocked to retard degradation.

[0045] The forward format element contains a processing motif adjacent to a conformational motif, and the reverse format element contains a conformational motif adjacent to a processing motif. This arrangement ensures that after processing, the sequence of interest is flanked on both ends by the conformational motifs. Thus, the sequence of interest is flanked by two conformations within the construct, each of which seals an end of the nucleic acid.

[0046] Detailed explanation of the figure 1 is an exemplary representation of a template of the invention (100). Shown are sequences encoding first and second processing motifs (101 and 102), first and second conformational motifs (103 and 104), a sequence of interest (105), recognition sites for an endonuclease including a cleavage site (106 and 107), and a nicking site within the template (108).

[0047] FIG. 2 is a representation of a different exemplary template (100), shown as a double-stranded circular nucleic acid construct with a nicking site (108) shown along with a backbone sequence (110) and sequences for the generation of a single-stranded nucleic acid construct (111), terminated at both ends with sequences encoding a format element (113). A close-up of the single-stranded nucleic acid generated by polymerase amplification of the template strand at the format element (113) is shown. It shows the recognition sites for the endonuclease, including the nicking site (178), the first processing motif (151), the first conformational motif (153), and the cleavage site (161). The first processing motif and the first conformational motif are adjacent and separated only by the cleavage site, which together form the format element (157).

[0048] FIG. 3 is a depiction of one method of amplifying a template (100) of the present invention, using rolling circle amplification of the template, resulting in a nascent nucleic acid strand (150). Shown on the nascent strand are a first processing motif (151), a second processing motif (152), a first conformational motif (153), and a second conformational motif (154), which, together with the sequence of interest, form the basis of the nucleic acid construct (156) and backbone sequence (155). Shown is a formatting element (157) that includes a cleavage site (not shown). The nascent strand is processed using the required enzyme (not shown) that recognizes the cleavage site, resulting in a single-stranded nucleic acid construct with a blocked end (160) with by-products resulting from the template backbone (158) and the processing motif (159).

[0049] Figure 4 shows two depictions of sections of the nascent strand from Figure 3, with processing steps indicated, resulting in the generation of a single-stranded nucleic acid construct with blocked ends (160), along with by-products. Depending on the type of conformational motif utilized, the conformation or structure of the 3' and 5' ends of the construct will differ.

[0050] FIG. 5 shows an alternative representation of a template (200). Shown are sequences encoding a first and second processing motif (201 and 202), a first and second conformational motif (203 and 204), a sequence of interest (205), a recognition site for an endonuclease that includes a cleavage site (206 and 207), and a nicking site (208) within the template. Also shown is a nascent strand of nucleic acid generated from the template (250). Shown on the nascent strand are a first processing motif (251), a second processing motif (252), a first conformational motif (253), and a second conformational motif (254), which together with the sequence of interest (255) form a nucleic acid construct. Cleavage sites (256 and 257) are formed within format elements (281 and 282). The nascent strand is processed using the required enzyme that recognizes the cleavage site, resulting in a single-stranded nucleic acid construct with blocked ends (260), along with a by-product (not shown).

[0051] 6 is a gel photograph (0.8% TAE agarose gel, 1× Gel Red stain) showing the results of the assay results of Example 2. The lanes on the gel are as follows: [Table 1]

[0052] 7 is a gel photograph (0.8% TAE agarose gel, 1× Gel Red stain) showing the results of the assay results of Example 3. The lanes on the gel are as follows: [Table 2]

[0053] FIG. 8A depicts the conformation of the AAV2 ITR. The AAV2 ITR is composed of two arm palindromes (B-B' and C-C') embedded in a larger stem palindrome (A-A'). The ITR can obtain two configurations (flip and flop). The flip (schematic) and flop configurations have the B-B' and C-C' palindrome closest to the 3' end, respectively. The D sequence remains single-stranded since it occurs only once at both ends of the genome. The boxed motif corresponds to the Rep binding element (RBE). FIG. 8B and 8C are representations of the linear single-stranded nucleic acid template of the invention (i), followed by the single-stranded product of the template both before (ii) and after (iii) cleavage. FIG. 8B contains a single-stranded D region similar to the wild-type AAV ITR. FIG. 8C includes the D region within a conformational motif by pairing it with a D′ region, so it is in the double-stranded portion.

[0054] Figure 9 is a representation of the template, plasmid map, used in Example 1. Shown are the sequence of interest, the conformational motif, the processing motif, the backbone, and the site of the processing enzyme (MlyI). Also shown are the sites of nicking endonucleases (e.g., BsrDI that may be nicked by mutants of Nb.BsrDI).

[0055] Figure 10 is a gel photograph (0.8% TAE agarose gel, 1x SafeView stain) showing two lanes, the first being a marker lane and the second showing the nucleic acid construct made using the template of Figure 9 according to Example 1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] The present invention satisfies the need for an efficient, cell-free, enzymatic, cost-effective, accurate and clean method for producing large quantities of single-stranded nucleic acid molecules in vitro. To extend the lifespan of single-stranded nucleic acid molecules for cell-based use, the inventors have devised an elegant method to protect the ends of single-stranded nucleic acid molecules from immediate degradation by capping these ends.

[0057] Blocked end An important feature of all linear nucleic acid molecules is that they are polymers containing nucleotide residues and have two distinct ends. The nature of the ends is determined by the nature of the nucleic acid backbone. For natural (non-synthetic) nucleic acid molecules, these two ends are the 5' (five prime) and 3' (three prime) ends. In natural nucleic acids (i.e., DNA or RNA), the 5' end is the end of the molecule that ends with a 5' phosphate group. By convention, nucleic acid sequences are written with the 5' end on the left and the 3' end on the right, and the order described herein follows that convention. The 3' end is the end of the molecule that ends with a 3' phosphate group. Generally, in natural nucleic acids, a phosphodiester bond is formed between the phosphate group of one nucleotide and the sugar of another nucleotide to form the backbone. Using the chemical convention for carbon numbering of nucleotides, the phosphate group is attached to the 5' carbon of the sugar, and thus becomes the 5' end of the nucleotide. A phosphodiester bond is formed between the 5' end of one nucleotide and the 3' hydroxyl group of another nucleotide, forming a polymer with one open 5' end and one open 3' end. The 5' end may therefore be considered to be the terminal residue with the 5' phosphate group. The 3' end may therefore be considered to be the terminal residue with the 3' hydroxyl group. In the case of DNA and RNA, these terminal residues are nucleotide residues.

[0058] In the present invention, the terminus of the linear single-stranded nucleotide is formed by the action of an endonuclease on an intermediate product of the method of the present invention. Thus, the terminal residue of the conformational motif becomes the terminal residue of the single-stranded nucleic acid product. Prior to cleavage, this residue effectively connected the conformational motif to the processing motif.

[0059] Nucleic acids can only be synthesized in vivo in a 5' to 3' direction because the polymerases that assemble the new strands generally rely on the energy generated by cleaving nucleoside triphosphate bonds and attaching new nucleoside monophosphates to 3'-hydroxyl (-OH) groups via phosphodiester bonds. The relative positions of entities along a strand of nucleic acid, including genes and various protein binding sites, are generally noted as either upstream (towards the 5' end) or downstream (towards the 3' end). In nature, due to the antiparallel nature of DNA, this means that the 3' end of the template strand is upstream of the gene and the 5' end is downstream.

[0060] In the case of fully synthetic non-natural (synthetic) nucleic acids, the ends can be labeled according to the backbone structure. For example, when examining peptide nucleic acids (PNAs), the sugar phosphate backbone is replaced with units of N-(2 aminoethyl)glycine. Each of the four natural bases is then connected to the backbone via a methylene carbonyl linker. PNAs have N- and C-termini, rather than 5' and 3' termini.

[0061] In the present invention, the ends of linear nucleic acid molecules are blocked, regardless of the nomenclature of these ends.Therefore, the terminal residues or terminal nucleotides of these ends are not free or exposed.In the case of natural nucleic acids such as DNA and RNA, these terminal residues are terminal nucleotides, 3' and 5' ends.In the case of synthetic nucleic acids, these ends have appropriate nomenclature.

[0062] Each blocked end is stabilized and therefore unavailable for immediate reaction with enzymes such as single-stranded nucleases. When nucleic acids are used in a cellular environment, the ends are kept away, shielded, or blocked from cellular components that may cause immediate degradation of single-stranded nucleic acids. Thus, the ends of single-stranded nucleic acid molecules do not function as they normally would without blocking. Blocking the ends confers enhanced stability to the molecule compared to a similar molecule without blocked ends. This is demonstrated by the inventors in Example 1, where the molecules of the present invention remain intact, whereas similar molecules without blocked ends are degraded.

[0063] The end is preferably blocked by the presence of a conformational motif. The conformational motif has a specific sequence. The sequence of the conformational motif is designed to be able to form intramolecular hydrogen bonds to form or adopt a specific conformation. When the conformation is adopted in the single-stranded nucleic acid construct, the terminal nucleotide is blocked by the motif, which means that it is secured.

[0064] The intramolecular hydrogen bond may be within the conformational motif sequence itself, or may be between a portion or parts of the conformational motif and at least one other sequence in the entire single-stranded nucleic acid molecule, such as the sequence of interest. The intramolecular hydrogen bond may or may not include the terminal nucleotide.

[0065] Hydrogen bonds are a non-covalent type of bond between or within molecules. These bonds are formed from a hydrogen atom covalently bonded to an electronegative atom (hydrogen acceptor) and another electronegative atom (hydrogen donor, i.e. only nitrogen, oxygen, and fluorine atoms function) of the same or a different molecule. They are the strongest type of dipole-dipole interaction. Hydrogen bonds are involved in the specific base pairing of the DNA double helix and are responsible for the stability of the DNA double helix structure.

[0066] Watson-Crick base pairing typically involves the formation of hydrogen bonds between the nitrogenous bases (nucleobases) of nucleotides. In the standard base pairings adenine-thymine (AT) in DNA, adenine-uracil (AU) in RNA, and both cytosine-guanine (CG) base pairs form hydrogen bonds. AT / U and CG base pairings function by forming double or triple hydrogen bonds between the amine and carbonyl groups of the complementary bases.

[0067] A wobble base pair is a base pairing between two nucleotides in a nucleic acid molecule, especially RNA, that does not follow the standard Watson-Crick base pairing rules. The four major wobble base pairs are guanine-uracil (GU), hypoxanthine-uracil (IU), hypoxanthine-adenine (IA), and hypoxanthine-cytosine (IC). The thermodynamic stability of wobble base pairs is comparable to that of Watson-Crick base pairs. Wobble base pairs are fundamental to RNA structure.

[0068] Alternative or non-canonical base pairing is also possible in nucleic acid structures, again held together by hydrogen bonds. These are generally more common in RNA, but are also possible in DNA and other nucleic acids. One example of non-canonical base pairing is Hoogsteen and reverse Hoogsteen base pairing. In these interactions, the purine bases adenine and guanine flip their normal orientation and form a new set of hydrogen bonds with their partners. Hoogsteen hydrogen bonds have been shown to exist in quadruplexes, such as the i-motif and G-quadruplex, which are discussed in more detail herein.

[0069] Combinations of various base pairing mechanisms can also be envisaged. For example, when hydrogen bonds are formed between the AT and GC base pairs of standard B-form DNA, some hydrogen bond donor and acceptor groups of the nucleobases remain unused. Each purine base has two such groups on the edge exposed to the major groove. Triple-stranded DNA can form intermolecularly between the duplex and a third oligonucleotide strand. The third strand bases can form Hoogsteen type hydrogen bonds with the purines of the B-form duplex.

[0070] Base pairs can also be formed between natural and unnatural bases and between pairs of unnatural bases.

[0071] Thus, base pairing is an example of intramolecular hydrogen bonding that allows the conformational motif to adopt the relevant conformation. When a conformational motif relies on base pairing to seal the terminal nucleotide, there may be a sequence within the motif that base pairs with a sequence elsewhere in the single-stranded nucleic acid construct (i.e., within the sequence of interest). Alternatively, a sequence within the conformational motif may be designed to base pair with at least one other sequence within the conformational motif such that hydrogen bonds are formed within the motif itself. All types of base pairs are contemplated, including those formed between nucleotides that are "non-complementary" according to standard Watson-Crick base pairing.

[0072] Intramolecular hydrogen bonds are also interactions that are not defined as classical base pairing, such as the planar arrangement of guanine residues within the G-tetrads of a G-quadruplex that are stabilized by Hoogsteen hydrogen bonds. These structures are discussed further below.

[0073] In addition, the stabilization of nucleic acid molecules may also depend on base stacking interactions. Pi-pi stacking (also called π-π stacking) refers to the attractive non-covalent interactions between aromatic rings because they contain pi bonds. These interactions are important in nucleobase stacking within nucleic acid molecules held together by hydrogen bonds. Thus, single-stranded nucleic acid constructs are likely to be further stabilized by base stacking interactions. Other interactions that stabilize nucleic acids are also possible, and these include pi-cation interactions, van der Waals interactions, and hydrophobic interactions.

[0074] In one embodiment, the conformational motif is designed to include a sequence that allows the base-pairing section to be formed. The base-pairing section may include a suitable number of nucleotides in the base-pairing section. In some aspects, the base-pairing section may be formed from a sequence of nucleotides. Since the conformation needs to be maintained, the length of the base-pairing section may be at least 5 base pairs. The base-pairing section may include at least 2 nucleotides, or 2-5 nucleotides, or 5 nucleotides, or may include more than 5 nucleotides, i.e., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 nucleotides or more. In some cases, the base-pairing section may include more nucleotides to safely seal the terminal nucleotide. Thus, the base-pairing section may be 1-50 or 1-100 nucleotides in length, or indeed 1-250 nucleotides or more.

[0075] The terminal nucleotide residue can intramolecularly hydrogen bond to another portion of the single stranded nucleic acid construct that contains the conformational motif. In one aspect, the terminal nucleotide forms a base pair with another nucleotide in the construct.

[0076] However, the terminal residue may be free of hydrogen bonding, or more specifically, base pairing. In this case, the conformational motif fixes or seals the terminal nucleotide by encompassing, surrounding or surrounding it so that the single-stranded nuclease cannot freely cleave it from the adjacent nucleotide in the construct (and then cleave the adjacent nucleotide, etc.). In other words, the terminus is sterically protected from degradation, since it is not possible for a larger entity to reach the terminus. As an example, the terminal nucleotide may be fixed within a quadruplex motif.

[0077] It may be only the terminal residues at each end of the single stranded nucleic acid molecule that are blocked. Alternatively, one or more adjacent residues may also be blocked. At least 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 50 or more residues may also be blocked, along with the terminal residues.

[0078] In a further aspect, each end may be sealed by the formation of a duplex involving at least the terminal residues of the ends of the molecule. The duplex is formed by base pairing between nucleotide sequences. These sequences may be adjacent (hairpin) or separate (such as stem loop).

[0079] A residue refers to a single constituent unit of a nucleic acid polymer, such as a nucleotide.

[0080] In a further aspect, it is preferred that a base-paired or double-stranded section that acts to seal the end or terminal nucleotide of the single-stranded nucleic acid construct is formed within the conformational motif.Thus, the conformational motif includes self-complementary sequences that can form base-paired or double-stranded sections.These may be flanked or separated by non-complementary sequences.

[0081] In other aspects, the base pair or double-stranded section that acts to seal the end or terminal nucleotide of the single-stranded nucleic acid construct is formed outside the conformation motif.Thus, it may comprise a part of the sequence of interest, or in fact a spacer sequence that can be introduced into the nucleic acid construct (i.e., between two coding regions in the "sequence of interest").Thus, the conformation that is achieved may be a lariat, which is a loop of single-stranded nucleic acid that comprises annealed complementary sequences or double-stranded sections that include the terminal residues.

[0082] In some interesting aspects discussed further herein, the ends may be sealed in conformations such as quadruplexes. These are quadruplex (four-stranded) structures, which may be involved in the structure of the telomeric ends of chromosomes. The underlying pattern is a tetrad, a planar arrangement of four residues stabilized by Hoogsteen hydrogen bonds and coordination to a central cation. Quadruplexes are formed by stacking multiple tetrads. Depending on how the sequence is initially folded into these arrangements, many different topologies may form. Quadruplex structures may be further stabilized by the presence of cations, especially potassium, which is located in the central channel between each pair of tetrads. Quadruplexes have been shown to be possible with DNA, RNA, LNA, and PNA, and may be intramolecular.

[0083] Exemplary quadruplexes include G-quadruplexes formed from G-rich sequences and i-motifs (intercalation motifs) formed by cytosine-rich sequences.

[0084] Thus, in one embodiment, the terminal nucleotides are blocked within a quadruplex, optionally a G-quadruplex or an i-motif.

[0085] Conformational motifs One of the desired products is a single-stranded nucleic acid molecule or construct, composed of any suitable nucleic acid, but preferably DNA or RNA, that contains a sequence of interest flanked on both sides by conformational motifs that seal the ends of the single strand.Thus, the single-stranded nucleic acid construct has a first (usually at the 5' end) and a second (usually at the 3' end) conformational motif.Each conformational motif may be unique, but they all share the property that they can seal the ends of the single strand.

[0086] The single stranded nucleic acid molecule or construct may comprise any suitable conformational motif, as discussed in relation to the blocked ends.

[0087] Conformational motifs include sequences that can form intramolecular hydrogen bonds. These hydrogen bonds can be any type of base pair or Hoogsteen type hydrogen bonds found in structures such as tetraplexes / quadraplexes.

[0088] In particular, a conformational motif can be a sequence that includes one or more sections of sequence that can base pair to another section of sequence, either within the conformational motif itself or elsewhere in the single stranded nucleic acid.

[0089] Thus, a conformational motif may only comprise two sections of sequence that are "complementary" and whose base pairs form an antiparallel or truly parallel duplex. This duplex may or may not include the terminal residues of the single-stranded nucleic acid (i.e., the 3' or 5' ends). In this case, the conformational motif may form a hairpin (where the two sections are adjacent) or a stem loop (where the two sections are separated by a spacer sequence leaving a single-stranded nucleic acid). It will be understood that such a structure may be achieved by including an inverted repeat sequence in the conformational motif. A palindromic sequence is a section of a double-stranded nucleic acid sequence where the sequence reading 5' to 3' forward in one section matches the sequence reading 5' to 3' forward in the complementary section that forms the duplex.

[0090] Thus, a conformational motif may include sequences necessary to form one or more of the following: a hairpin, a stem loop, or a pseudoknot. All of these conformations have in common two sections of sequence that can form a duplex. Alternative structures include a lariat or lasso, which also contain a section of sequence that can form a duplex.

[0091] The conformational motif can be a hybrid of different conformations, such as a G-quadruplex, with additional sequences designed to form a duplex to seal the ends by direct base pairing; all that is required is that the conformational motif is capable of securing the terminal nucleotides.

[0092] Organisms with single-stranded DNA or RNA genomes, or whose genetic material may exist as a single strand for part of their life cycle, have evolved to protect the free ends of nucleic acids by using specific structures or by other means, including the arrangement of proteins. Indeed, mammalian genomes have evolved the use of telomeres to protect the ends of chromosomes that may have single-stranded overhangs.

[0093] For example, AAV uses ITRs to protect the ends of its single-stranded DNA genome. Adeno-associated viruses (AAV) are non-pathogenic members of the Parvoviridae family. Wild-type AAV genomes contain inverted terminal repeats (ITRs) that are typically composed of 145 nucleotides at both ends. The terminal 125 nucleotides of each ITR self-anneal to form a palindromic double-stranded T-shaped hairpin structure, with the small palindromic B-B' and C-C' regions forming the cross-arms and the large palindromic A-A' region forming the stem. Each structure is followed by a unique ~20 nucleotide D (or D') region. The generation of recombinant AAV (rAAV) is not affected by truncations within the ITRs, resulting in lengths of 137 nucleotides or less. In nature, the ITRs function as origins of replication and are composed of two arm palindromes (Figure 8A, B-B' and C-C') embedded in a larger stem palindrome (A-A'). ITRs can acquire two configurations: flip and flop. The flip (Figure 8A, shown for AAV2) and flop configurations have B-B' and C-C' palindrome nearest the 3' end, respectively. The boxed motif corresponds to the Rep binding element (RBE) to which the AAV Rep protein binds. The RBE may consist of a tetranucleotide repeat with the consensus sequence 5'-GNGC-3'.

[0094] It has previously been shown that the presence of the D region of single-stranded DNA (as shown in Figure 8B) can be detrimental to the expression of transgenes carried by recombinant AAV vectors (Ping et al, Mol Biotechnol DOI 10.1007 / s12033-014-9832-3). The D region is thought to provide a binding site for a human protein that preferentially binds to this region and prevents the synthesis of the second strand (Qing et al, Proc. Natl. Acad. Sci. USA, Vol. 94, pp. 10879-10884, September 1997, Medical Sciences and Kwon et al, Human Gene Therapy, DOI:10.1089 / hum.2020.018). Therefore, if it is intended to express the transgene in a cell, it may not be desirable to include a D region in the single-stranded DNA of the present invention, which can be done by removing the sequence or providing it within a conformational motif such that the single-stranded DNA pairs with the D' region as shown in Figure 8C. Thus, not only is the D region present, but also the paired D' region. Such pairing may provide additional stabilization of the ITR-type structure. Furthermore, the presence of a double-stranded D region may allow the binding of transcription factors (such as RFX) and enhance nuclear transport (Julien et al, Sci Rep. 2018 Jan 9;8(1):210. doi:10.1038 / s41598-017-18604-3.) An additional benefit of the presence of a D region may be that the presence of this region may weaken the humoral immune response of the host. Kwon et al showed that the D sequence may inhibit the expression of MHC-II genes. Thus, if the D region is present but in double-stranded form, it may have the advantage of dampening the host immune response and avoiding the inhibition of second strand synthesis seen when the D region is in single-stranded form, while at the same time providing a mechanism for nuclear transport. Thus, the presence of a double-stranded D region within a nucleic acid construct, particularly within a conformational motif, is desirable.

[0095] Thus, the present invention extends to linear single-stranded nucleic acid molecules with blocked ends, where at least one end comprises an ITR structure that includes a double-stranded D region. The D region may be duplexed with a D' region. As used herein, the D' region is sufficiently complementary to the D region to allow for the formation of a duplex between the two sequences. The D region may be a naturally occurring D region sequence (shown in Figure 8A-AGGAACCCCTAGTGATGGAG as SEQ ID NO:2, various serotype variants as SEQ ID NOs:3-6) or a sequence with sufficient homology, such as at least 80, 85, 90, 95 or 99% homology. The linear single-stranded DNA of the present invention may include one or two ITR ends as described herein. These ends may be the same or different. The advantage of such a structure is that any transgene is expressed while the host's immune system is temporarily suppressed.

[0096] Thus, the conformation motif of the single-stranded nucleic acid construct can be an ITR sequence taken from any AAV serotype. It can be a sequence derived based on the ITR from any AAV serotype, for example, one or more of the elements can be modified, changed or replaced. Depending on the application in which the single-stranded nucleic acid construct is used, the RBE can be removed or the length of any palindrome can be changed. The conformation motif can be a completely different sequence from the natural AAV ITR sequence, but still maintain a similar structure. Those skilled in the art will appreciate how to use appropriate self-complementary sequences to design sequences that form two-arm palindrome.

[0097] Other viral genomes also rely on blocked ends at the ends of their linear genomes: HIV has at least a 5' blocked end.

[0098] Alternatively, the use of folded structures such as G-quadruplexes and intercalated motifs (i-motifs) can be considered. I-motifs and G-quadruplexes are four-stranded quadruplex structures formed by DNA, with i-motifs formed by cytosine-rich DNA regions and G-quadruplexes formed by guanine-rich DNA forms. I-motifs are particularly stable at pH values ​​below physiological values, making them applicable in nanotechnology and nanomedicine, and have been used as biosensors, nanomachines and molecular switches.

[0099] The sequences of G-quadruplexes vary and can be defined by a putative formula: (G 3+ N 1-n G 3+ N 1-n G 3+ N 1-n G 3+ ) where N is any nucleotide, including guanine. The number of residues between the guanines defines the length of the loop. Loops larger than 7 nucleotides have been observed.

[0100] Thus, the conformational motifs adopt conformations that are held by hydrogen bonds and can be further stabilized by interactions such as base stacking. These conformations can actually be further stabilized by the presence of small molecules or ions, examples of which are shown below.

[0101] Quadruplexes (alternatively called tetraplexes) can form complexes around a central ion, for example. Many ligands, both small molecules and proteins, can bind to quadruplexes. These ligands can be naturally occurring or synthetic. All characterized G-quadruplex binding proteins have been found to share a 20 amino acid long motif / domain (RGRGR GRGGG SGGSG GRGRG, SEQ ID NO: 7) called NIQI (Novel Interesting Quadruplex Interaction Motif), which is similar to the RG-rich domain (RRGDG RRRGG GGRGQ GGRGR GGGFKG, SEQ ID NO: 8) of the previously mentioned FMR1 G-quadruplex binding protein. Cationic porphyrins have been shown to bind G-quadruplexes intercalatively. It may be important to match the quartet-stacked quadruplex with the loops of the nucleic acid that hold it together. π-π interactions may be an important determinant of ligand binding. Ligands should have a higher affinity for parallel folded quadruplexes. Ligands that bind to other conformational motifs to stabilize them are also contemplated.

[0102] Conformation motifs block the ends of single-stranded nucleic acid molecules, generally forming a specific structure. Conformation motifs can be designed so that this structure has its own function and also blocks the ends. For example, aptamers can be designed to be formed by conformation motifs, or ribozymes, deoxyribozymes, and riboswitches. Aptamers bind to specific targets due to electrostatic interactions, hydrophobic interactions, and their complementary shapes. It is possible to engineer aptamer sequences through repeated rounds of in vitro selection or SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even larger entities such as cells, tissues, and organisms. Alternatively, conformation motifs can be designed to include sequences that facilitate passage through cell or nuclear membranes. Additionally or alternatively, conformation motifs can be designed to allow the formation of oligomeric complexes using nucleic acid constructs, which can be useful in nanotechnology and the like.

[0103] The conformation of a nucleic acid can be affected by changes in conditions, and the sequence of the conformational motif should be selected such that the conformation is adopted under the conditions (i.e., pH, temperature, salt concentration, pressure, protein concentration, sugar concentration, osmolarity, etc.) in which the nucleic acid construct will be used.

[0104] The nucleic acid constructs can be used in many different conditions, such as physiological conditions or conditions that are advantageous for use in technology, for example in electronic devices.

[0105] Physiological conditions may be those external or internal environmental conditions that may occur in nature for the organism or cell system, and which are suitable for the conformational motif to adopt the associated conformation.

[0106] If the nucleic acid construct is to be used for non-cellular purposes, i.e. in nanotechnology, conformation may be achieved in relevant buffers, or indeed in pure water, as appropriate.

[0107] Thus, the conformational motifs may be in single-stranded format in the concatemer precursor molecule, and these may be conditions that do not adopt a conformation or are actually possible. It will be understood that in the concatemer precursor, the terminal residues are adjacent to the processing motif. It is the adjacent nature of the motifs that allows for the generation of linear single-stranded nucleic acid molecules with blocked ends.

[0108] The desired array The single stranded nucleic acid construct also comprises a sequence of interest. It will be understood that the sequence of interest may comprise more than one sequence, and may in fact comprise many sequences, for example, several gene sequences may be contained within the "sequence of interest", each of which may have associated promoter and enhancer elements, if necessary.

[0109] The sequence of interest may also include a spacer sequence that includes a sequence complementary to the sequence of the conformational motif, allowing a base-paired section to be formed to block the end or terminal nucleotides.

[0110] This sequence of interest can be any suitable sequence or can include any number of sequences. The sequence itself can have a function, such as forming an aptamer, a nucleic acid enzyme, a ribozyme, a deoxyribozyme, a riboswitch, a small interfering RNA, etc. The sequence of interest can code for a product that can be an aptamer, a protein, a peptide, or an RNA, such as a small interfering RNA. The sequence of interest can include an expression cassette that includes one or more promoter or enhancer elements and a gene or other coding sequence that encodes an mRNA or protein of interest. The expression cassette can include a eukaryotic promoter operably linked to a sequence that encodes a protein of interest, and optionally an enhancer and / or a eukaryotic transcription termination sequence.

[0111] Alternatively, the sequence of interest can be designed to be a carrier sequence.The sequence of interest can therefore be sufficiently complementary to another separate sequence that can anneal to it, so that the entire single-stranded nucleic acid carrier is effectively used as a delivery mechanism for another molecule by forming a duplex with the single-stranded section.The separate oligonucleotide can be completely synthetic.In this context, the single-stranded product serves as a "carrier" molecule.

[0112] The sequence of interest can be used to generate DNA for expression in host cells, particularly to generate DNA vaccines.DNA vaccines usually code for modified forms of the DNA of infectious organisms.DNA vaccines are administered to subjects, where they then express selected proteins of infectious organisms and initiate immune responses against those proteins that are usually protective.DNA vaccines can also code for tumor antigens in cancer immunotherapy approaches.

[0113] The sequence of interest can generate other types of therapeutic DNA molecules, such as those used in gene therapy.For example, such DNA molecules can be used to express functional genes when a subject has a genetic disorder caused by a malfunctioning version of the gene.Examples of such diseases are well known in the art.

[0114] The sequence of interest may be able to function as a donor nucleic acid for gene editing purposes in both animals and plants.Exemplary methods of gene editing include CRISPR gene editing and transcription activator-like effector nuclease (TALEN)-based methods.

[0115] The novel structures of the present invention may also have non-medical applications in materials science, nanotechnology, data storage, etc., and sequences of interest may be selected accordingly. Nucleic acids may be used as biobatteries, security markings of objects, or biomolecular electronic components.

[0116] Particularly for therapeutic applications, it is preferred that the single-stranded nucleic acid construct with blocked ends lacks a bacterial origin of replication, lacks resistance genes (i.e. for antibiotics), lacks CpG islands (except for DNA vaccines where the same may be useful), lacks cytosine and adenine methylation, and lacks sequences that identify the nucleic acid as foreign to the host cell (if the construct is intended for a cell).

[0117] The single-stranded nucleic acid construct may be a natural nucleic acid molecule, such as DNA or RNA. The single-stranded nucleic acid construct is preferably DNA. The single-stranded nucleic acid construct may also be a non-natural nucleic acid molecule. Examples of non-natural nucleic acid molecules or heterologous nucleic acids (XNA) include 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA) and FANA. Hachimoji DNA is a synthetic nucleic acid analog that uses four synthetic nucleotides in addition to the four or five nucleotides present in the natural nucleic acids DNA and RNA. Enzymes have been engineered, mutated or developed to recognize synthetic nucleic acid molecules, and therefore the methods and products of the present invention apply equally to these analogs, or hybrids of synthetic and natural nucleic acids and chimeras thereof.

[0118] Preparation of single-stranded nucleic acid molecules / constructs Single-stranded nucleic acid constructs can be generated using a unique method by rolling circle amplification of distinct templates followed by processing of the single-stranded nucleic acid concatemers resulting from this amplification.

[0119] Methods for producing single-stranded nucleic acid constructs with blocked ends rely on the amplification of a template nucleic acid ("sequence unit") by rolling circle amplification with an associated polymerase enzyme, resulting in the generation of a long single-stranded nucleic acid with multiple repeats of the sequence unit encoded by the template. This concatemeric single-stranded nucleic acid can then be processed into a product single-stranded nucleic acid with blocked ends.

[0120] The amplification process requires the addition of substrates (i.e., suitable nucleosides for nucleic acid production), and any cofactors (salts, ions, etc.). Suitable conditions include the presence of a buffer and a temperature at which the enzyme can operate. Suitable conditions for rolling circle amplification may be isothermal.

[0121] Amplification is the generation of multiple copies of a nucleic acid template or the generation of multiple copies of a nucleic acid sequence that is complementary to a nucleic acid template. In the methods of the present invention, amplification preferably refers to the generation of multiple copies of a nucleic acid sequence that is complementary to a nucleic acid template.

[0122] If the template is double-stranded, techniques are preferably used to ensure that the strand complementary to the desired product is used as the template. This can be achieved in a number of ways, discussed further below.

[0123] As used, a nucleoside is a compound in which a nucleic acid base (nucleobase) is linked to a sugar moiety. The nucleic acid base can be a natural or modified / synthetic nucleic acid base. The nucleic acid base can include, among others, a purine base (e.g., adenine or guanine), a pyrimidine (e.g., cytosine, uracil, or thymine), or a deazapurine base. The nucleic acid base can be a ribose or deoxyribose sugar moiety. The sugar moiety can include a natural sugar, a sugar substitute, a substituted sugar, or a modified sugar. The nucleoside can include 2'-hydroxyl, 2'-deoxy, or 2',3'-dideoxy form of the sugar moiety.

[0124] Nucleotide or nucleotide base refers to nucleoside phosphate. It includes natural, synthetic or modified nucleotides, or alternative replacement moieties (e.g., inosine). Nucleoside phosphate can be nucleoside monophosphate (NMP), nucleoside diphosphate (NDP), or nucleoside triphosphate (NTP). The sugar moiety of nucleoside phosphate can be a pentose sugar, such as ribose. Nucleotide can be, but is not limited to, deoxyribonucleoside triphosphate (dNTP) or ribonucleoside triphosphate (rNTP).

[0125] Nucleotide analogues are compounds that are structurally similar to naturally occurring nucleotides.Nucleotide analogues may have modified phosphate backbones, sugar moieties, nucleic acid bases, or combinations thereof.It will be understood that the use of such analogues results in nucleic acids that may have different base pairing properties, and the interactions that occur when such bases are stacked may be different from those found in natural nucleic acids.

[0126] Unlike amplification such as PCR, which requires temperature cycling, the amplification reaction is preferably isothermal (constant temperature).This method can be used to amplify any suitable template, preferably circular nucleic acid template.Nucleic acid template can be provided in any suitable amount to the reaction, including a minimal amount.

[0127] Preferably, the nucleic acid template is amplified using RCA.

[0128] The one or more polymerase enzymes used in the amplification can be proofreading or non-proofreading nucleic acid polymerases. The nucleic acid polymerases used can be strand-displacing nucleic acid polymerases. The nucleic acid polymerases can be thermophilic or mesophilic nucleic acid polymerases.

[0129] The method may require a highly processive strand-displacing polymerase to amplify the nucleic acid template under conditions for high-fidelity amplification. Polymerase fidelity is the result of accurate replication of the template. In addition to effectively discriminating between correct and incorrect nucleotide incorporation, some polymerases possess 3' to 5' exonuclease activity. This proofreading activity is used to excise the misincorporated base, which is then replaced by the correct base. High-fidelity amplification utilizes polymerases that combine a low misincorporation rate with proofreading activity to provide faithful replication of the template.

[0130] The amplification reaction may use a polymerase that generates a single-stranded amplified nucleic acid after amplification. Thus, the polymerase is capable of strand displacement synthesis.

[0131] In some embodiments, Phi29 DNA polymerase or a Phi29-like polymerase can be used to amplify the template. Alternatively, a combination of Phi29 DNA polymerase and another polymerase can be used.

[0132] The amplification reaction may use low concentrations of primers in one version of the method. The inventors have found that low concentrations of primers are advantageous because they allow the amplification reaction to produce only single-stranded nucleic acid. A primer is a short linear oligonucleotide that hybridizes to a sequence in a template to prime the nucleic acid synthesis reaction. A primer can be any nucleic acid, such as RNA, DNA, non-natural nucleic acid, or a mixture thereof. A primer can include natural, synthetic, or modified nucleotides.

[0133] Alternatively, assuming the template is a double-stranded circular template, a nicking enzyme can be used to create a nick in one strand of the double-stranded template, leaving an entry point for the polymerase to initiate the nucleic acid synthesis reaction using the nicked strand of the template itself.

[0134] Thus, the nucleic acid template is amplified by contacting the template with at least a polymerase and nucleotides and incubating the reaction mixture under conditions suitable for nucleic acid amplification. Amplification of the nucleic acid template can be carried out under isothermal conditions. Additional components can include one or more of a nicking enzyme (nickase), a cofactor (e.g., magnesium ions), a primer, and / or a buffer.

[0135] Rolling circle amplification of a circular template produces linear single-stranded concatemers with multiple adjacent repeats encoded by the template (each referred to herein as a sequence unit). Due to the nature of the template, this means that each sequence unit contains a sequence of interest flanked by formatting elements. This means that there are formatting elements on both ends of the sequence of interest. Each sequence unit may also contain backbone sequence.

[0136] This method relies on sequences in the template encoding formatting elements, one at each end of the sequence encoding the sequence of interest. The formatting elements are two adjacent sequences encoding processing and conformation motifs. The forward formatting element contains a processing motif adjacent to a conformation motif, and the reverse formatting element contains a conformation motif adjacent to a processing motif. The processing motifs contain the recognition site and associated cleavage site for an endonuclease.

[0137] Concatamers can be processed into nucleic acid constructs using endonucleases, where the cleavage sites release the terminal residues of the conformational motifs.

[0138] When the cleavage site of the concatemeric nucleic acid is cleaved by the required endonuclease, this releases the conformational motif from the processing motif, allowing the ends of the single-stranded nucleic acid molecules to be sealed under appropriate conditions.

[0139] The amplification and processing reactions can occur simultaneously, i.e., the endonuclease can be present to process the concatemers as soon as they are formed, or there can be a delay in adding the endonuclease until the amplification has progressed further or is actually completed.

[0140] Thus, the method for generating single-stranded nucleic acid constructs is elegant, efficient, and not limited by the length of the sequence of interest.

[0141] Templates In the template, the sequence encoding the sequence of interest is flanked on either side by sequences encoding the formatting elements, one in the forward direction and the other in the reverse direction. The encoded sequences are nested such that the sequence of interest is adjacent to the conformational motif, which in turn is directly adjacent to the processing motif, and the conformational motif and the processing motif together form the formatting element. Such nesting can be represented as shown in FIG. 1. Thus, the sequences of the processing motif and the conformational motif are adjacent. In other words, the formatting elements on either side of the sequence of interest are in the opposite or mirror image direction, which ensures that the conformational motif is closest to the sequence of interest, while the processing motif is the outermost part of the formatting element.

[0142] The formatting element is unique in the generation of single-stranded nucleic acid molecules, but is not present in the final product in its complete form, since the processing motif is cleaved from the conformational motif. The action of the endonuclease during processing ensures that the cleavage site of the processing motif is cleaved, thus discarding the processing motif. This is therefore a mechanism by which a partially removed useful product is generated, ensuring that the final product contains a minimum of unnecessary sequences, providing more room for the sequence of interest. Thus, the processing motif and the adjacent conformational motif are effectively bound together until the cleavage site is cleaved, releasing the terminal residue of the product. The combination of the processing motif adjacent to the conformational motif is effectively separated by the cleavage site of the endonuclease, allowing the direct generation of single-stranded nucleic acids with blocked ends from longer single-stranded nucleic acid molecules in a one-step process using the endonuclease. The processing motif is removed from the single-stranded nucleic acid by processing with a restriction enzyme, and is not present in the single-stranded nucleic acid with blocked ends.

[0143] The format elements are effectively cleaved by the action of the endonuclease and are therefore partially removed from the final product.

[0144] Processing motif A processing motif comprises a sequence capable of forming a base-paired section that includes a recognition site for an endonuclease and an associated cleavage site, it being understood that the cleavage site may be distant from the recognition site, but both must generally be double-stranded.

[0145] In one format, the processing motif can include at least one region of sequence that can bind to another sequence within the processing motif, thus forming a base-pairing section, and these sections can be considered self-complementary in sequence. These sequences can be adjacent or separated by a spacer element. Such motifs can be designed by including a stretch of complementary sequence in a single-stranded nucleic acid. It will be understood that although both sequences are present on the same strand of the nucleic acid, the design of the molecule ensures that one sequence is in the correct orientation to bind intramolecularly to the other sequence. For example, in DNA, the sequences must run antiparallel in order for base pairs to form. Such motifs are common, for example, among single-stranded genomes of viruses.

[0146] The base-pairing sections of the processing motif may be adjacent such that the sections form a hairpin, etc. The nucleic acid may form an antiparallel double-stranded hairpin-like structure. The hairpin structure is composed of a double-stranded base-pairing region called a stem. Alternatively, the base-pairing sections of the processing motif may include a spacer sequence between the two stretches of base-pairable sequence such that a structure such as a stem loop is formed. The spacer may be of any suitable length. The hairpin may be formed from a nucleic acid sequence that is a palindrome, as defined herein.

[0147] Base-paired or double-stranded sections of a nucleic acid molecule can also have complementary sequences, with base-paired and duplex being further defined herein.

[0148] The base-paired portion of the processing motif contains a recognition site and an associated cleavage site for an endonuclease, which is preferably formed at the foot of the base-paired section so that the entire processing motif can be cleaved from the single strand using the required endonuclease.

[0149] Base pairing occurs between at least two sections of sequence within a single strand. This base pairing can be canonical (i.e., Watson-Crick classical base pairs, which are adenine (A)-thymine (T) in DNA, adenine (A)-uracil (U) in RNA, and cytosine (C)-guanine (G) in both) or non-canonical (i.e., Hoogsteen base pairs, or interactions between carbon-hydrogen and oxygen / nitrogen groups, etc.). These are described elsewhere.

[0150] The template contains one or more sequences that encode processing motifs having any of these properties. The processing motifs can be different sequences.

[0151] The template may include a sequence encoding a first processing motif and a sequence encoding a second processing motif. The first and second processing motifs encoded by the template are positioned at the outer ends of the conformational motif (and within the format element) such that both ends of the sequence of interest terminate in format elements of opposite orientation (forward and reverse).

[0152] Given the nature of the requirement of the processing motif in the single-stranded nucleic acid concatemer (before processing), the sequences of the first and second processing motifs may be the same or different. If they are the same, a restriction site will be formed at the foot of the base-paired section, so that the entire processing motif can be cleaved from the single strand using the required endonuclease. Thus, regardless of the orientation of the processing motif with respect to the sequence of interest (before or after), the entire processing motif can be cleaved from the nucleic acid because the cleavage site is at the foot of the base-paired section, which can also be described as the final base pair of the paired section, or its base.

[0153] Alternatively, the first and second processing motifs of the single-stranded nucleic acid concatemer (before processing) may be different, such that the recognition sites for the endonucleases, including the cleavage sites, are also different, allowing the use of different endonucleases when processing the single-stranded nucleic acid concatemers of the invention.

[0154] Thus, the template may contain sequences encoding the same or different first and second processing motifs.

[0155] Endonucleases are enzymes that cleave phosphodiester bonds in polynucleotide chains, whether they are proteinaceous or composed of nucleic acids such as DNA. In the present invention, a cleaved double-stranded nucleic acid is required to generate a nucleic acid molecule with blocked ends. Therefore, a combination of two endonucleases may be required, each cleaving a single strand. Alternatively, a single enzyme that cleaves both strands can be used. The endonuclease can be, for example, a nicking endonuclease, a homing endonuclease, a guide endonuclease such as Cas9, or a restriction endonuclease. The nicking endonuclease can be a modified restriction endonuclease that is modified to cleave only a single strand.

[0156] In one embodiment, the endonuclease is a restriction endonuclease.

[0157] Restriction endonucleases are enzymes that cleave double-stranded nucleic acids at a cleavage site within or near a specific recognition site. To cleave, all restriction endonucleases make two cuts, passing once through each backbone (i.e., each strand) of the duplex. Since restriction endonucleases require the presence of double-stranded nucleic acid to recognize the recognition site, such a structure is necessary to allow the endonuclease to cleave the nucleic acid. Therefore, the inventors propose the construction of base-paired sections in single-stranded nucleic acids, preferably using self-complementary sequences, so that the single-stranded molecule forms a double-stranded structure that includes the recognition and cleavage sites.

[0158] Restriction endonucleases recognize specific sequences of nucleotides and create double-stranded breaks in the duplex. Recognition sites can also be classified by the number of bases, usually between 4 and 8. Many, but not all, recognition sites are palindromic, and this property is very useful when designing processing motifs, as it aids in the design of sequences and makes them easier to place into base-paired sections. In the single-stranded format, each section that can form a palindrome when base-paired with each other is called an inverted repeat. These two sequences may be separated by a spacer sequence in the single-stranded nucleic acid.

[0159] Restriction endonucleases may be blunt cutters (i.e., cut straight through the base-paired section) or may cut in an offset manner (i.e., cut is offset through the base-paired section). The cleavage site may be within or near the recognition site, so the cleavage site does not have to be part of the recognition site. Thus, the cleavage site is associated with the recognition site, but does not necessarily form a part of it.

[0160] Thousands of restriction endonucleases are known, both naturally occurring and engineered, along with their recognition and cleavage sites. Any suitable recognition and cleavage site can be included in the processing motif. Exemplary restriction endonucleases commonly used in cloning and the like are HhaI, HindIII, NotI, EcoRI, ClaI, BamHI, BglII, DraI, EcoRV, Pst1, SalI, SmaI, SchI and XmaI. Many are commercially available from suppliers such as New England Biolabs and ThermoFisher Scientific.

[0161] For cleavage using an endonuclease to release a conformational motif from a format element of a single-stranded nucleic acid concatemer, it is preferred that the cleavage site be adjacent to the conformational motif of the template, such that the terminal nucleotide of the conformational motif forms the terminus and blocked end of the product single-stranded nucleic acid molecule.

[0162] Encoded within the template are formatting elements, some of which are sequences encoding conformational motifs that are designed to fold into a final single-stranded nucleic acid molecule with blocked ends, which conformational motifs block the ends of the single-stranded nucleic acid molecule (i.e., the 5' and 3' ends for DNA and RNA).

[0163] Conformational motifs include sequences that can form base-paired sections or duplexes within single stranded nucleic acid molecules or with capping oligonucleotides. This base-paired section or duplex can be formed within the concatemer prior to processing with an endonuclease, or can be formed after processing with an endonuclease after the processing motif has been removed from the concatemer. With reference to the figures, these are depicted with the conformational motifs that form the base-paired sections of the concatemeric nucleic acid (see Figures 2, 3, 4, and 5) for ease of reference. These structures may not form until the processing motif is cleaved by an endonuclease. If a capping oligonucleotide is required, the conformational motif will fold appropriately once the capping oligonucleotide is added, as base pairing to this entity may cause the necessary folding.

[0164] A duplex can be formed by base pairing between at least two sections of sequence within a single strand. This base pairing can be canonical (i.e., Watson-Crick classical base pairs, which are adenine (A)-thymine (T) in DNA, adenine (A)-uracil (U) in RNA, and cytosine (C)-guanine (G) in both) or non-canonical (i.e., Hoogsteen base pairs, or interactions between carbon-hydrogen and oxygen / nitrogen groups, etc.). Hoogsteen base pairs allow the formation of specific structures of single-stranded nucleic acid G-rich segments called G-quadruplexes, or C-rich segments called i-motifs. G-quadruplexes usually require four G triplets separated by a short spacer. This allows the assembly of planar quartets composed of stacked associations of Hoogsteen-bound guanine molecules.

[0165] Thus, a conformational motif may comprise a section of a sequence that is self-complementary or complementary to another sequence within a single stranded nucleic acid molecule, ie, the sequence of interest or a spacer sequence within the sequence of interest.

[0166] A conformational motif may comprise a sequence for forming two or more base-paired sections or duplexes, each of which is separated by a spacer sequence of single-stranded nucleic acid, or the base-paired sections or duplexes may form part of a larger structure which may include any one or more of the following: a hairpin, a single-stranded region, a bulge loop, an internal loop, a multi-branched loop or a junction.

[0167] When a conformational motif forms at least one base-paired section or duplex, the terminal residues of the single-stranded nucleic acid molecule are blocked. The terminal nucleotides (or residues) at either end of the single-stranded DNA are hidden / protected. This makes the terminal residues less readily available to single-stranded exonucleases etc.

[0168] The terminal nucleotides of a single-stranded nucleic acid molecule are blocked by being included within a base-paired section or duplex of a conformational motif, and thus lacking a free single-stranded end, or by folding within the topology of the conformational motif, so that the end is fixed rather than free for further interactions.

[0169] In single-stranded nucleic acid products, it is preferred that the ends (terminal nucleotides) are not in single-stranded form: these ends are stabilized by the presence of base pairing between each terminal residue and another portion of the single-stranded nucleic acid.

[0170] The conformational motifs from the concatemeric nucleic acid molecules, when processed, form one end of a single stranded nucleic acid construct, the terminal residue being blocked by the conformational motif.

[0171] In a single stranded nucleic acid construct, each end is blocked by a conformational motif.

[0172] Preferred conformational motifs according to the present invention include hairpins, stem loops, junctions, pseudoknots, ITRs, modified ITRs, synthetic ITRs, i-motifs and sequences capable of folding as a G-quadruplex.

[0173] A hairpin is a structure of a nucleic acid, such as DNA or RNA, that results from base pairing between adjacent complementary sequences of a single strand of the nucleic acid. The adjacent complementary sequences may be separated by a number of nucleotides, e.g., 1-10 or 1-5 nucleotides. An example of this is shown in Figure 2. When a loop of non-complementary sequence is included between two sections of complementary sequence, this forms a hairpin loop or stem loop. The loop, like the stem or double-stranded section, can be of any suitable length. Other similar structures include the lasso.

[0174] The conformational motifs at each end can fold into the same specific structure (i.e., a hairpin, stem-loop, ITR, etc.) or each can be independently designed to fold into a different structure (i.e., the first end is a hairpin and the second end is an ITR).

[0175] As previously discussed, conformational motifs can have additional functions: they can form functional structures such as aptamers, or they can be designed to provide a mechanism for binding single-stranded nucleic acid constructs together in an oligomeric conformation.

[0176] The template also codes for a sequence of interest. In the concatemer and single-stranded nucleic acid construct with blocked ends, the sequence of interest can be any desired nucleic acid sequence of any suitable length. The sequence of interest can be a functional sequence (i.e., directly acts as an aptamer, etc., without further transcription or translation). Alternatively, the sequence of interest can code for a functional sequence. Functional sequences include aptamers, catalytic entities by nucleic acid enzymes, including ribozymes, non-coding RNAs (ncRNAs), including microRNAs (miRNAs), short interfering RNAs (siRNAs), and piwi-interacting RNAs (piRNAs).

[0177] The sequence of interest may be able to function as donor nucleic acid for gene editing in both animals and plants.Exemplary methods of gene editing include CRISPR gene editing and transcription activator-like effector nuclease (TALEN)-based methods.When the sequence of interest is donor nucleic acid, it may be necessary to include a sequence or element to allow the excision of donor nucleic acid by the required mechanism.

[0178] The sequence of interest can be a transgene, such as a gene or genetic material for expression in a cell. The transgene is operably linked to a promoter sequence in an expression cassette.

[0179] The sequence of interest may include a sequence that codes for a therapeutic product. The therapeutic product may be a DNA aptamer, a protein, a peptide, or an RNA molecule such as a small interfering RNA. To provide therapeutic utility, such a sequence of interest may include an expression cassette that includes one or more promoter or enhancer elements and a gene or other coding sequence that codes for an mRNA or protein of interest. The expression cassette may include a eukaryotic promoter that is operably linked to the sequence that codes for the protein of interest, and optionally an enhancer and / or a eukaryotic transcription termination sequence.

[0180] The sequence of interest can be used to generate DNA for expression in a host cell, particularly to generate a DNA vaccine. A DNA vaccine usually encodes a modified form of the DNA of an infectious organism. The DNA vaccine is administered to a subject, where it expresses a selected protein of the infectious organism and initiates an immune response against that protein, which is usually protective. A DNA vaccine may also encode a tumor antigen in a cancer immunotherapy approach. Any DNA vaccine can be used as the sequence of interest.

[0181] The process of the present invention can also produce other types of therapeutic DNA molecules, such as DNA molecules used in gene therapy. For example, such DNA molecules can be used to express a functional gene when a subject has a genetic disorder caused by a malfunctioning version of that gene. Examples of such diseases are well known in the art.

[0182] The portion of the template encoding the sequence or conformational motif of interest preferably lacks a bacterial origin of replication, lacks resistance genes (i.e., in the case of antibiotics), lacks CpG islands (except for DNA vaccines, where the same may be useful), and lacks methylation of cytosines and adenines or other markers of foreign DNA. However, these entities may be present outside of the sequence and conformational motif of interest as the remainder of the template is processed and removed from the product.

[0183] The template is preferably circular or capable of being circularized. The template may be double-stranded or single-stranded.

[0184] If the template is double-stranded, it is preferred to include a sequence for a nicking enzyme before the first processing motif. These enzymes, also known as nicking endonucleases, hydrolyze only one strand of the duplex, generating a nucleic acid molecule that is "nicked" rather than cut. This provides a starting point for rolling circle amplification without the need for additional primers, and ensures that only one strand of the nucleic acid concatemer is generated in the amplification reaction. Such enzymes are commercially available, for example from New England Biolabs and Thermo Fisher Scientific. These enzymes are sufficiently specific that recognition and cleavage sites can be engineered into the relevant strand of the template so that the correct strand is used directly as the template.

[0185] The template can be any suitable nucleic acid, either natural, such as DNA or RNA, or man-made as discussed above. Preferably, the template is DNA.

[0186] Template Amplification To generate a single stranded nucleic acid construct, the template must be enzymatically amplified.

[0187] The template can be amplified with one or more polymerase enzymes that can use the template to synthesize a complementary nucleic acid copy if sufficient raw materials or substrates (such as nucleotides) and cofactors (such as metal ions) are provided to amplify the nucleic acid.

[0188] Any suitable polymerase enzyme can be used for this amplification step, and it is possible to use one enzyme or a combination of enzymes.

[0189] The enzyme may be a DNA or RNA polymerase depending on the nature of the template, or an artificial, modified, engineered, or mutated polymerase for using synthetic templates or for producing synthetic single-stranded nucleic acids.

[0190] Amplification preferably proceeds via a strand displacement method. This is an isothermal method that does not require repeated cycles of heating and cooling (as in PCR), but the polymerase enzyme can displace any strand annealed to the template. Strand displacing polymerases are known, including Phi29, Deep Vent®, BST DNA polymerase I, and their variants. This means that multiple polymerases can act on the same template simultaneously, each displacing a nascent strand generated by the previous polymerase.

[0191] The most preferred strand displacement amplification technique is rolling circle amplification (RCA), in which a strand-displacing polymerase continuously progresses around a circular template while extending the nascent oligonucleotide, thereby generating long concatemeric strands of nucleic acid.

[0192] Preferably, the amplification reaction can be initiated on a double-stranded circular template by nicking the template with a nicking endonuclease. Such enzymes are discussed above. By nicking one strand of the double-stranded template, this opens the template for the polymerase to bind and can utilize the free 3' end created to extend this strand into a concatemeric nucleic acid by processing back and forth around the circular template.

[0193] The use of nicking sites and nicking endonucleases on the template allows for a method to generate only single-stranded concatemers from RCA, as the enzyme is used to cleave only one backbone strand, preventing amplification of the opposite strand.

[0194] Therefore, the use of a nicking site within the template is preferred as it allows for the generation of the desired product and prevents the undesired amplification of the complementary strand of a double-stranded template.

[0195] Alternatively, the inventors have found that amplification can be forced to proceed to create large amounts of only one of the strands of a double-stranded template using very small amounts of specific primers that are designed to anneal to the desired template strand (and not its complementary strand). In this aspect, only picomolar amounts of primer are required. Thus, primers can be supplied in amounts from 1 pM to 100 nM.

[0196] If the template is single-stranded, a primer can be used to initiate rolling circle amplification. Preferably, the primer is designed to anneal only to the template and not to the concatemer nucleic acid molecule, thus ensuring that only one species of concatemer is generated.

[0197] Therefore, we devised a method to ensure that RCA amplifies the template and produces only the desired concatemers that are the correct species for the generation of single-stranded nucleic acid constructs, and not the complementary strand. Making the complementary strand would result in 50% waste amplification reactions and would also make the synthesis of single-stranded constructs much more difficult. This is because the presence of complementary concatemers essentially results in the formation of double-stranded nucleic acid.

[0198] The template is contacted with at least one polymerase. One, two, three, four, or five different polymerases can be used. The polymerase can be any suitable polymerase that synthesizes a polymer of nucleic acid. The polymerase can be a DNA or RNA polymerase. Any polymerase can be used, including any commercially available polymerase. Two, three, four, five, or more different polymerases can be used, for example, one that provides a proofreading function and one or more that do not. Polymerases with different mechanisms can be used, for example, strand-displacing polymerases and polymerases that replicate nucleic acids by other methods. A suitable example of a DNA polymerase that does not have strand-displacing activity is T4 DNA polymerase.

[0199] The polymerase may be very stable, so that its activity is not substantially reduced by long incubation under processing conditions. Thus, the enzyme preferably has a long half-life under a set of processing conditions, including but not limited to temperature and pH. The polymerase also preferably has one or more properties suitable for manufacturing processes. The polymerase preferably has high fidelity, for example by having proofreading activity. Furthermore, the polymerase preferably exhibits high processivity, high strand displacement activity, and low Km for nucleotides and nucleic acids. The polymerase may be capable of using circular and / or linear DNA as a template. The polymerase may be capable of using double-stranded or single-stranded nucleic acids as a template. The polymerase preferably does not exhibit exonuclease activity unrelated to its proofreading activity.

[0200] Those skilled in the art will appreciate that commercially available polymerases, such as Phi29 (New England Biolabs, Inc., Ipswich, Mass., US), Deep Vent® (New England Biolabs, Inc.), Bacillus stearothermophilus (Bst) DNA polymerase I (New England Biolabs, Inc.), Klenow fragment of DNA polymerase I (New England Biolabs, Inc.), M-MuLV reverse transcriptase (New England Biolabs, Inc.), Vent® (exo-minus) DNA polymerase (New England Biolabs, Inc.), Vent® DNA polymerase (New England Biolabs, Inc.), Deep Vent® (exo-) DNA polymerase (New England Biolabs, Inc.), large fragment of Bst DNA polymerase (New England Biolabs, Inc.), and the like, may be used. It can be determined whether a given polymerase exhibits characteristics as defined above by comparing it to the properties exhibited by a high fidelity fusion DNA polymerase (e.g., Pyrococcus-Yke, New England Biolabs, Mass.), Pfu DNA polymerase from Pyrococcus furiosus (Strategene, Lajolla, Calif.), Sequenase™ variants of T7 DNA polymerase, T7 DNA polymerase, T4 DNA polymerase, DNA polymerase from Pyrococcus species GB-D (New England Biolabs, Mass.), or DNA polymerase from Thermococcus litoralis (New England Biolabs, Mass.).

[0201] Alternatively, the polymerase can be a DNA-dependent RNA polymerase. Exemplary enzymes include T3 RNA polymerase, T7 RNA polymerase, Hi-T7™ RNA polymerase, SP6 RNA polymerase, E. coli poly(A) polymerase, E. coli RNA polymerase, and E. coli RNA polymerase, holoenzyme (all available from NEB).

[0202] When high processivity is referred to, it usually refers to the average number of nucleotides added by a polymerase enzyme per association / dissociation with a template, i.e., the length of primer extension resulting from a single association event.

[0203] Strand displacement polymerases are preferred. Preferred strand displacement polymerases are Phi 29, Deep Vent and Bst DNA polymerase I, or any mutants thereof. "Strand displacement" describes the ability of a polymerase to displace a complementary strand when it encounters a region of double-stranded DNA during synthesis. Thus, the template is amplified by displacing the complementary strand and synthesizing a new complementary strand. Thus, during strand displacement replication, the newly replicated strand is displaced to make way for the polymerase to further replicate the complementary strand. The amplification reaction is initiated when the free end of a primer or single-stranded template anneals to a complementary sequence on the template (both are priming events). As nucleic acid synthesis proceeds, if it encounters an additional primer or other strand annealed to the template, the polymerase will displace it and continue elongating that strand. It should be understood that strand displacement amplification methods differ from PCR-based methods in that cycles of denaturation are not essential for efficient amplification, since the double-stranded template does not present an obstacle to continued synthesis of new strands. Strand displacement amplification may only require one initial heating cycle to denature the initial template, if double-stranded, and to allow primers, if used, to anneal to their primer binding sites. Following this, no further heating or cooling is required, so the amplification may be described as isothermal. In contrast, PCR requires a cycle of denaturation (i.e., raising the temperature to 94°C or higher) during the amplification process to melt the double-stranded DNA and provide new single-stranded template. During strand displacement, the polymerase displaces a strand of nucleic acid that has already been synthesized.

[0204] The strand-displacing polymerase used in the methods of the invention preferably has a processivity of at least 20 kb, more preferably at least 30 kb, at least 50 kb, or at least 70 kb or more. In one embodiment, the strand-displacing DNA polymerase has a processivity comparable to or greater than that of phi29 DNA polymerase.

[0205] Contact of the template with either the polymerase and nickase or the primer can occur under conditions that promote annealing of the primer to the template. Conditions include the presence of single-stranded DNA that allows hybridization of the primer. Conditions also include temperature and buffer that allows annealing of the primer to the template. Depending on the nature of the primer, appropriate annealing / hybridization conditions can be selected. Examples of preferred annealing conditions used in the present invention include 30 mM Tris-HCl pH 7.5, 20 mM KCl, 8 mM MgCl 2 Annealing can be performed following denaturation using heat by gradually cooling to the desired reaction temperature.

[0206] The template and polymerase also contact the nucleotides. The combination of the template, polymerase and nucleotides forms a reaction mixture. The reaction mixture may also include one or more primers, or a nicking enzyme (nickase). The reaction mixture may also independently include one or more metal cations or any other cofactors required for nucleic acid synthesis.

[0207] A nucleotide is a monomer or single unit of a nucleic acid; a nucleotide is composed of a nitrogenous base, a five-carbon sugar (ribose or deoxyribose), and at least one phosphate group. Any suitable nucleotide can be used.

[0208] The nucleotides can be present as free acids, their salts or chelates, or a mixture of free acids and / or salts or chelates.

[0209] The nucleotides can be present as monovalent metal ion nucleotide salts or divalent metal ion nucleotide salts.

[0210] The nitrogenous bases can be adenine (A), guanine (G), thymine (T), cytosine (C), and / or uracil (U). The nitrogenous bases can also be modified bases such as 5-methylcytosine (m5C), pseudouridine (Ψ), dihydrouridine (D), inosine (I), and / or 7-methylguanosine (m7G).

[0211] Preferably, the five carbon sugar is deoxyribose, such that the nucleotide is a deoxynucleotide.

[0212] Nucleotides may be in the form of deoxynucleoside triphosphates, denoted by dNTP. This is a preferred embodiment of the present invention. Suitable dNTPs may include dATP (deoxyadenosine triphosphate), dGTP (deoxyguanosine triphosphate), dTTP (deoxythymidine triphosphate), dUTP (deoxyuridine triphosphate), dCTP (deoxycytidine triphosphate), dITP (deoxyinosine triphosphate), dXTP (deoxyxanthosine triphosphate), and derivatives and modified versions thereof. Preferably, dNTPs include one or more of dATP, dGTP, dTTP or dCTP, or modified versions or derivatives thereof. Preferably, mixtures of dATP, dGTP, dTTP and dCTP or modified versions thereof are used.

[0213] The nucleotides may be in solution or provided in lyophilized form, with a solution of nucleotides being preferred.

[0214] The nucleotides can be provided in a mixture of one or more suitable bases, including any newly designed artificial base, preferably one or more of adenine (A), guanine (G), thymine (T), cytosine (C). In the process of synthesizing nucleic acids, two, three, or preferably all four nucleotides (A, G, T, and C) are used.

[0215] Concatemer The single-stranded concatemers produced can also be processed into new, single-stranded nucleic acids with blocked ends that can contain the sequence of interest.

[0216] A concatemer is a nucleic acid molecule that has repeating units of sequence units present in the template. Each sequence unit contains a sequence of interest flanked on both sides by formatting elements, as described above. The sequence units may also contain backbone sequences encoded by the template that are not ultimately present in the nucleic acid construct of the present invention.

[0217] Concatameric nucleic acid molecules can contain multiple sequence units, for example, 10, 50, 100, 200, 500, or 1000 or more sequence units in a contiguous tandem. Concatameric molecules can be at least 5 kb in size, at least 50 kb, at least 100 kB, or up to 200 kB in length.

[0218] Processing of concatemeric nucleic acid molecules Once the template is amplified, or even during amplification, the concatemeric nucleic acids can be processed into single-stranded nucleic acid constructs using the necessary endonucleases that cleave one or more processing sites.

[0219] Therefore, it is preferable that the processing motif can form base-pairing portions while in the form of a concatemeric nucleic acid. Thus, the processing motif can be designed so that base-pairing is formed under conditions suitable for isothermal amplification. When these base-pairing portions are formed in the concatemeric nucleic acid, recognition sites for endonucleases are formed, along with the necessary cleavage sites. This elegant system allows the processing of the concatemer, despite the fact that it is only a single strand of nucleic acid. It is the design of the template that allows the formation of processing sites in the concatemeric nucleic acid, allowing a single step of processing this concatemer by the addition of one or more endonucleases.

[0220] Endonuclease can be added when the amplification reaction is completed, or can be added while the amplification reaction is in progress or at the beginning. To ensure that concatemeric nucleic acid is processed quickly, it is preferable that the amplification reaction is in progress before the endonuclease is added. Alternatively, the amplification process can be completed (i.e., the template is exhausted, the nucleotides are exhausted, the reaction mixture is too viscous) before the endonuclease is added.

[0221] Upon endonuclease cleavage, the conformational motifs act to cleave the concatemers into single-stranded nucleic acid constructs with blocked ends, and by-products are generated that consist of the template backbone in association with processing motifs. Because the by-products have unblocked ends, they can be removed using single-stranded exonucleases.

[0222] The invention will now be described with reference to the following non-limiting examples. EXAMPLES

[0223] Example 1: Generation of Nucleic Acid Constructs: Template: Template A (Figure 9, SEQ ID NO:1). The template contains a nicking site, a processing motif adjacent to a conformational motif, a sequence of interest, a second conformational motif adjacent to a second processing motif, and a backbone of similar size to the sequence of interest, which contains an additional endonuclease target site that cleaves only dsDNA.

[0224] The sequence of template A is shown as SEQ ID NO:1 in the associated sequence listing.

[0225] Nicking reaction in 20 μl ●4 μl of template (stock concentration 1 μg / μl) 13μl of water 2 μl CutSmart Buffer (NEB) 1 μl of nickase (Nb.BsrDI, NEB) Incubate at 37°C for 180 minutes, then at 80°C for 20 minutes.

[0226] Amplification reaction in 1000 μl ●4μl template (stock concentration 0.2μg / μl) 100μl Buffer-10x stock solution: -300mM Tris pH7.9 -300mM KCl -50 mM(NH 4 ) 2 SO 4 -100mM MgCl 2 837 μl ddH 2 O 20 μl of dNTP (stock solution 100 mM (Bioline)) ●35μl of SSB (stock solution 5μg / μl (E.coli SSB, in-house produced)) 2 μl of inorganic pyrophosphatase (stock solution 2 U / μl (enzyme)) 2 μl phi29 DNA polymerase (stock solution 100 U / μl (Enzymatics)) → Incubate at 30℃ for 16 hours

[0227] Processing Reaction ● 1000μl amplification reaction 20 μl of MlyI (stock solution 10 U / μl) → Incubate at 37℃ for 180 minutes

[0228] result: The gel photograph shown in FIG.

[0229] This gel shows the digestion products of the RCA reaction. Wells on the left: Thermo Scientific Gene Ruler 1 kb Plus DNA ladder (sizes in bp on the left). Wells on the right: RCA processed with MlyI (expected size in nt (nucleotides) on the right). Backbone and product bands of equal size do not stain brightly because they are predominantly single stranded. The "signature" lower band that would indicate the product is double stranded is not seen (the MlyI site is in the backbone and cuts the dsDNA dropping the backbone bands to 1597 and 407 base pairs).

[0230] Example 2: Testing the stability of terminal nucleotides of nucleic acid constructs using exonucleases This example tests whether novel nucleic acid constructs with blocked ends provide significant exonuclease resistance compared to nucleic acids whose ends do not form defined structures (standard single-stranded DNA).

[0231] Exonuclease Stability Test: In this study, five product molecules with different conformational motifs were generated: i. ssDNA without conformational motifs (and therefore no fixed terminal nucleotides), ii. ssDNA with a trinucleotide loop (GAA) conformation motif that anchors the terminal nucleotides at both the 3' and 5' ends of the base-paired duplex stretch; iii. G-quadruplex conformation motif (TTAGGG) 4 and an additional sequence that forms a section of intramolecular base pairing to the sequence of interest and includes a terminal nucleotide within the section of double-stranded nucleic acid; iv. ssDNA with a G-quadruplex conformation motif at both the 3' and 5' ends without additional base-pairing sections, hence the quadruplex (TTAGGG). 4 , which relies on fixing each terminal nucleotide by including it within v. ssDNA with a pseudoknot conformation motif at both the 3' and 5' ends lacking additional base-pairing sections, thus relying on fixing each terminal nucleotide by incorporating it within a pseudoknot.

[0232] Nucleic acid molecules were diluted to 100ng / μl in 100mM KCl and heat denatured (95°C, cooled to room temperature) to allow the conformational motifs to conform properly. 10μl of each construct was used for subsequent exonuclease testing in a final volume of 50μl in 1x Exonuclease VII Reaction Buffer (NEB; 50mM Tris-HCl, 50mM sodium phosphate, 8mM EDTA, 10mM 2-mercaptoethanol, pH 8.0). Reactions were incubated for 30 min at 37°C in the presence or absence of 100U / ml Exonuclease VII (NEB). Products were resolved on an agarose gel containing GelRed dye (Figure 6). [Table 3] [Table 4]

[0233] result: ssDNA lacking conformational motifs anchoring the 3′ and 5′ ends was almost completely digested in the presence of exonuclease VII within the short window of the experiment (Fig. 6 , lanes 1–2).

[0234] Any ssDNA that contains conformational motifs for locking the 3' and 5' terminal nucleotides (as described above in (ii) to (v)), i.e., single-stranded nucleic acid constructs with blocked ends, are more resistant to exonuclease digestion than ssDNA.

[0235] The construct depicted as (ii) (lanes 3-4) was end-blocked by including it within a base-paired duplex stretch of sequence, which demonstrated resistance to exonucleases.

[0236] Using the G-quadruplex conformation motif, two different nucleic acid constructs were created. The construct described in (iv) (lanes 7-8) blocked the ends by including them within a G-quadruplex. The construct described in (iii) (lanes 5-6) contains an additional section of double-stranded nucleic acid in which the terminal nucleotides are involved in base pairing. In this experiment, the addition of the extra double-stranded sequence appeared to aid resistance to exonucleases. This demonstrates that, based on the desired properties of the blocked ends, the conformation can be engineered to suit the specific conditions in which the nucleic acid construct may be used.

[0237] The construct depicted as (v) (lanes 9-10) had the ends blocked by including them within pseudoknots, which appeared to be moderately resistant to exonucleases under the conditions tested.

[0238] These data indicate that end capping can retard exonucleolytic degradation and that construct structure can be engineered to enhance the stability of nucleic acid constructs by altering the sequence of conformational motifs.

[0239] Example 3: Testing the stability of terminal nucleotides of nucleic acid constructs in the presence of cell extracts This experiment was designed to test whether novel nucleic acid constructs with blocked ends offered significant resistance in the presence of cell extracts compared to nucleic acids whose ends do not form defined conformations (in these examples, standard single-stranded DNA).

[0240] Preparation of cell extracts: HEK293T cells (Clontech Z2180N) grown in Eagle's minimum essential medium were incubated at 37°C and 5% CO 2 (supplemented with 10% FBS, glutamine, non-essential amino acids, and antibiotics). Three fully confluent 10 cm plates were washed with PBS. Cells were harvested and lysed using 10 ml of 1x cell lysis buffer (Promega E397A). Approximately 2,000,000 cells per ml of suspension were obtained. After incubation at room temperature for 5 min, the suspension was clarified by centrifugation (4000 rpm for 5 min). Glycerol was added to 20% and the cell extract was aliquoted and frozen at -80°C.

[0241] Cell Extract Stability Testing: All five nucleic acid constructs (prepared in Example 2) were diluted to 100 ng / μl in 100 mM KCl and heat denatured (95°C, then cooled to room temperature) to allow the conformational motifs to form the appropriate conformation. 2 and 10 mM Tris pH 7.5, with 5% of the thawed cell extract added. Samples were incubated for 24 or 72 hours, and products were resolved on an agarose gel containing GelRed dye (Figure 7). [Table 5] [Table 6]

[0242] result: ssDNA lacking conformational motifs sealing the 3′ and 5′ ends was gradually digested to near completion in the presence of 5% cell extract (lanes 1, 6, and 11), and small amounts were detected after 72 h of incubation.

[0243] All other nucleic acid constructs with blocked ends provided significantly greater stability in the presence of extract.

[0244] Under the conditions tested, blocking the 3' and 5' ends by inclusion within a section or stretch of double-stranded nucleic acid formed by base pairing appeared to provide the greatest amount of resistance to degradation. Results for constructs (ii) and (iii), lanes 2, 7, 12, and lanes 3, 8, 13, respectively, showed the greatest stability.

[0245] However, the remaining constructs showed some resistance, indicating that it is possible to secure the terminal residues without directly participating in base pairing. The G-quadruplex version shown in (iv) showed relatively strong stability (lanes 4, 9, 14), but the level of resistance to degradation of the molecules with the pseudoknotted conformational motif (lanes 5, 10, 15) was the lowest among the blocked-end structures.

[0246] To exclude the possibility that certain bands appeared as artifacts from cell extracts, a control containing 5% extract without added DNA was incubated for 72 h (lane 16).

Claims

1. A nucleic acid template for cell-free in vitro production of a single-stranded nucleic acid construct having blocked ends, comprising at 5' to 3' of the single-stranded nucleic acid: i) the first processing motif and adjacent ii) a first conformational motif; iii) a sequence of interest; iv) a second conformational motif adjacent thereto; v) a sequence encoding an element of a second processing motif, The processing motif comprises a self-complementary sequence capable of forming a base-pairing section comprising a recognition site and associated cleavage site of an endonuclease, and the conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond; A nucleic acid template, each blocked end comprising a terminal nucleotide.

2. The nucleic acid template of claim 1 , wherein the cleavage site within a processing motif is adjacent to a conformational motif.

3. 3. The nucleic acid template of claim 1 or 2, wherein the cleavage site for the endonuclease within the processing motif is at the terminal base pair of the base-paired section.

4. 4. The nucleic acid template of claim 1, wherein the conformational motif comprises a sequence capable of forming an intramolecular hydrogen bond that acts to anchor a terminal nucleotide to the end of the single-stranded nucleic acid construct.

5. The single stranded nucleic acid construct comprises: i) aptamers, ii) a nucleic acid enzyme.

6. The nucleic acid template of any one of claims 1 to 5, wherein the intramolecular hydrogen bonds are formed between nucleotide bases in the sequence of the conformational motif, enabling the conformational motif to adopt a conformation.

7. 7. The nucleic acid template of claim 6, wherein the intramolecular hydrogen bonds between the nucleotide bases comprise Watson-Crick base pairs, Hoogsteen base pairs, or non-canonical base pairing.

8. The conformational motif is i) quadruplex, ii) hairpins; iii) a cross shape; iv) a stem loop, and / or v) pseudoknot, or a combination of two or more of the conformations thereof.

9. The nucleic acid template of any one of claims 1 to 8, wherein the blocked end comprises intramolecular base pairing of the terminal nucleotide.

10. The nucleic acid template of any one of claims 1 to 9, wherein the blocked end comprises including the terminal nucleotide in an ITR structure having a double-stranded D region.

11. 1. A method for producing a single stranded nucleic acid molecule having blocked ends, comprising: (a) Amplification of a circular template using a polymerase capable of rolling circle amplification, the template comprising: i) the first processing motif and adjacent ii) a first conformational motif; iii) a sequence of interest; iv) a second conformational motif adjacent thereto; v) a sequence encoding an element of a second processing motif, amplifying, wherein the processing motif comprises a self-complementary sequence capable of forming a base-pairing section comprising a recognition site for an endonuclease comprising a cleavage site, said conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond, said amplification producing single-stranded nucleic acid concatemers; (b) processing said single-stranded nucleic acid concatemers using one or more endonucleases that recognize said cleavage sites of one or more of said processing motifs.

12. A method for producing a single-stranded nucleic acid molecule with blocked ends according to claim 11, wherein the template is as described in any one of claims 1 to 10.

13. A single stranded nucleic acid concatemer having two or more repeats of a sequence unit, said sequence unit being i) the first processing motif and adjacent ii) a first conformational motif; iii) a sequence of interest; iv) a second conformational motif adjacent thereto; v) a second processing motif, A single-stranded nucleic acid concatemer, wherein the processing motif comprises a self-complementary sequence capable of forming a base-pairing section that comprises an endonuclease recognition site and associated cleavage site, and said conformational motif comprises at least one sequence capable of forming an intramolecular hydrogen bond.

14. 12. A method for producing a single-stranded nucleic acid molecule having blocked ends according to claim 11, wherein at least one blocked end forms a G-quadruplex.

15. 12. A method for producing a single-stranded nucleic acid molecule having blocked ends according to claim 11, wherein at least one blocked end forms an ITR structure having a double-stranded D section.

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