Novel self-cleaving DNAzymes and selection processes

Abstract

The present invention provides a novel in vitro method for selecting DNAzymes having self-cleavage activity, novel DNAzymes, and their use for generating single-stranded DNA.

Description

[Background technology]

[0001] Biomolecular and therapeutic applications are meeting the demand for the development of progressively more sophisticated and complex DNA origami. DNA origami is a technique that enables the fabrication of nanostructures with designer-defined shapes and dimensions at the nanometer scale, relying on the programmability and self-assembly properties of DNA. In recent years, this has motivated a shift towards high-precision DNA origami design that includes functional domains (Gerling T. et al., 2015) and enables higher-level assemblies (Sigl, C. et al., 2021, Pumm, AK. et al., 2022).

[0002] EP3516055B1 describes a phage-mediated method for the scalable biotechnological generation of single-stranded DNA (ssDNA) using self-cleaved DNA sequences.

[0003] DNAzymes are DNA molecules that form structures capable of catalyzing chemical reactions (Breaker et al., 1997). Some DNA molecules catalyze self-processing reactions (Carmi et al., 1996). Such DNAzymes can be used to create DNA constructs that, when exposed to specific reaction conditions, become modified based on their intrinsic catalytic activity. For example, there are manipulated self-cleaved DNAzymes using oxidation (Carmi et al., 1996), depurine reactions (Sheppard et al., 2000), or hydrolysis (see, e.g., Chandra et al., 2009) mechanisms, created using various directed evolutionary strategies.

[0004] Two classes of engineered self-cleaving DNAzymes that hydrolyze DNA rapidly and with high sequence specificity have been described (Gu et al., 2013). One such DNAzyme, named I-R3, has a small catalytic core consisting of 17 nucleotides adjacent to either one or two double-stranded substructures. A representative of this class of DNAzymes is obtained by fermenting Zn at a near-neutral pH in millimolar concentrations. 2+ When incubated in the presence of approximately 1 minute -1 Observation rate constant (k) of DNA hydrolysis obs It exhibits a half-life of approximately 40 seconds. This DNAzyme cleaves the phosphoester bond between the 3' oxygen and the phosphorus center of the ApA bond, generating a 3' cleavage fragment with a 5' phosphate group.

[0005] When used for ssDNA generation, the I-R3 DNAzyme generates a target ssDNA strand with a constant end sequence motif (the "scar" sequence AG at 5' and ACGTTGA at 3') derived from the DNAzyme sequence itself. However, attempts to accommodate a complementary sequence of the 7nt-long 3' motif would introduce multiple long repeat sequences into the DNA origami scaffold strand. This may impair the addressability of the DNA during the structural assembly reaction, resulting in the formation of structures that are little to no structure generated and / or incorrectly folded. Alternatively, leaving ssDNA overhangs at the break points would be incompatible with functional domains that utilize blunt-end stacking interactions for higher-order assemblies. Furthermore, homologous recombination repair (HDR) applications may be negatively affected by non-homologous ssDNA overhangs.

[0006] Qiao Zhang et al., 2022, described the II-R2 / 3 DNAzyme, which exhibits complete generality of site-directed DNA cleavage while also removing overhang motifs. However, the significantly larger core size (59 nt) of II-R2 / 3 compared to the core size (40 nt) of IR3, along with the insufficient cleavage yield observed with II-R2 / 3 (capped at approximately 90%), significantly impairs its usefulness for successfully mass-producing multiple (more than 30) arbitrary ssDNAs required for DNA origami.

[0007] Most reported in vitro selection strategies for DNAzymes can be assigned to three main groups. The first group encompasses entirely new DNA and RNA self-cleaving DNAzymes designed to perform cleavage within stationary sequences at a certain distance from the catalytic core (Breaker, R. et al., 1994; Carmi, N. et al., 1996). The second group uses two stationary adjacent sequences as binding arms to orient the DNAzyme core to a predetermined region of the substrate (Chandra, M. et al., 2009; Lee, Y. et al.; 2017). The third group consists of in vitro selection methods based on the circularization of DNA libraries (Gu, H. et al., 2013; Zhang C. et al., 2021). By leveraging the freedom of selection of cleavage sites within the evolving catalytic core, this approach has yielded one of the most active DNA-cleaving DNAzymes.

[0008] Despite the abundance of reported methods, none allow for the intentional generation of scarless autocleavage DNAzymes from random space. Available approaches either rely on existing DNAzymes with desired cleavage sites (Qiao Zhang et al., 2022) or apply selective pressure to cleavage sites within known substrate sequences (Xiao Y. et al., 2012). [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] There remains a demand for improved DNAzymes that minimize "scarring" at the 5' and 3' ends of ssDNA while maintaining high cleavage efficiency. Furthermore, there remains a demand for novel DNAzyme selection processes that enable the selection of DNAzymes from random space, independent of specific cleavage mechanisms, existing DNAzymes, and / or specific substrate sequences. [Means for solving the problem]

[0010] In one aspect, the present invention relates to a method for selecting a DNAzyme having self-cleavage activity, for example, an in vitro method, wherein the method is (a) To provide a plurality of single-stranded DNA strands, Each single-stranded DNA strand contains a core region of random nucleotides adjacent to its 5' constant region 1 and its 3' constant region 2. Each constant region includes a primer binding portion, (b) Subjecting multiple single-stranded DNA strands to self-cleavage conditions, (c) Isolating cleavage product 2, and optionally isolating cleavage product 1, The cleavage product 2 includes a steady-state region 2 and a core region. The cleavage product 2 contains DNA self-cleavage activity, and The cleavage product 1 includes a steady-state region 1, Preferably, a break in the single-stranded DNA strand occurs between the catalyst core and the steady region 1. or The cleavage product 2 includes a steady-state region 2, a core region, and a portion of the steady-state region 1. The cleavage product 2 contains DNA self-cleavage activity, and The cleavage product 1 includes the remaining portion of the steady-state region 1. Preferably, when a break in the single-stranded DNA strand occurs within the constant region 1, isolation is performed. (d) To generate a complement of cleavage product 2, (e) Optionally, separate the complement of cleavage product 2, (f)Ligate the 3'-end of the complement of cleavage product 2 to the complement of cleavage product 1, thereby obtaining the complement of the single-stranded DNA strand of step (a) having DNA self-cleavage activity, (g)Optionally, separate the complement of the single-stranded DNA strand, (h)Amplify the complement of the single-stranded DNA strand, thereby obtaining a DNA strand containing DNA self-cleavage activity, (i)Optionally, separate the DNA strand containing self-cleavage activity, (j)Optionally, subject the DNA strand containing self-cleavage activity to an error-prone (EP-PCR) step, (k)Optionally, subject the DNA strand containing self-cleavage activity of step (h), optionally step (i) or step (j) to one or more rounds of steps (b)-(h), and optionally one or more rounds of steps (i) and / or (j), to provide a method.

[0011] In a further aspect, the present invention relates to a DNAzyme obtainable by the method according to the present invention, preferably wherein the DNAzyme contains self-cleavage activity between the catalytic core and the constant region 1 or within the constant region 1.

[0012] In a further aspect, the present invention relates to a DNAzyme having the following sequence: 5'- N & G Y $ Y # GT N $ Y # ACGC Y # Y $ YGTCTTATCGGTT Y $ Y $ N # N -3' (SEQ ID NO: 1), In the sequence, A is a nucleotide having the base adenine, C is a nucleotide having the base cytosine, G is a nucleotide having the base guanine, T is a nucleotide that has a thymidine base. N is independently A, C, G, or T. Y is independently C or T, $ However, independently, preferably T, # However, independently, preferably C, & However, independently, preferably G, The DNAzyme contains self-cleavage activity and optionally includes additional nucleotide N # However, this relates to a DNAzyme located between nucleotide positions 29 and 30 of sequence number 1, where the nucleotide numbering is in the direction from 5' to 3' of sequence number 1.

[0013] In a further embodiment, the present invention relates to the use of the DNAzyme of the present invention in the generation of single-stranded DNA molecules, preferably the use in which the single-stranded DNAzyme is used in DNA nanotechnology and / or the single-stranded DNAzyme is used in genome editing. [Brief explanation of the drawing]

[0014] [Figure 1] This document presents a general scheme for a novel in vitro selection process for self-cleaving DNAzymes. The in vitro selection procedure can be divided into four main steps: cleavage reaction (I), generation of reverse complement of the active fraction (II), repair of the 5' stationary sequence by ligation (III), and PCR amplification (IV). Cleavage sites can be selected within the 5' stationary sequence, including cleavage between the 5' stationary sequence and the core (the latter indicated by a scissors symbol). The ligation adapter sequence must be designed as appropriate. To obtain a robust cleaver regardless of its mode of action, a strategy was implemented that allows cleavage of any catalyst type, as long as it results in DNA cleavage at the intended site. Therefore, instead of starting work directly with the enrichment pool, sequence data was transferred to a new DNA strand in the form of reverse complement. [Figure 2]This paper describes a specific embodiment of a novel in vitro partitioning process for a self-cleaving DNAzyme that applies evolutionary pressure to the cleavage of the first nucleotide (light gray) of the 5' terminal constant region 1. The initial DNAzyme library sequence was constructed in a fairly conventional manner, with a randomized core region (black) adjacent to two constant regions 1 and 2 (gray). The latter were partially complementary to each other, forming a terminal hybridization stem for the core loop, thus mimicking a DNAzyme cassette setup. The non-hybridization portion served as a primer-binding region for the amplification step. The initial library consisted of four sublibraries, each featuring one of four possible base pairs at a pre-selected cleavage site, intended to avoid bias toward a single fixed nucleotide at the start of selection. Aiming to generate a DNAzyme more compact than the already available I-R3 and II-R2 / 3, we opted for the N32 core region. [Figure 3] This shows the activity of parental DNAzyme 12-11 and its reselected mutants. A. Fraction cleaved 24 hours after incubation, B. Progression of the cleavage reaction over time, C. Cleavage yield versus time data fitted to a pseudo-first-order rate model. [Figure 4] The sequences of the self-cleaving DNAzymes and activity data tested for 12-11 DNAzymes that undergo self-cleavage reactions under the condition of one of the four nucleotides at the cleavage site, and for selected mutants, are shown. [Figure 5] Confirmation of cleavage sites of 12-11 DNAzymes by DMS assay and mass spectrometry. A: Sequence of double-labeled oligonucleotides of 12-11 DNAzymes, with the catalyst core underlined and cleavage sites marked. B: Assigned DMS ladder of double-labeled 12-11 DNAzymes and their cleavage products (1 and 2) after degradation by 15% PAGE and imaging by excitation of Cy3 and Cy5 terminal tags. C: Products of 12-11 cleavage detected by ESI-MS. [Modes for carrying out the invention]

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains.

[0016] As used herein, the terms “comprising” or “comprises” mean “including, but not limited to.” These terms are intended to be open-ended in identifying the presence of any described feature, element, integer, step, or component, but not in any way in which one or more other features, elements, integers, steps, components, or groups thereof are excluded from the presence or addition of such features, elements, integers, steps, components, or groups thereof. Accordingly, the terms “comprising” or “comprises” include the more restrictive terms “consisting of” and “essentially consisting of.” In one embodiment, the terms “comprising” or “comprises” as used throughout this application, particularly in the claims, may be replaced by the terms “consisting of” or “essentially consisting of.”

[0017] In the context describing the present invention (particularly in the context of the following claims), the terms “a,” “an,” and “the,” as well as similar references, should be interpreted as encompassing both singular and plural forms unless otherwise indicated herein or unless the context clearly contradicts this interpretation. For example, the term “steady region” includes multiple steady regions (including mixtures thereof). Where the plural form is used for compounds and salts, etc., it should be interpreted as also meaning a single compound or salt, etc.

[0018] In the context of this disclosure, the term "DNA" refers to deoxyribonucleic acid, which consists of single strands of monomeric units called nucleotides, each nucleotide comprising a nitrogen-containing nucleic acid base, a 2-deoxyribose sugar moiety, and a phosphate group, with each nucleotide being single-stranded linked by a phosphate group that links the 5' OH group of the 2-deoxyribose sugar moiety to the 3' OH group of an adjacent 2-deoxyribose sugar moiety. In certain embodiments, the nitrogen-containing nucleic acid base is independently selected from cytosine [C], guanine [G], adenine [A], and thymine [T]. In certain embodiments, one or more of these nucleic acid bases are non-standard bases, in particular modified adenosine, especially N6-carbamoylmethyladenine or N6-methiadenine; modified guanine, especially 7-deazaguanine or 7-methylguanine; modified cytosine, N4-methylcytosine, 5-carboxylcytosine, 5-formylcytosine, 5-glycosylhydroxymethylcytosine, 5-hydroxycytosine, or 5-methylcytosine; modified thymidine, especially α-glutamylthymidine or α-ptresinylthymine; uracil or a modification thereof, especially uracil, base J, 5-dihydroxypentauracil; or 5-hydroxymethyldeoxyuracil; deoxyarcheosin; and non-standard bases selected from the list of 2,6-diaminopurine. A single strand or portion of DNA can interact with a complementary strand of DNA through complementary nucleic acid base interactions to form a double helix. Cytosine and guanine, and adenine and thymine, are complementary to each other by forming two (A / T) and three (G / C) hydrogen bonds between their nucleic acid bases, respectively. A double helix can be formed by two single strands of DNA that are completely complementary to each other, as in genomic DNA, or by single strands of DNA that are partially complementary to each other, including situations where one single strand of DNA is partially complementary to two or more other single-stranded DNA strands.Double strands can also be formed by two fully or partially self-complementary sequences of single strands of DNA, resulting in the formation of higher-order motifs such as hairpins, loops, hybridization stems, or triple-stranded DNA, pseudoknots, or kissing hairpins.

[0019] In the context of this disclosure, the term “DNAzyme,” also called “deoxyribozyme,” “DNA enzyme,” or “catalytic DNA,” refers to a DNA oligonucleotide capable of performing a specific chemical reaction. This specific chemical reaction may be cleavage, such as autocleavage. There is little evidence of naturally occurring DNAzymes. While not bound by any theory, this is likely due to the limited number of functional groups compared to proteins and the limited physical flexibility of the natural double-stranded structure. Therefore, DNAzymes are typically single-stranded DNA sequences that optionally possess self-complementary regions that confer specific structures that may influence their activity in certain cases. DNAzymes are generated by high-throughput in vitro selection techniques from a pool of many random DNA spaces that can be screened for specific chemical reactions or catalytic activity. By applying selective pressure through the adaptation of selection conditions during the selection process, e.g., incubation time, salt concentration, pH, and the presence of cofactors, DNA sequences can be selected according to specific chemical reactions, e.g., hydrolytic cleavage, and / or specific locations. The selection process can be further enhanced by introducing genetic modifications to the DNA sequences within the initial pool of DNA sequences, or after several selection rounds to evolve DNA strands with increased activity that dominate the pool after multiple selection steps. Typically, genetic modifications are introduced by recombination or point mutations. To introduce point mutations, the pool can be amplified using error-prone PCR (EP-PCR) to generate many different strands of various random single mutations.

[0020] As used herein, “pseudogene nucleic acid” is a nucleic acid comprising at least one target DNA oligo or polynucleotide sequence and two self-cleaved DNA sequences adjacent to each target DNA oligo or polynucleotide sequence. Preferably, the pseudogene nucleic acid comprises one or more target DNA oligos or polynucleotide sequences, for example, two, three, four, or more than about 50 target DNA oligos or polynucleotide sequences.

[0021] As used herein, "cleavage activity" refers to the observed rate constant (k obs ) Value and final yield (Y) of the self-cleavage reaction max It is expressed by the value. The reaction yield (Y) is the yield at a given time point, as the reaction initiation is calculated as the percentage of DNAzyme molecules that have undergone self-cleavage. obs This is a pseudo-first-order kinetic model: Y=Y max (1-e -kobs*t The final yield of the reaction is calculated by conforming to the formula. Preferably, after a long reaction time Y, preferably within 24 hours from the start of the cleavage reaction, for example, at one or more time points 1, 2, 3, 5, 7, 9, 10, 15, 20, or 24 hours from the start of the cleavage reaction, more preferably determined from the percentage of cleaved DNAzyme molecules 24 hours from the start of the cleavage reaction.

[0022] The inventors designed a novel in vitro selection method that applies evolutionary pressure to the location of the cleavage site (Figures 1 and 2). The proposed in vitro selection procedure can be divided into four main steps (Figure 1): cleavage reaction (I), generation of reverse complement of the active fraction (II), repair of the 5' constant sequence by ligation (III), and PCR amplification (IV). Traditionally, the capture step of the active DNAzyme sequence utilizes a reaction with a specific chemical group in the expected catalytic product (Wang et al., 2014). The DNA cleavage reaction can unfold after different catalytic pathways that provide a multitude of possible end groups to the cleavage site (Xiao et al., 2012, Lee et al., 2017). Often, this makes it possible to isolate sequences that catalyze the reaction in a particular mode. With the ambition of obtaining a robust cleavage agent regardless of its mode of action, the inventors implemented a strategy that allows any catalyst type as long as it results in DNA cleavage at the intended site. Therefore, instead of starting work directly with the enrichment pool, sequence data was transferred to a new DNA strand in the form of reverse complement. The isolated DNA after cleavage served as a template for primer extension via the 3' constant sequence. The reaction yield indirectly indicated the library cleavage efficiency in the current round. This was then used to track the enrichment progress by selection and adjust the incubation time for the cleavage reaction. Most importantly, the DNA strands generated after primer extension always terminated at a 3'-hydroxyl group, regardless of the original mode of action of the DNAzyme. This was readily used to restore the complete library structure by adapter ligation. Subsequently, PCR amplification was used to generate a full-size ssDNA pool for the next round. The selection procedure was repeated until the library was sufficiently enriched.

[0023] This novel selection approach enabled the successful evolution of a new self-cleaving DNAzyme that functions robustly with high cleavage yields, is extremely compact (31 nucleotides (nt) / 32 nt), thus reducing the amount of discarded DNA during ssDNA generation, and is insensitive to the upstream DNA sequence context, i.e., generates a target sequence without scarring at the 3' end.

[0024] In one aspect, the present invention relates to a method for selecting a DNAzyme having self-cleavage activity, for example, an in vitro method, wherein the method is (a) To provide a plurality of single-stranded DNA strands, Each single-stranded DNA strand contains a core region of random nucleotides adjacent to its 5' constant region 1 and its 3' constant region 2. Each constant region includes a primer binding portion, (b) subjecting multiple DNA strands to self-cleavage conditions, (c) Isolating cleavage product 2, and optionally isolating cleavage product 1, The cleavage product 2 includes a steady-state region 2 and a core region. The cleavage product 2 contains DNA self-cleavage activity, and The cleavage product 1 includes a steady-state region 1, Preferably, a break in the single-stranded DNA strand occurs between the catalyst core and the steady region 1. or The cleavage product 2 includes a steady-state region 2, a core region, and a portion of the steady-state region 1. The cleavage product 2 contains DNA self-cleavage activity, and The cleavage product 1 includes the remaining portion of the steady-state region 1. Preferably, when a break in the single-stranded DNA strand occurs within the constant region 1, isolation is performed. (d) To generate a complement of cleavage product 2, (e) Optionally, separate the complement of cleavage product 2, (f) Ligating the 3' end of the complement of cleavage product 2 to the complement of cleavage product 1, thereby obtaining the single-stranded DNA complement of step (a) that has DNA self-cleavage activity, (g) Optionally, separate the complement of a single-stranded DNA strand, (h) Amplifying the complement of a single-stranded DNA strand, thereby obtaining a DNA strand containing DNA self-cleavage activity, (i) Selectively isolate DNA strands containing self-cleavage activity, (j) Optionally, subject a DNA strand containing self-cleavage activity to the error-prone (EP-PCR) step, (k) The present invention relates to a method comprising (k) optionally subjecting a DNA strand containing the self-cleavage activity of step (h), optionally step (i), or step (j) to one or more rounds of steps (b) to (h), and optionally step (i) and / or (j).

[0025] In one embodiment, steps (a) to (k) are consecutive steps.

[0026] In one embodiment, the multiple single-stranded DNA strands in step (a) may be further divided into subpools. For example, the multiple single-stranded DNA strands may be further divided into subpools, each pool characterized by one of four possible nucleotides A, T, G, or C at a pre-selected cleavage site intended to avoid bias toward one fixed nucleotide at the start of selection.

[0027] In one embodiment, in step (a), preferably in the first selection round, each single-stranded DNA strand consists of a constant region 1 at its 5' end and a core region of random nucleotides adjacent to the constant region 2 at its 3' end.

[0028] The number of nucleotides in the core region of the single-stranded DNA strand in step (a) may be user-defined and may contain any number of nucleotides. It may be preferable to set the number as small as possible, as long as the cleavage activity is not impaired, as this can significantly reduce the amount of waste DNA during the generation process, thereby saving costs. In one embodiment, the core region of the single-stranded DNA strand in step (a) contains about 20 to 100 nucleotides, or about 25 to 50 nucleotides, or about 30 to 40 nucleotides, or about 32 to 35 nucleotides. In one embodiment, the nucleotide positions in the core region are independently represented by any nucleotide (N).

[0029] In the context of this disclosure, the term “catalytic core region” of a DNAzyme refers to a specific nucleotide sequence exhibiting a particular catalytic ability, where each nucleotide position is composed of nucleotides that are conserved to varying degrees of identity. In one embodiment, 19 of the 31 nucleotides in the catalytic core region are highly conserved in terms of their identity and sequence context. In one embodiment, the nucleotide positions in the catalytic core region are independently represented by pyrimidine (Y). In one embodiment, the nucleotide positions in the catalytic core region are independently represented by any nucleotide (N). The catalytic core region is subject to directed evolution (in vitro selection) toward a particular catalytic ability. For example, at the start of an in vitro selection process, the core region is represented by random nucleotides and is composed of a portion of multiple sequences synthetically prepared by methods known to those skilled in the art, including but not limited to solid-phase synthesis, enzymatic DNA synthesis, and enzymatic ligation. The incorporation frequency of nucleotides to each nucleotide position is entirely flexible and can be adjusted by the designer. For example, the core region of the initial library may be completely randomized or may include positions where one of the nucleotides has a higher incorporation probability. Furthermore, a sequence of nucleotides can be constrained to a certain predefined identity, thereby allowing the formation of loop or hairpin structures within the core region. In one embodiment, every position in the core region of the initial pool had the potential to incorporate A, TC, or G nucleotides. In one embodiment, each position in the core region of the initial library had a 70% incorporation probability of a nucleotide with defined identity. To expand the chemical repertoire of catalysts or to enhance DNAzyme properties, the use of unnaturally modified nucleotides in the synthesis of the initial library is also possible.

[0030] As used herein, the term “random nucleotide” refers to the random nature of individual nucleotides, including but not limited to the random composition of nucleotides, preferably A, T, G, or C, the random sequence of individual nucleotides, and / or non-standard bases or 2' or other sugar modifications as described herein. Essentially, modifications to individual nucleotides should not interfere with base pairing and / or phosphodiester bonding properties between single nucleotides.

[0031] In one embodiment, all single-stranded DNA strands in step (a) include the same constant region 1 and the same constant region 2. Preferably, constant region 1 and constant region 2 are different, which allows different primers to bind to constant region 1 or constant region 2 and / or to form complementary subregions. For example, constant region 1 of the single-stranded DNA strand in step (a) includes a subregion complementary to a portion of constant region 2, thereby forming a hybridization stem adjacent to the core region (Figure 2). In one embodiment, the complementary subregions in each constant region do not overlap with the primer-binding subregion.

[0032] In one embodiment, the 5' end of the constant region 1 of the single-stranded DNA strand in step (a) is labeled. Such labels are known to those skilled in the art and include, but are not limited to, fluorophores such as cyanine, e.g., Cy3 or Cy5. Preferably, the molecular size of the label is small enough not to interfere with the cleavage activity and / or subsequent steps of the selection method. In one embodiment, the label on the single-stranded DNA strand in step (a) is not present in the first round of the selection method. In this case, the label can be introduced using primer 1 in step (h).

[0033] The self-cleavage conditions in step (b) may vary and may depend, for example, on a specific cleavage reaction or concentration during in vitro selection. The cleavage conditions may be adapted between different selection rounds to change the selection pressure. Such conditions may include incubation time, temperature, pH, buffer, and cofactors. In one embodiment, the self-cleavage conditions in step (b) include one or more ions, preferably positively charged ions. The ions may be monovalent, divalent, and / or polyvalent ions. A specific example is Na + Zn 2+ Mg 2+ Mn 2+ Cu 2+ , or Ca 2+ In one embodiment, the ions are Zn 2+ , or Zn 2+ and Mg 2+ A combination of, or Zn 2+ Ca 2+ , and Mg 2+ The combination is preferably Zn 2+ The ion concentration can range from approximately 100 nM mM to 100 mM, or from approximately 1 mM to 50 mM, or from approximately 5 mM to 25 mM.

[0034] In one embodiment, the self-cutting conditions in step (b) include temperatures in the range of about 10 to 65°C, or about 15 to 45°C, or about 20 to 40°C, or about 25 to 35°C.

[0035] In one embodiment, the self-cleavage conditions in step (b) include a pH in the range of about 6.8 to 8.2, or about 6.8 to 8.0, or about 7.0 to 7.5, or about 7.1 to 7.4.

[0036] In one embodiment, the pH is in the range of approximately 7.1 to 7.2, and the temperature is in the range of approximately 21 to 25°C.

[0037] In one embodiment, the self-cleavage conditions in step (b) include a buffer. The buffer is known to those skilled in the art and is preferably selected with respect to a specific pH or pH range during the cleavage reaction. For example, the buffer may be selected from (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES), tris(hydroxymethyl)aminomethane (Tris), 3-(N-morpholino)propanesulfonic acid (MOPS), 2-(N-morpholino)ethanesulfonic acid (MES), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), or phosphate-buffered saline (PBS).

[0038] In one embodiment, the self-cutting conditions in step (b) include an incubation time of about 15 minutes to 24 hours, or about 30 minutes to 20 hours, or about 60 minutes to 18 hours. The incubation time may be constant during different selection rounds. Alternatively, the incubation time may be extended or shortened between different selection rounds. In one embodiment, the incubation time is progressively shortened in subsequent selection rounds after the first selection round. In one embodiment, the self-cutting conditions in step (b) include an incubation time of 15 to 20 hours, preferably 18 hours, in the first round, and an incubation time that is progressively shortened in subsequent rounds, preferably shortened to 20 minutes in round 9.

[0039] The cleavage product in step (c) can be isolated by any method known in the art, preferably by polyacrylamide gel electrophoresis (PAGE) or magnetic beads. For the purposes of the present invention, isolation in step (c) and optionally tracking (if the single-stranded DNA strand in step (a) is labeled) preferably involves PAGE, e.g., urea-PAGE. Since PAGE separates molecules according to their molecular weight, PAGE can be used to apply selective pressure to the cleavage site.

[0040] In step (c), at least cleavage product 1 and cleavage product 2 are obtained. The composition of the cleavage product may vary depending on the exact cleavage site(s). Step (c) may involve isolating a specific cleavage product and concentrating a single-stranded DNA strand at the use-defined cleavage site. In one embodiment, a cleavage product is isolated in which the cleavage site is between the catalyst core and the constant region 1. In this embodiment, cleavage product 2 comprises the constant region 2, the core region, and DNA self-cleavage activity, while cleavage product 1 comprises the constant region 1. In another embodiment, a cleavage product is isolated in which the cleavage site is within the constant region 1. In this embodiment, cleavage product 2 comprises the constant region 2, the core region, DNA self-cleavage activity, and a portion of the constant region 1, while cleavage product 1 comprises the remainder of the constant region 1.

[0041] In one embodiment, the self-cleavage activity is present in the catalyst core region.

[0042] In one embodiment, step (c) optionally isolates the cleavage product 1, which includes either the steady region 1 or the remainder of the steady region 1.

[0043] In one embodiment, the cleavage product 1 isolated in step (c) is further analyzed to identify the cleavage site. Methods for analyzing the cleavage site are known in the art and include, but are not limited to, DMS ladder or mass spectrometry, as described in the examples.

[0044] In step (d), a complement of the cleavage product 2 is generated. Any method known in the art for generating the complementary sequence can be used. In one embodiment, the generation in step (d) comprises a linear amplification reaction, preferably, primer 2 binding to the primer-binding region of constant region 2. Extension of the binding primer 2 is preferably carried out via polymerase chain reaction (PCR), preferably using a hot-start polymerase. Amplification conditions are known to those skilled in the art and may include, but are not limited to, 2 minutes at 95°C [30 seconds at 95°C, 30 seconds at 61°C, 30 seconds at 72°C] 10 times, followed by 5 minutes at 72°C. PCR in the presence of only one primer results in linear amplification but allows for the capture of a minimum amount of the complement of the single-stranded DNA strand having cleavage activity.

[0045] In one embodiment, the complements produced in step (d) contain a hydroxyl group at their 3' end. This allows for their recovery to their full-length complements regardless of the ligation cleavage reaction of the complement containing steady region 1 or the complement containing the remainder of steady region 1 in step (f).

[0046] In one embodiment, the primer 2 includes a tag that allows the complementary to be separated from its template, e.g., the cleavage product 2, and preferably the tag is linked via a linker. In one embodiment, the tag is linked to the 5' end of the primer 2. The properties of the tag are not important and are known to those skilled in the art and include, but are not limited to, poly T-tails or magnetic beads. Preferred linkers are known to those skilled in the art and include, but are not limited to, carbon linkers, preferably C6 linkers.

[0047] Primer 2 may also be labeled. In one embodiment, the label is ligated to the 5' end of primer 2. In another embodiment, the label is ligated to a tag. In one embodiment, the label is a fluorescent substance such as cyanine, e.g., Cy3 or Cy5, and combinations thereof. In one embodiment, the label on primer 2, if present, is different from the label on the 5' end of the constant region 1 of the DNA strand in step (a).

[0048] This method may involve separation in steps (e), (g), and (i). Separation may be necessary if subsequent steps of this method are impaired or impossible. Separation may be carried out by any method known in the art, including but not limited to PAGE or magnetic beads, preferably via PAGE. Those skilled in the art may select the tags of primer 2 depending on the separation method used thereafter. For example, if PAGE is used for separation, the tags of primer 2 may be poly-T tails to distinguish the size of the complements from their mold, e.g., the cleavage product 2.

[0049] The ligation in step (f) repairs the full-length complement of the single-stranded DNA from step (a) that had cleavage activity in step (b).

[0050] In one embodiment, ligation in step (f) is carried out via the 3' hydroxyl group of the complement of cleavage product 2 and / or via the 5' phosphate group of the complement of cleavage product 1.

[0051] In one embodiment, ligation in step (f) is performed enzymatically using a ligase such as T4 RNA ligase 1.

[0052] In step (h), a DNA strand containing the full-length sense strand of the complement obtained in step (f) is obtained, thereby repairing a single-stranded DNA strand with DNA self-cleavage activity that can be used in one or more further selection rounds.

[0053] In step (h), the complement of the single-stranded DNA from step (a) that had cleavage activity in step (b) is amplified. In one embodiment, amplification involves PCR, preferably using a hot-start polymerase. In one embodiment, PCR is performed using a labeled and / or tagged primer 2 bound to the primer-binding region of constant region 2 and a primer 1 bound to the complement of the primer-binding region of constant region 1. In a preferred embodiment, PCR is a two-step PCR. In the first step, the complement from step (f) is used as a template to obtain a PCR-I reaction product using primer 2 bound to the primer-binding region of constant region 2 and primer 1 bound to the complement of the primer-binding region of constant region 1. The conditions for the first step may be 2 minutes at 95°C, followed by 10 repetitions of [30 seconds at 95°C, 30 seconds at 63°C, 40 seconds at 72°C], and then 5 minutes at 72°C. The PCR-I reaction product can be used directly as a template for the second step, which is similarly performed using primer 2 and primer 1. The conditions for the second stage may be 2 minutes at 95°C, followed by 15 repetitions of [30 seconds at 95°C, 30 seconds at 63°C, and 40 seconds at 72°C], and then 5 minutes at 72°C. In one embodiment, primer 1 in step (h) is labeled to obtain a DNA strand containing self-cleavage activity with a labeled 5' end. Preferably, the labeling of primer 1 is different from the labeling of primer 2, if present.

[0054] To increase sequence diversity, an error-prone PCR (EP-PCR) step may be introduced. Preferably, the EP-PCR step is introduced in round 2, or in later rounds such as round 3, round 4, round 5, round 6, round 7, round 8, round 9, round 10, round 11, or round 12. In one embodiment, the EP-PCR of step (j) is performed after 9 rounds, followed by approximately 3 rounds without EP-PCR, using the DNA strand containing the self-cleavage activity of step (h). In one embodiment, the EP-PCR of step (j) introduces a mutagenesis rate in the range of approximately 2.0–9.0% or approximately 2.5–8.0% compared to the total number of nucleotides present in the core region.

[0055] In one embodiment, a DNA strand containing self-cleavage activity is subjected to one or more steps (a) to (h), and optionally to (i) and (j). In one embodiment, the DNA strand containing self-cleavage activity is subjected to one or more rounds until the cleavage activity yields a final yield Ymax of about 70%, about 80%, about 90%, about 92%, about 95%, about 96%, or about 97%, or about 98%, or about 99%. In one embodiment, the final yield Ymax is calculated by Y = Ymax(1-e-kobs*t), where Y is the reaction yield and t is within 24 hours after the start of the reaction, preferably about 24 hours after the start of the reaction.

[0056] In one embodiment, the DNA strand containing self-cleavage activity is subjected to about 8 selection rounds, or about 9 selection rounds, or about 10 selection rounds, or about 11 selection rounds, or about 12 selection rounds, or about 13 selection rounds, or about 14 selection rounds, or about 15 selection rounds, preferably about 12 selection rounds.

[0057] In another aspect, the present invention relates to a DNAzyme that can be obtained by a method according to the present invention, preferably the DNAzyme having self-cleavage activity between or within the catalytic core and the constant region 1.

[0058] In another embodiment, the present invention relates to a DNAzyme comprising the following sequence: 5'- N & GY $ Y # GT N $ Y # ACGC Y # Y $ YGTCTTATCGGTT Y $ Y $ N # Includes N -3' (sequence number 1), In the array, A is a nucleotide that has the base adenine. C is a nucleotide that has the base cytosine, G is a nucleotide that has the base guanine. T is a nucleotide that has a thymidine base. N is independently A, C, G, or T. Y is independently C or T, $ However, independently, preferably T, # However, independently, preferably C, & However, independently, preferably G, The DNAzyme contains self-cleavage activity and optionally includes additional nucleotide N # However, this relates to a DNAzyme located between nucleotide positions 29 and 30 of sequence number 1, where the nucleotide numbering is in the direction from 5' to 3' of sequence number 1.

[0059] Therefore, in a further embodiment, the DNAzyme has the following sequence: 5'- N & GY $ Y # GT N $ Y # ACGC Y # Y $ YGTCTTATCGGTT Y $ N # Y $ N # Includes N -3' (Sequence ID 2), In the array, A is a nucleotide that has the base adenine. C is a nucleotide that has the base cytosine, G is a nucleotide that has the base guanine. T is a nucleotide that has a thymidine base. N is independently A, C, G, or T. Y is independently C or T, $ However, independently, preferably T, #However, independently, preferably C, & However, independently, preferably G, This DNAzyme contains self-cleavage activity.

[0060] In another embodiment, the DNAzyme is selected from any one of the sequences provided in the table below, in the 5' to 3' direction. [Table 1-1] [Table 1-2]

[0061] In another embodiment, the DNAzyme is an A-cleaving agent, i.e., it cleaves after nucleotide A, for example, after the last nucleotide A in the 5' constant region of a single-stranded DNA strand, such as a DNAzyme having a sequence selected from SEQ ID NOs. 24-30, preferably SEQ ID NOs. 24-30 or 47.

[0062] In another embodiment, the DNAzyme is a T-cleavage agent, i.e., it cleaves after nucleotide T, for example, after the last nucleotide T in the 5' constant region of a single-stranded DNA strand, such as a DNAzyme having a sequence selected from SEQ ID NOs: 31-33.

[0063] In another embodiment, the DNAzyme is a C-cleavage agent, i.e., it cleaves after nucleotide C, for example, after the last nucleotide C in the 5' constant region of a single-stranded DNA strand, such as a DNAzyme having a sequence selected from SEQ ID NOs. 34-37.

[0064] In another embodiment, the DNAzyme is a G-cleavage agent, i.e., it cleaves after the nucleotide G, for example, after the last nucleotide G in the 5' constant region of a single-stranded DNA strand, such as a DNAzyme having a sequence selected from SEQ ID NOs. 38-46, preferably SEQ ID NOs. 38-47.

[0065] In one embodiment, the self-cleavage activity is approximately 60% or more, approximately 70% or more, approximately 80% or more, approximately 90% or more, approximately 92% or more, approximately 95% or more, approximately 96% or more, or approximately 97% or more, or approximately 98% or more, or approximately 99% or more in the final yield Y max This results in, preferably, a final yield Y max This is calculated by Y = Ymax(1-e-kobs*t), where Y is the reaction yield and t is within 24 hours after the start of the reaction, preferably about 24 hours after the start of the reaction.

[0066] In one embodiment, the DNAzyme is 0.005 min -1 ~0.240 minutes -1 , or 0.35 minutes -1 ~0.140 minutes -1 , or 0.050 minutes -1 ~0.100 minutes -1 k within the range obs It has, preferably, k obs However, Y=Y max (1-e -kobs*t The formula is calculated by ), where Y is the reaction yield and t is within 24 hours after the start of the reaction, preferably about 24 hours after the start of the reaction.

[0067] In one embodiment, the final yield Y max At approximately 25-40°C and / or pH approximately 6.8-7.3, Zn 2+ The final yield is determined by the DNAzyme reaction in the presence of Zn at a pH of 6.8–7.1 and / or a temperature of 35°C. 2+ It is determined from DNAzyme reactions in the presence of [the substance].

[0068] In one embodiment, the self-cleavage activity is hydrolytic cleavage activity, which, for example, results in a hydroxyl group at the 3' end and a phosphate group at the 5' end.

[0069] In a further embodiment, the present invention relates to the use of the DNAzyme of the present invention in the generation of single-stranded DNA molecules.

[0070] In one embodiment, the use is (a) To provide a pseudogene nucleic acid, wherein the pseudogene nucleic acid comprises at least one target DNA oligo or polynucleotide sequence and two self-cleaved DNA sequences adjacent to each target DNA oligo or polynucleotide sequence, (b) Incorporating pseudogene nucleic acid into a vector, transforming bacterial cells with the vector, and generating precursor ssDNA from the vector under bacterial culture conditions, wherein the precursor ssDNA contains pseudogene nucleic acid, (c) Isolating precursor ssDNA from bacterial cultures, (d) Digesting the precursor ssDNA under reaction conditions in which the self-cleaved DNA sequence becomes active, (e) Separating target single-stranded DNA oligos or polynucleotides (multiple) to obtain target single-stranded DNA oligos or polynucleotides (multiple), At least one of the two self-cleaved DNA sequences is a DNAzyme of the present invention, for example, sequence numbers 1, 2, or 24-47, or optionally 24-46.

[0071] In one embodiment, the two DNAzymes of the present invention are identical or different. In one embodiment, one of the two self-cleaving DNA sequences is a DNAzyme of the present invention, for example, any one of sequence numbers 1, 2, 24-47, or optionally 24-46, and the other is another DNAzyme having self-cleaving activity. The self-cleaving activity of the other DNAzyme is preferably by the same conditions as the self-cleaving activity of the DNAzyme of the present invention, preferably by the same monovalent or divalent ions as the DNAzyme of the present invention, more preferably Zn 2+It is activated by [the other DNAzyme]. In certain embodiments, the other DNAzyme is selected from I-R3 or its variants, e.g., those described in Gu et al., 2013; II-R1 or its variants, e.g., those described in Qiao Zhang et al., 2022; II-R2 or its variants, e.g., those described in Qiao Zhang et al., 2022; II-R3 or its variants, e.g., those described in Qiao Zhang et al., 2022; 13PD1 or its variants, e.g., those described in Qiao Zhang et al., 2022; and combinations thereof.

[0072] For example, for ssDNA generation, phagemids consist of user-defined sequences in which self-cleaved DNA sequences (DNAzymes) are arranged alternately. After ssDNA is generated on a large scale in phage culture, the target ssDNA strand can be digested, separated, and readily used for DNA origami assembly (or other applications using ssDNA, such as homologous recombination repair for CRISPR / CAS gene editing).

[0073] Single-stranded DNA molecules can be used in DNA nanotechnology, for example, by assembling them, and / or folding them into DNA origami structures, (tile-based) DNA nanostructures, or crystalline DNA nanomaterials, as described in the prior art, e.g., WO2018 / 054571, particularly on pages 10-14 and the examples.

[0074] Such nanostructures may be used in medicine or therapy. For example, DNA-based nanostructures can be used to encapsulate viruses or viral particles. Such nanostructures are described in the prior art, e.g., WO2021 / 165528.

[0075] As a further example, DNA-based nanostructures can be used in gene therapy. For example, using an ssDNA molecule, a nucleic acid nanostructure can be constructed comprising at least one scaffolding strand and multiple staple strands, wherein the nanostructure, preferably the at least one scaffolding strand, comprises at least one nucleic acid sequence encoding a gene, preferably the gene being a gene involved in a pathological pathway, a gene suitable for vaccination, a gene for genome editing, and / or a CRISPR-based gene. In one embodiment, the gene is selected from prokaryotic genes, viral genes, and eukaryotic genes. In one embodiment, the gene is selected from prokaryotic genes such as CRISPR-based genes and eukaryotic genes such as human genes. In a preferred embodiment, the gene is a eukaryotic gene, preferably a mammalian gene, e.g., a human gene. Such nucleic acid nanostructures are known in the art, for example from EP22207474. Alternatively, an ssDNA molecule can be used as an aptamer of another molecule, or can bind to or process another molecule, preferably a molecule involved in a pathological pathway.

[0076] Single-stranded DNA molecules can also be used for diagnostic purposes. For example, ssDNA molecules can bind to, detect, or process other molecules, preferably those involved in pathological pathways. ssDNA may also be used in studies to bind to, detect, or process other molecules.

[0077] ssDNA may also be used as a probe for sequencing or DNA synthesis, such as polymerase chain reaction. Such methods are known to those skilled in the art and include classical sequencing methods such as Maxam-Gilbert sequencing and Sanger sequencing, as well as next-generation sequencing methods such as Illumina sequencing and pyrosequencing.

[0078] ssDNA can also be used in genome editing. For example, ssDNA molecules can be used in CRISPR / Cas-based genome editing, for example, for homologous recombination repair (HDR). Such methods are known to those skilled in the art, for example, from Bai et al., 2020 or Quadros et al., 2017. For example, HRD can be used in gene therapy to introduce mutations or to replace and correct mutated genetic information in a subject.

[0079] In one embodiment, the present invention is a method for recombinant generation of ssDNA molecules, (a) A step of providing a pseudogene nucleic acid, The present invention provides a nucleic acid in which the pseudogene nucleic acid comprises at least one target DNA oligo or polynucleotide sequence and two self-cleaved DNA sequences adjacent to each target DNA oligo or polynucleotide sequence. (b) A step of incorporating a pseudogene nucleic acid into a vector, transforming bacterial cells with the vector, and generating precursor ssDNA from the vector under bacterial culture conditions, The precursor ssDNA includes a pseudogene nucleic acid, and the steps are as follows: (c) A step of isolating precursor ssDNA from a bacterial culture, (d) A step of digesting the precursor ssDNA under reaction conditions in which the self-cleaved DNA sequence becomes active, (e) The step of separating the target single-stranded DNA oligo or polynucleotide(s) to obtain the target single-stranded DNA oligo or polynucleotide(s), The present invention provides a method in which at least one of two self-cleaved DNA sequences is a DNAzyme of the present invention, for example, one of the DNAzymes from sequence numbers 1, 2, or 24-47, or optionally 24-46.

[0080] In one embodiment, the two DNAzymes of the present invention are identical or different. In one embodiment, one of the two self-cleaving DNA sequences is a DNAzyme of the present invention, for example, any one of sequence numbers 1, 2, 24-47, or optionally 24-46, and the other is another DNAzyme having self-cleaving activity. The self-cleaving activity of the other DNAzyme is preferably by the same conditions as the self-cleaving activity of the DNAzyme of the present invention, preferably by the same monovalent or divalent ions as the DNAzyme of the present invention, more preferably Zn 2+ It is activated by [the other DNAzyme]. In certain embodiments, the other DNAzyme is selected from I-R3 or its variants, e.g., those described in Gu et al., 2013; II-R1 or its variants, e.g., those described in Qiao Zhang et al., 2022; II-R2 or its variants, e.g., those described in Qiao Zhang et al., 2022; II-R3 or its variants, e.g., those described in Qiao Zhang et al., 2022; 13PD1 or its variants, e.g., those described in Qiao Zhang et al., 2022; and combinations thereof.

[0081] The present invention is further characterized by the following embodiments. 1. A method for selecting a DNAzyme having self-cleavage activity, wherein the method is (a) To provide a plurality of single-stranded DNA strands, Each single-stranded DNA strand contains a core region of random nucleotides adjacent to its 5' constant region 1 and its 3' constant region 2. Each constant region includes a primer binding portion, (b) Subjecting multiple single-stranded DNA strands to self-cleavage conditions, (c) Isolating cleavage product 2, and optionally isolating cleavage product 1, The cleavage product 2 includes a steady-state region 2 and a core region. The cleavage product 2 contains DNA self-cleavage activity, and The cleavage product 1 includes a steady-state region 1, Preferably, a break in the single-stranded DNA strand occurs between the catalyst core and the steady region 1. or The cleavage product 2 includes a steady-state region 2, a core region, and a portion of the steady-state region 1. The cleavage product 2 contains DNA self-cleavage activity, and The cleavage product 1 includes the remaining portion of the steady-state region 1. Preferably, when a break in the single-stranded DNA strand occurs within the constant region 1, isolation is performed. (d) To generate a complement of cleavage product 2, (e) Optionally, separate the complement of cleavage product 2, (f) Ligating the 3' end of the complement of cleavage product 2 to the complement of cleavage product 1, thereby obtaining the single-stranded DNA complement of step (a) that has DNA self-cleavage activity, (g) Optionally, separate the complement of a single-stranded DNA strand, (h) Amplifying the complement of a single-stranded DNA strand, thereby obtaining a DNA strand containing DNA self-cleavage activity, (i) Selectively isolate DNA strands containing self-cleavage activity, (j) Optionally, subject a DNA strand containing self-cleavage activity to the error-prone (EP-PCR) step, (k) A method comprising, optionally, subjecting a DNA strand having the self-cleavage activity of step (h), optionally step (i), or step (j) to one or more rounds of steps (b) to (h), and optionally step (i) and / or (j). 2. The method according to Embodiment 1, wherein the constant region 1 of the single-stranded DNA strand in step (a) includes a portion complementary to a portion of the constant region 2, thereby forming a hybridization stem adjacent to the core region. 3. The method according to Embodiment 1 or 2, wherein the core region of the single-stranded DNA strand in step (a) comprises approximately 14 to 100 nucleotides, or approximately 20 to 100 nucleotides, or approximately 25 to 50 nucleotides, or approximately 30 to 40 nucleotides, or approximately 32 to 35 nucleotides. 4. The method according to any one of the prior embodiments, wherein the 5' end of constant region 1 of the DNA strand in step (a) is labeled. 5. The self-cleavage condition in step (b) is one or more ions, preferably monovalent, divalent, and / or trivalent, and / or polyvalent ions, for example, Na + Zn 2+ Cu 2+ Mn 2+ Mg 2+ Ca 2+ The method according to any one of the prior embodiments, comprising, and / or PLL-g-Dex. 6. The method according to any one of the prior embodiments, wherein the self-cutting conditions in step (b) include a temperature in the range of about 10 to 62°C, or about 15 to 45°C, or about 20 to 40°C, or about 25 to 35°C. 7. The method according to any one of the prior embodiments, wherein the self-cleavage conditions in step (b) include a pH in the range of about 6.8 to 8.2, or about 6.8 to 8.0, or about 7.0 to 7.5. 8. The method according to any one of the prior embodiments, wherein the self-cleavage conditions in step (b) include a buffer such as (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES). 9. The method according to any one of the prior embodiments, wherein the self-cutting conditions in step (b) include an incubation time of about 30 minutes to 20 hours, preferably about 1 to 20 hours, preferably about 2 to 20 hours, preferably about 3 to 20 hours, preferably about 5 to 20 hours, preferably about 10 to 20 hours, preferably about 15 to 20 hours, preferably about 18 hours in the first round. 10. The method according to any one of the prior embodiments, wherein the self-cutting conditions in step (b) include an incubation time of about 30 minutes to 20 hours, preferably about 15 to 20 hours, preferably about 18 hours, in the first round, and an incubation time that is progressively shortened in subsequent rounds. 11. The method according to any one of the prior embodiments, wherein the isolation in step (c) comprises polyacrylamide gel electrophoresis (PAGE). 12. The method according to any one of the prior embodiments, wherein in step (c), the cleavage product 2 comprises a core region, a constant region 2, and self-cleavage activity, and the cleavage product 1 comprises a constant region 1, and the cleavage of a single-stranded DNA strand occurs between the catalytic core and the constant region 1. 13. The method according to any one of the prior embodiments, wherein the cleavage product 1 isolated in step (c) is further analyzed to confirm the cleavage site. 14. The method according to any of the prior embodiments, wherein the generation in step (d) includes linear PCR amplification. 15. The method according to any of the prior embodiments, wherein the generation in step (d) includes linear PCR amplification using a primer 2 bound to the primer binding region of the constant region 2. 16. The method according to Embodiment 15, wherein the primer 2 includes a tag, preferably a tag attached to the 5' end of the primer 2. 17. The method according to Embodiment 16, wherein the tag is selected from the group consisting of poly T-tails and magnetic beads, and preferably the tag is linked to the primer 2 via a linker. 18. The method according to any of the prior embodiments, wherein the separation in steps (e), (g), and (i) is carried out via PAGE or via magnetic beads, preferably via PAGE. 19. The method according to any of the prior embodiments, wherein the ligation in step (f) is carried out via the 3' hydroxyl group of the complement of the cleavage product 2. 20. The method according to any of the prior embodiments, wherein the ligation in step (f) is carried out via the phosphate group at the 5' end of the complement of the cleavage product 1. 21. The method according to any one of the preceding embodiments, wherein the ligation in step (f) is carried out enzymatically. 22. The method according to any one of the preceding embodiments, wherein the amplification in step (h) comprises polymerase chain reaction (PCR). 23. The method according to any one of the preceding embodiments, wherein the amplification in step (h) comprises two-step PCR. 24. The method according to any one of the preceding embodiments, wherein the amplification in step (h) comprises polymerase chain reaction (PCR) using primer 2 that binds to the primer-binding region of constant region 2 and primer 1 that binds to the complement of the primer-binding region of constant region 1. 25. The method according to embodiment 24, wherein primer 2 in step (h) is tagged and / or primer 1 in step (h) is labeled in order to obtain a DNA strand containing self-cleaving activity having a labeled 5' end. 26. The method according to any one of the preceding embodiments, wherein the EP-PCR in step (j) is carried out using the DNA strand containing self-cleaving activity in step (h) after 9 rounds, followed by about 3 rounds without EP-PCR. 27. The method according to any one of the preceding embodiments, wherein the EP-PCR in step (j) aims for a mutagenesis rate in the range of about 2.0 - 9.0% or about 2.5 - 8.0% compared to the total number of nucleotides present in the core region. 28. A DNAzyme obtainable by the method according to any one of embodiments 1 to 27, preferably, the DNAzyme contains self-cleaving activity between the catalytic core and constant region 1 or within constant region 1. 29. A DNAzyme, 5'-N & G Y $ Y # GT N $ Y # ACGC Y # Y $ YGTCTTATCGGTT Y $ Y $ N #N - 3’ (SEQ ID NO: 1) (optionally, additional nucleotide N # is included between nucleotides 29 and 30 of SEQ ID NO: 1, and nucleotide numbering is in the 5’ to 3’ direction of SEQ ID NO: 1), or 5’ - N & G Y $ Y # GT N $ Y # ACGC Y # Y $ YGTCTTATCGGTT Y $ N # Y $ N # N - 3’ (SEQ ID NO: 2), and includes a sequence selected from in the sequence, A is a nucleotide having base adenine, C is a nucleotide having base cytosine, G is a nucleotide having base guanine, T is a nucleotide having base thymidine, N is independently A, C, G, or T, Y is independently C or T, S is independently G or C, W is independently A or T, $ is independently preferably T, # is independently preferably C, & is independently preferably G, The DNAzyme, which contains self - cleavage activity. 30. The DNAzyme according to embodiment 28 or 29, wherein the DNAzyme has a sequence of SEQ ID NOs: 24 - 47, optionally a sequence selected from SEQ ID NOs: 24 - 46. 31. The DNAzyme, wherein it is 0.005 minutes -1 ~0.240 minutes -1 or 0.35 minutes -1 ~0.140 minutes -1 or 0.050 minutes -1~0.100 minutes -1 k within the range obs It has, preferably, k obs However, Y=Y max (1-e -kobs*t A DNAzyme according to any one of Embodiments 28 to 30, calculated by the formula, where Y is the reaction yield and t is within 24 hours after the start of the reaction, preferably about 24 hours after the start of the reaction. 32. The final yield Y has an autocleavage activity of approximately 92%, 95%, 96%, or 98%. max This results in a final yield of Y max However, Y=Y max (1-e -kobs*t A DNAzyme according to any one of Embodiments 28 to 31, calculated by the formula, where Y is the reaction yield and t is within 24 hours after the start of the reaction, preferably about 24 hours after the start of the reaction. 33. A DNAzyme according to any one of embodiments 28 to 32, wherein the self-cleavage activity is hydrolytic cleavage. 34. Use of a DNAzyme according to any of embodiments 28 to 33 in the generation of a single-stranded DNA molecule. 35. The use of a single-stranded DNAzyme in DNA nanotechnology, as described in Embodiment 34. 36. The use of the single-stranded DNAzyme as described in Embodiment 34, in which it is used for genome editing. 37. The use according to Embodiment 36, wherein genome editing is homologous recombination repair (HDR).

[0082] Example 1 In vitro selection method for self-cleaved DNAzymes Our initial DNAzyme library sequences were constructed in a fairly conventional manner, with the randomized core region adjacent to two stationary sequences (Figure 2). However, the latter were partially complementary to each other, forming a terminal hybridization stem for the core loop, thus mimicking a DNAzyme cassette setup. The non-hybridization portion served as a primer-binding region for the amplification step. The initial library consisted of four sublibraries, each featuring one of four possible base pairs at a pre-selected cleavage site intended to avoid bias toward a single fixed nucleotide at the start of selection. 20 It is known that a core region of a sufficiently short length is sufficient to evolve DNA-cleaving deoxyribozymes (Velez et al., 2012). Aiming to generate DNAzymes that are more compact than the already available I-R3 and II-R2 / 3, N 32 The core region was chosen. After each step, products were separated, isolated, and tracked using high-resolution urea-PAGE. In addition, selective pressure on the cleavage sites was performed using PAGE. The isolated DNA after cleavage served as a template for primer extension via the 3' constant sequence. A circular PCR protocol in the presence of only one primer resulted in linear amplification, successfully capturing a minimum amount of active DNAzyme species and providing 5'Cy5 labeling to the product. The reaction yield indirectly indicated the library cleavage efficiency in the current round. This was therefore used to track enrichment progress by selection and adjust incubation times for cleavage reactions. Most importantly, what was generated after primer-extended DNA strands always terminated at the 3'-hydroxyl group, regardless of the original mode of action of the DNAzyme. This was readily used to restore the complete library structure by adapter ligation. The reaction used oligonucleotides consisting of the reverse complement of the 5' constant sequence and T4 RNA ligase 1. Subsequently, two-step PCR amplification was used to generate a full-size ssDNA pool for the next round. The selection process was repeated until the library was sufficiently concentrated.

[0083] result Identification of a new self-cleaving deoxyribozyme The first significant activity of the DNAzyme pool was detected in round 5. To apply selective pressure to the highly active species, we progressively reduced the incubation time of the cleavage step from 18 hours to 20 minutes in round 9. The primer extension yield leveled off at this stage, and activity assays performed on the corresponding DNA pools were consistent with this observation. To increase sequence diversity, we introduced an error-prone PCR (EP-PCR) step after round 9 and continued selection over three further rounds.

[0084] Libraries from both the 8th and 12th rounds were cloned, and 76 identified unique sequences were screened for activity. Members of four major families exhibited activity (41 out of 76 tested sequences). Based on the initial testing of each of these four families, one most prominent candidate—12-35, 8-35, 12-11, and 12-8—was selected for further testing. Sequence analysis revealed that directional evolution did not dramatically affect the populations of several families between rounds 8 and 12, including the 12-8 family. Simultaneously, the occupancy of the pool by the 8-35 family decreased under selective pressure. This was likely due to competition with the 12-11 family, which emerged after EP-PCR. The 12-11 family evolved and became dominant in round 12, as represented by one identified inactive sequence, 8-19, in round 8.

[0085] The reaction sites of RNA-cleaved DNAzymes were identified using RNAse T1 digestion and alkaline hydrolysis ladders prior to the analysis. We adapted DMS profiling to generate similar characteristic digestion DNA ladders and confirmed the cleavage sites of the DNAzymes tested. All four catalysts performed cleavage first in the core region, as designed. The reaction products were also analyzed using mass spectrometry. DNAzyme 12-11 followed a hydrolytic pathway cleavage link between nucleotides G1 and G2 in the core region, producing different 3'-hydroxyl and 5'-phosphate-terminated products. Simultaneously, 12-35, 8-35, and 12-8 demonstrated various oxidation mechanisms that resulted in nucleoside removal, even though buffering conditions in the selective reaction preferred hydrolytic cleavage. Nucleoside removal was associated with redox-inactive Zn 2+ This has already been reported for guanosine in the presence of ions (Lee et al., 2017). Similarly, 12-35 autocleavage resulted in the removal of guanosine G1, while 8-35 removed cytosine C1, and 12-8 excised cytosine C1 or adjacent adenosine A2. These findings confirm the ability of our in vitro selection method to evolve DNAzymes with diverse modes of action.

[0086] Characterization of a new self-cleaving deoxyribozyme 12-11 Initial tests from 12-11 revealed high sensitivity of DNAzymes to the pH of the cleavage buffer. The optimal pH at 25°C ranged from 7.1 to 7.2. Furthermore, thorough activity screening at different incubation temperatures revealed a "gradient" activity profile, with optimal activity shifting from 7.4 at 10°C to 6.8 at 50°C, and the highest yields were achieved in the 25-40°C range (over 90%). The other three DNAzymes also exhibited unique pH-T dependent patterns and maximum yield conditions, but their objectively low final yields (less than 60%) made them unsuitable for our applications.

[0087] Incubation in the presence of different divalent and monovalent ions is performed on Zn 12-11. 2+The dependence was revealed. Magnesium and calcium played a regulatory role, and both the rate and yield of self-cleavage improved in the presence of these ions. However, Mg 2+ As long as Ca exists, 2+ It can be removed without significant loss. When calcium was excluded, the reaction rates of zinc and magnesium were maximized at approximately 3 mM and 10 mM, respectively. We used Na at various concentrations. + The activity of 12-11 was screened with HEPES. Changes in HEPES concentration were observed in Na + The effect was not as significant as that of increasing the concentration, but it resulted in a substantial decrease in reaction yield. Reducing the sodium concentration to 50 mM universally resulted in a slight improvement in yield compared to the standard reaction.

[0088] DNAzyme 12-11 demonstrated robustness, remaining active in various buffer systems despite the need to establish a pH-T activity profile for each buffer. A correlation was observed between the DNAzyme activity profile and the dpKa / dT values ​​of the buffer systems. These findings suggest that 12-11 possesses a specific, intrinsic optimal pH-T profile, and that the pH of the buffer system is more important than its chemical properties in the final reaction, thus expanding the compatibility of 12-11 with systems using buffers other than HEPES.

[0089] Enhancement of 12-11 deoxyribozymes by reselection As identified in the initial in vitro selection procedure, DNAzyme 12-11 possessed the intended cleavage site and yielded a decent yield, albeit over long incubation periods. Nevertheless, the catalyst was sensitive to upstream nucleotide identity of the reaction site, which imposed sequence restriction on the generated ssDNA. To achieve complete universality of the DNAzyme cleavage site, secondary in vitro selection was performed using 12-11 sequences dopped to 70%. In particular, obtaining variants with faster reaction rates and higher final yields was another goal to be achieved in this re-selection.

[0090] First, following the same protocol as the initial selection, the libraries were pre-enriched until the DNA pool clearly showed activity (round R4). Next, an EP-PCR step with a low mutation rate was introduced, followed by the selection being divided into four distinct directed evolution experiments: RA, RT, RC, and RG. Each featured a ligation adapter terminated at one of four nucleotides, allowing for selective pressure on specific nucleotides at the cleavage site in subsequent rounds. Upon completion of selection, activity assays were performed to evaluate the fractions of active species in the DNA pools from rounds R4–R10. All four libraries showed high enrichment at different selection stages, with cleavage fractions exceeding 50%. The decrease in selection following the activity peak was most likely associated with the final accumulation of parasitic sequences that tend to overpower the DNAzyme sequence during the PCR amplification step. The DNA pools RA6, RT9, RC9, and RG10 were analyzed by HTS. The top 15 most frequent sequences from each library identified using HTS were screened for activity, and the candidate list was narrowed down to 16 sequences combined with the highest cleavage yield.

[0091] method In Vitro Selection The first selection round was initiated using a 500 pmol initial library consisting of four equimolar sublibraries SL1-SL4. Subsequent rounds were initiated using 5-10 pmol of ssDNA recovered after the PCR amplification step. Starting from round 2, the enriched ssDNA pool was also end-labeled with Cy5 fluorescent dye via labeled primers during the PCR amplification step. The cleavage reaction was carried out at 25°C in cleavage buffer I containing 50 mM HEPES (pH 7.5 at 25°C), 100 mM NaCl, 10 mM MgCl2, 2 mM ZnCl2, and 2 mM CaCl2. The duration of the cleavage reaction in the first round was 18 hours, which was adjusted in subsequent rounds to increase the selective pressure. The cleaved fractions were separated using 10% urea-PAGE, and the weight marker WM1 was used to aid in the localization of the active DNA fraction in the gel. The removed gel was pulverized, and the DNA was extracted by incubation with 1×TEN buffer (10 mM Tris, 1 mM EDTA, 300 mM NaCl). The DNA was then recovered by precipitation with ethanol and stored as a dry pellet.

[0092] The obtained DNA was used as a template for a 20 μL single-primer aPCR linear amplification reaction using poly T-tail primer P1 (2 minutes at 95°C, followed by 10 repetitions of [30 seconds at 95°C, 30 seconds at 61°C, 30 seconds at 72°C], and 5 minutes at 72°C). Primer P1 was also terminally labeled with Cy5 to enable tracking of the reaction product in the PAGE gel. The reverse-phase complement product of the active DNA zyme pool was isolated by urea-PAGE assisted by the weight marker WM2 as described above.

[0093] Ligation was performed to repair the cleaved constant sequence. The reaction was carried out on a 20 μL scale using T4 RNA ligase 1 (New England Biolabs) with 75 pmol of adapter LA1 according to the manufacturer's instructions. The ligated DNA was isolated by urea-PAGE using Cy5 labeling carried over from the previous step, and aided by the weight marker WM3 as described above.

[0094] PCR amplification of the enriched DNA pool was performed in two steps. In the first step, the isolated ligation product was used as a template for 20 μL of PCR-I (2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 63°C, 40 seconds at 72°C, followed by 10 repetitions and 5 minutes at 72°C) using primers P1 and P2. Upon completion, 2.5 μL of the PCR-I reaction was directly used as a template for 20 μL of PCR-II (2 minutes at 95°C, 30 seconds at 95°C, 30 seconds at 63°C, 40 seconds at 72°C, followed by 15 repetitions and 5 minutes at 72°C) using primers P1 and P2. The amplification reactions (as well as the single-primer aPCR described above) were performed using Phusion Hot Start Flex Polymerase (New England Biolabs) with 5x Phusion GC Buffer according to the manufacturer's instructions. The remaining PCR-I reaction product was diluted to 100 μL with 1 × TEN buffer, purified using phenol-chloroform extraction, and subsequently precipitated with ethanol. The dried pellet was stored at -20°C and served as a backup for the current selection round. The amplified sense ssDNA DNAzyme pool was isolated from the PCR II reaction product by urea-PAGE assisted by the weight marker WM4 as described above. Polymerase may occasionally result in nucleotide insertions and deletions, which can lead to certain variability at the 3' end of the core region of one or more nucleotides, i.e., shorter or longer DNAzyme sequences compared to the initial core region sequence without affecting cleavage effectiveness.

[0095] Error-prone PCR (EP-PCR) was used to increase sequence diversity in a specific selection round. EP-PCR was performed on a 50 μL scale using the JBS dNTP-Mutagenesis Kit (Jena Bioscience) according to the manufacturer's instructions. This reaction used 2.5 μL of PCR I reaction material as a template and was performed for 15 cycles (after round 9) or 5 cycles (after round 3), aiming for mutagenesis rates of 8.0% or 2.5%, respectively. To remove mutagenic dNTP analogs, the EP-PCR reaction mixture was purified using the QIAquick PCR Purification Kit (Qiagen). The resulting DNA was then converted to the native nucleotide composition using a PCR II reaction material performed for only 5 cycles.

[0096] Re-select Re-selection of 12-11 DNAzymes was performed in the same manner as the initial selection, with some modifications. The initial pool sequences were based on the 12-11 DNAzymes identified in the initial selection. The first selection round was initiated using a 400 pmol degenerate library containing 70% doped 12-11 catalytic core sequences. The cleavage reaction was performed at 25°C in cleavage buffer II containing 50 mM HEPES (pH 7.0 at 25°C), 50 mM NaCl, 10 mM MgCl2, 2 mM ZnCl2, and 2 mM CaCl2. Ligation reactions were performed using adapter LA1 for the first three rounds. In round 4, the re-selection procedure was divided into four separate steps. The aPCR products purified by PAGE were evenly divided and ligated using four separate ligation reactions with adapters LA2-LA5 until the end of selection (we had pre-enriched them).

[0097] The consensus sequence was manually constructed by considering parent sequences 12-11 and tested DNAzyme sequences that had superior cleavage activity (at least 90% final yield), taking into account an average of 1000 hits in the selection pool.

[0098] DMS ladder for identifying the cutting site DNA-zyme oligonucleotides double-labeled with terminal Cy3 and Cy5 dyes (50 pmol) were dissolved in 20 μL of 1×TE buffer. Oxidation was initiated by adding 4 μL of fresh 10% dimethyl sulfate (DMS) aqueous solution. The reaction mixture was incubated at 25°C for 6 minutes, then terminated with 125 μL of stop solution (200 mM 2-mercaptoethanol, 300 mM NaCl). To completely quench and remove the DMS, the solution was incubated at 37°C for 10 minutes, followed by precipitation with ethanol. The dry pellet was dissolved in 50 μL of fresh 10% piperidine aqueous solution and incubated at 95°C for 25 minutes to cleave the oxidized DNA. The solution was evaporated under vacuum. To remove trace amounts of piperidine, the DNA pellet was redissolved in 50 μL of water, the solution was evaporated, and this step was repeated once more. The resulting DMS ladder was separated using high-resolution urea-PAGE after the corresponding cleavage reaction to identify the cleavage sites.

[0099] DNAzyme oligonucleotides double-labeled with terminal Cy3 and Cy5 dyes (50 pmol) were dissolved in 20 μL of 1×TE buffer. Oxidation was initiated by adding 4 μL of fresh 10% dimethyl sulfate (DMS). The reaction mixture was incubated at 25°C for 6 minutes, then terminated with 125 μL of stop solution (200 mM 2-mercaptoethanol, 300 mM NaCl). To completely quench and remove the DMS, the solution was incubated at 37°C for 10 minutes, followed by precipitation with ethanol. The dry pellet was dissolved in 50 μL of fresh 10% piperidine and incubated at 95°C for 25 minutes to cleave the oxidized DNA. The solution was evaporated under vacuum. To remove trace amounts of piperidine, the DNA pellet was redissolved in 50 μL of water, the solution was evaporated, and this step was repeated once more. The resulting DMS ladder was separated using high-resolution urea-PAGE after the corresponding cleavage reaction to identify the cleavage sites.

[0100] Kinetic analysis of DNAzyme autocleavage activity The reaction mixture was prepared using 5× cleavage buffer (1x concentration: 50 mM HEPES [pH 7.00 at 25°C], 25 mM NaCl, 10 mM MgCl2) and 5 pmol DNAzyme. The reaction was initiated by adding ZnCl2 to the desired final concentration and final reaction volume of 10 μL, and incubated at 35°C immediately beforehand. A 1 μL aliquot was collected at the point of formulation throughout the reaction process and immediately quenched with 3 μL of stop solution (80% formamide, 1× TBE, 50 mM EDTA). The resulting sample was degraded by 12% urea-PAGE, and the gel was imaged with a Typhoon FLA 9500 laser scanner. Cleavage yield was quantified using concentration measurement with Image Lab software. obs The values ​​are calculated using the yield versus time data in the first-order kinetics Y=Y max (1-e kobs*t This was obtained by directly fitting it to the formula. In the formula, Y is the yield at the current time, and Y max This is the final yield, and k obs θ is the observed reaction rate, and t is time. The final yield was determined by evaluating the yield after a longer incubation period, preferably 24 hours.

[0101] To quantify the cleavage yield, cleaved and uncleaved DNA molecules were separated by PAGE, as these two species differ in electrophoretic mobility due to their different sizes. Detection was performed using DNAzyme molecules end-labeled with detectable fluorophore molecules.

[0102] Quantitative analysis was performed by generating gel images using a laser scanner (TyphoonFLA9000), and then the pixel values ​​of the obtained DNA bands were measured by density using software (ImageLab-BioRad). In this way, numerical values ​​representing the amount of cleaved and uncleaved DNAzyme molecules were obtained in this experiment. [Table 2-1] Table 2-2 Table 3-1 Table 3-2

[0103] Table 4-1 Table 4-2

Claims

1. A method for selecting a DNAzyme having self-cleavage activity, wherein the method is (a) To provide a plurality of single-stranded DNA strands, Each single-stranded DNA strand contains a core region of random nucleotides adjacent to its 5' terminal constant region 1 and its 3' terminal constant region 2. Each constant region includes a primer binding portion, (b) Subjecting the plurality of single-stranded DNA strands to self-cleavage conditions, (c) Isolating cleavage product 2, and optionally isolating cleavage product 1, The cutting product 2 includes the steady region 2 and the core region. The cleavage product 2 contains DNA self-cleavage activity, and The cutting product 1 includes the steady-state region 1, or The cutting product 2 includes the steady-state region 2, the core region, and a portion of the steady-state region 1. The cleavage product 2 contains DNA self-cleavage activity, and The cleavage product 1 includes the remaining portion of the steady-state region 1, and is isolated. (d) To generate a complement of the cleavage product 2, (e) Optionally, separate the complement of the cutting product 2, (f) Ligating the 3' end of the complement of the cleavage product 2 to the complement of the cleavage product 1, thereby obtaining the single-stranded DNA complement of step (a) having DNA self-cleavage activity, (g) Optionally, separate the complement of the single-stranded DNA strand, (h) Amplifying the complement of the single-stranded DNA strand to obtain a DNA strand containing the DNA self-cleavage activity, (i) optionally, to separate the DNA strand containing the self-cleavage activity, (j) Optionally, subject the DNA strand containing the self-cleavage activity to an error-prone (EP-PCR) step, (k) A method comprising, optionally, subjecting the DNA strand containing the self-cleavage activity of step (h), optionally step (i), or step (j) to one or more rounds of steps (b) to (h), and optionally one or more rounds of step (i) and / or (j).

2. The method according to claim 1, wherein the constant region 1 of the single-stranded DNA strand in step (a) includes a portion complementary to a portion of the constant region 2, thereby forming a hybridization stem adjacent to the core region.

3. The method according to claim 1 or 2, wherein the core region of the single-stranded DNA strand in step (a) comprises about 14 to 100 nucleotides, or about 20 to 100 nucleotides, or about 25 to 50 nucleotides, or about 30 to 40 nucleotides, or about 32 to 35 nucleotides.

4. The self-cleavage condition in step (b) is one or more ions, preferably monovalent, divalent, and / or trivalent, and / or polyvalent ions, for example, Na ＋ , Zn ２＋ Mg ２＋ ,Cd ２＋ Mn ２＋ Ca ２＋ The method according to any one of claims 1 to 3, comprising, and / or PLL-g-Dex.

5. The method according to any one of claims 1 to 4, wherein the self-cutting conditions in step (b) include an incubation period of about 15 to 20 hours, preferably about 18 hours, in the first round.

6. The method according to any one of claims 1 to 5, wherein the isolation in step (c) comprises polyacrylamide gel electrophoresis (PAGE).

7. The method according to any one of claims 1 to 6, wherein the cleavage product 2 in step (c) comprises the core region, the constant region 2, and self-cleavage activity, the cleavage product 1 comprises the constant region 1, and the cleavage of the single-stranded DNA strand occurs between the catalyst core and the constant region 1.

8. The method according to any one of claims 1 to 7, wherein the generation in step (d) comprises linear polymerase chain (PCR) amplification using a primer 2 which is preferably bound to the primer binding portion of the constant region 2, and preferably the primer 2 includes a tag that enables distinction from the cleavage product 2, and / or the primer 2 includes a label.

9. The method according to any one of claims 1 to 8, wherein the amplification in step (h) includes PCR, preferably two-step PCR.

10. A DNAzyme that can be obtained by the method of any one of claims 1 to 9, wherein the DNAzyme contains self-cleavage activity between or within the catalyst core and the constant region 1.

11. DNAzyme, sequence: 5'-N & GY ＄ Y ＃ GTN ＄ Y ＃ ACGCY ＃ Y ＄ YGTCTTATCGGTT Y ＄ Y ＄ N ＃ including N-3' (SEQ ID NO: 1), optionally with additional nucleotide N ＃ being included between positions 29 and 30 of the nucleotides of SEQ ID NO: 1, with nucleotide numbering in the 5' to 3' direction of SEQ ID NO: 1, In the array, A is a nucleotide that has the base adenine. C is a nucleotide that has the base cytosine, G is a nucleotide that has the base guanine. T is a nucleotide that has a thymidine base, N is independently A, C, G, or T. Y is independently C or T, ＄ However, independently, preferably T, ＃ However, independently, preferably C, & However, independently, preferably G, A DNAzyme comprising self-cleavage activity.

12. The self-cleavage activity is approximately 92%, 95%, 96%, or 98% in the final yield Y. ｍａｘ This results in, preferably, the final yield Y ｍａｘ However, Y = Y ｍａｘ (1-e －ｋｏｂｓ＊ｔ The DNAzyme according to claim 10 or 11, calculated by the formula, where Y is the reaction yield, kobs is the observed rate constant, and t is within 24 hours after the start of the reaction, preferably about 24 hours after the start of the reaction.

13. The DNAzyme according to any one of claims 10 to 12, wherein the self-cleavage activity is hydrolytic cleavage.

14. Use of a DNAzyme according to any one of claims 10 to 13 in the generation of a single-stranded DNA molecule.

15. The use according to claim 14, wherein the single-stranded DNAzyme is used in DNA nanotechnology, diagnostics, DNA synthesis, or gene therapy, preferably genome editing, more preferably homologous recombination repair (HDR).

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