Method for preparing single-stranded circular DNA with precise site-specific modification

Abstract

The invention provides a method for preparing single-stranded circular DNA. According to the method, through the design of the third DNA fragment and the first DNA fragment, chemical modification of any accurate site in the single-stranded circular DNA can be realized, so that the single-stranded circular DNA obtains higher stability, better binding capacity, improved immunogenicity and other properties, and the application of the single-stranded circular DNA in the field of biomedicine is effectively widened. According to the preparation method, the double-stranded assembly formed by complementation of the single-stranded circular DNA and the fragmented single-stranded nucleic acid chain can be obtained, and the single-stranded circular DNA can be obtained only by enzyme digestion reaction of the double-stranded assembly, so that the synthesis steps of the current single-stranded circular DNA are effectively simplified, the synthesis efficiency is improved, and the preparation method is suitable for industrial large-scale preparation.

Description

A method for preparing single-stranded circular DNA with precise site-specific modification Technical Field The present invention belongs to the field of biomedicine technology, and in particular, relates to a method for preparing single-stranded circular DNA. Background Art In recent years, DNA-based gene therapy has shown remarkable application potential in biomedical fields such as inherited metabolic diseases, antiviral vaccine preparation, tumor treatment and regenerative medicine. Currently, many studies have reported DNA-based anti-tumor drugs. In addition, antisense oligonucleotides (ASOs) based on DNA molecules have also been reported. [1] 、 small RNA antagonists (miRNA antagomir) [2] , CRISPR gene editing technology [3] , DNA expression vector [4] Treatment technologies are also developing rapidly, and the application of DNA drugs in biology is gaining more and more attention. DNA drugs are often easily degraded by enzymes in the body due to their own physical and chemical properties, and have problems such as poor stability, short efficacy and low bioavailability. Since single-stranded circular DNA (circDNA) does not have free ends, it is not easily digested by DNA exonucleases and exhibits higher stability. In addition, large-sized single-stranded circular DNA has a longer in vivo circulation time and better bioavailability because its molecular weight is higher than the glomerular filtration threshold. At present, studies have reported on miRNA sponge drugs based on small RNA antagonists, which mainly use single-stranded circular RNA (circRNA) as the drug skeleton. DNA is more stable than RNA, and when it binds to RNA, it can recruit RNase H in cells to degrade targeted RNA molecules. It can be predicted that it can show better effects than circRNA in its function as a miRNA sponge. Therefore, single-stranded circular DNA drugs have great potential in biomedical applications in gene expression regulation, especially in anti-miRNA. Current research shows that the introduction of chemical modifications (such as base modification, ribose modification, and phosphate backbone modification) can inhibit DNA from being recognized by nucleases and the immune system, thereby improving its stability in the body and reducing immunogenicity, while also improving its ability to bind to target molecules such as RNA and DNA, making it exhibit better therapeutic effects. Therefore, the synthesis of single-stranded circular DNA with precise site chemical modifications is particularly important in the development of related drugs. At present, the synthesis methods of single-stranded circular DNA are mainly divided into template-assisted short-chain DNA step-by-step addition cyclization method [5] and circular ligation of long single-stranded DNA based on secondary structure design [6] . The former repeatedly adds the short-chain DNA to be circularized and connected during the assembly process of a sufficient amount of template chain, so that it always maintains a low concentration, prompting it to assemble into a circular structure, and then connect to form a single-stranded circular DNA. However, the gradual addition method makes its steps cumbersome, which brings difficulties to operation and large-scale preparation. Another method is to shorten the head-to-tail distance of the raw DNA chain through secondary structure design, so as to use cyclase to directly connect the long single-stranded DNA head to tail to form a single-stranded circular DNA. Circularization efficiency and purification method are still limiting factors for its application. In addition, the length of the short-chain DNA raw material of the two methods limits the length and molecular weight of the target single-stranded circular DNA, and the general synthetic length range is below 200nt. Simultaneously due to the limitation of molecular weight, there are certain limitations in preparing single-stranded circular DNA with a longer length containing multiple functional sequences or with multiple repeat sequences. In addition, there are certain difficulties in the cyclization method based on long single chains for the introduction of chemical modification, which also causes obstacles to the in-depth study of the effect of chemical modification at specific sites and the drug development based on single-stranded circular DNA. Summary of the invention Given the limitations of existing technology, the synthesis of single-stranded circular DNA containing modified groups at specific sites and single-stranded circular DNA with a length greater than 200 nt are both huge challenges in the current synthesis field, and it is even more difficult to achieve a simple and convenient synthesis of these target products. The present invention provides a method for preparing single-stranded circular DNA, which can synthesize single-stranded circular DNA with any length (such as a length greater than 200 nt) and sequence simply and conveniently based on the precise assembly of a third DNA fragment and the subsequent connection process. Since the third DNA fragment is relatively short, those skilled in the art can synthesize it by phosphoramidite solid phase synthesis method. 9,10 The conventional methods in the art enable the third DNA fragment to carry a precisely positioned chemical modification at any site, so that the single-stranded circular DNA finally synthesized carries a modification at a precise site. The method of the present invention can prepare a single-stranded circular DNA with precise modification at any site, overcoming the technical obstacle of being unable to synthesize long-chain DNA with precise site modification at present, and has the advantages of low synthesis difficulty, high accuracy and low cost. At the same time, the method of the present invention can utilize enzyme cutting and enrichment to quickly purify the product, thereby reducing the economic cost and time cost of preparation. In summary, the method of the present invention uses the nucleic acid assembly principle to control the assembly structure of short-chain DNA, uses the enzyme ligation method to efficiently connect short-chain DNA fragments to form single-chain circular DNA molecules, and uses the enzyme digestion and sedimentation method to perform efficient purification to improve the yield of the target product. In some aspects, the invention provides a nucleic acid assembly that can be used to prepare long lengths of DNA with precisely targeted chemical modifications at any site. In some embodiments, the nucleic acid combination comprises at least 2 third DNA fragments, at least 2 first DNA fragments, and optionally, at least 1 second DNA fragment. The third DNA fragment is used to connect to form a long single-stranded DNA. Preferably, in some embodiments, the third DNA fragment is at most 20, preferably 4-10, and more preferably 4-8. Preferably, in some embodiments, the number of the second DNA fragment is 1 less than the third DNA fragment. Preferably, in some embodiments, the nucleic acid combination comprises 1 second DNA fragment. In some embodiments, the third DNA fragment is a 5' phosphorylated third DNA fragment. In some embodiments, the third DNA fragment comprises a 5' spacer, a functional sequence region and a 3' spacer. Preferably, in some embodiments, the third DNA fragment comprises a 5' spacer, a functional sequence region and a 3' spacer from the 5' end to the 3' end. In some embodiments, the 5' spacer sequence of each third DNA fragment is different, the 3' spacer sequence of each third DNA fragment is different, and / or the 5' spacer sequence and the 3' spacer sequence of the same third DNA fragment are different. Preferably, in some embodiments, the 5' spacer sequence of each third DNA fragment is different, the 3' spacer sequence of each third DNA fragment is different, and the 5' spacer sequence and the 3' spacer sequence of the same third DNA fragment are different. In some embodiments, the length of the 5' spacer and / or the 3' spacer is 8-20nt. In some embodiments, the functional sequence region is a sequence that interacts with miRNA, mRNA, pre-mRNA, lncRNA, circRNA, DNA or protein. Preferably, in some embodiments, the functional sequence region is a sequence that interacts with miRNA and / or mRNA and / or pre-mRNA. In some embodiments, the third DNA fragment is 16-100 nt in length. In some embodiments, the first DNA fragment comprises a sequence complementary to the 5' spacer region of the third DNA fragment and a sequence complementary to the 3' spacer region of another of the third DNA fragments. Preferably, in some embodiments, the first DNA fragment comprises a sequence completely complementary to the 5' spacer region of the third DNA fragment and a sequence completely complementary to the 3' spacer region of another of the third DNA fragments. In some embodiments, the first DNA fragment is 16-40 nt in length. In some embodiments, the second DNA fragment comprises a sequence that is completely or partially complementary to the sequence at the 5' end of the long single-stranded DNA, and a sequence that is completely or partially complementary to the sequence at the 3' end of the long single-stranded DNA. Preferably, in some embodiments, the second DNA fragment comprises a sequence that is complementary to a 4-35nt sequence at the 5' end of the long single-stranded DNA, and a sequence that is complementary to a 4-35nt sequence at the 3' end of the long single-stranded DNA. In some embodiments, the second DNA fragment is 20-80 nt in length, preferably 64 nt. In some aspects, the present invention provides a method for preparing single-stranded circular DNA, comprising the following steps: Step (1): connecting the third DNA fragment to obtain a long single-stranded DNA; Step (2): Connect two long single-stranded DNAs to obtain single-stranded circular DNA. In some embodiments, the third DNA fragment is 5'-phosphorylated before step (1), ie, a phosphate group is added to the 5' end of the third DNA fragment. In some embodiments, step (1) comprises the following steps: Step (1.1): mixing the third DNA fragment and the first DNA fragment and then annealing them; Step (1.2): connecting the product of step (1.1); Optionally, in some embodiments, step (1) includes step (1.3): purifying the product of step (1.2). In some embodiments, step (1.2) uses T4 DNA ligase for ligation. In some embodiments, step (1.3) comprises an ultrafiltration step. Optionally, in some embodiments, purification is performed using an ultrafiltration tube or using size-sorting magnetic beads. In some embodiments, step (2) comprises the following steps: Step (2.1): mixing the product of step (1) with the second DNA fragment and then annealing; Step (2.2): connecting the product of step (2.1); Optionally, in some embodiments, step (2) includes step (2.3): purifying the product of step (2.2). In some embodiments, step (2.2) uses a circularizing ligase for ligation. In some embodiments, step (2.3) comprises the following steps: Step (2.3.1): adding DNase exonuclease for enzyme digestion; Step (2.3.2): Purification, optionally, in some embodiments, is performed using ethanol and using an ultrafiltration method. In some embodiments, the length of the single-stranded circular DNA is 180 nt to 1000 nt. Preferably, in some embodiments, the length of the single-stranded circular DNA is 240 nt to 1000 nt. In some aspects, the present invention provides a use of a nucleic acid combination of the present invention in preparing a single-stranded circular DNA. The single-stranded circular DNA may contain any DNA modification (such as any DNA modification of interest) at any site. In some aspects, the present invention provides a single-stranded circular DNA of the present invention, and its use in treating, preventing or diagnosing a disease or in preparing a drug for treating or preventing a disease or a diagnostic or detection reagent. In some embodiments, the single-stranded circular DNA performs a regulatory function on the target nucleic acid. Preferably, in some embodiments, the function of pre-mRNA, mRNA, miRNA, LncRNA, circRNA and tRNA is regulated in the subject. In some embodiments, the single-stranded circular DNA can act as a sponge for miRNA to adsorb targeted miRNA or antisense nucleotides to induce degradation of targeted mRNA. Preferably, in some embodiments, the single-stranded circular DNA is a miRNA inhibitor and an antisense nucleotide drug. In some aspects, the present invention provides a use of a second DNA fragment for preparing single-stranded circular DNA. The second DNA fragment comprises sequences complementary to the sequences at the 5' end and the 3' end of the aforementioned long single-stranded DNA. In some embodiments, the 5' and 3' end sequences of the second DNA fragment comprise sequences that are completely or partially complementary to the 5' end sequence of the aforementioned long single-stranded DNA. In some embodiments, the non-5'-non-3' sequence of the second DNA fragment comprises a sequence that is completely or partially complementary to the 3' sequence of the aforementioned long single-stranded DNA. The method of the present invention can be used as a platform for preparing single-stranded circular DNA and can be used to prepare single-stranded circular DNA of any length and with any precise DNA modification at any site. The preparation of single-stranded circular DNA by the method of the present invention has the following advantages: (1) It is possible to prepare single-stranded circular DNA with any DNA modification of interest at any site, that is, it is possible to achieve precise modification of single-stranded circular DNA; (2) The length and sequence of the prepared single-stranded circular DNA are not limited, that is, single-stranded circular DNA of any length (e.g., a length greater than 200 nt) or sequence can be prepared; (3) The prepared single-stranded circular DNA has high purity, high yield, high efficiency, greatly reduces the impurity molecules produced in traditional methods, and is low in cost, suitable for industrial production; (4) A single-stranded circular DNA with symmetry can be prepared, which can play a better role as a miRNA sponge, for example; (5) The preparation process is simple and can be prepared by a one-pot synthesis method. BRIEF DESCRIPTION OF THE DRAWINGS FIG1 shows a schematic diagram of the synthesis of single-stranded circular DNA with precise site chemical modification; FIG2 shows the synthesis characterization of unmodified 254 nt single-stranded circular DNA; FIG3 shows the purification characterization and circular structure identification of unmodified 254 nt single-stranded circular DNA; FIG4 shows the cell transfection and anti-miRNA effects of unmodified 254 nt single-stranded circular DNA; FIG5 shows the synthesis characterization of unmodified 240 nt and 264 nt single-stranded circular DNA; FIG6 shows the effect characterization of RNase H targeted cleavage of RNA mediated by unmodified 264 nt single-stranded circular DNA; FIG. 7 shows a schematic diagram of the sequence of a 254 nt single-stranded circular DNA modified with 5mC at a precise site. DETAILED DESCRIPTION Listed below are definitions of various terms used to describe the compositions and nucleic acid combinations disclosed herein. These definitions apply to the terms used throughout this specification and claims, except where otherwise limited in specific cases, where these terms are used either individually or as part of a larger group. As used herein, the terms "optional" and "optionally" include both the cases of being selected or not being selected. For example, "optionally modified" includes both the cases of being modified and not being modified. As used herein, the terms "a," "an," and "the" are generally construed to cover both the singular and the plural. The term "include" used herein means the phrase "including (but not limited to)", and can be used interchangeably therewith. The term "comprising" used herein means the phrase "including (but not limited to)", and can be used interchangeably therewith. The technical solutions using "include" or "comprising" in this patent can be further defined as "consisting of" or "composed of". Throughout this specification, reference to "one embodiment," "some embodiments," "embodiments," "specific embodiments," "related embodiments," "an embodiment," "another embodiment," or "further embodiments," or combinations thereof, means that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. As used herein, "strip", such as "1 strip", refers to one, a type or a section of corresponding nucleic acid molecules. For example, "at least 2 third DNA fragments" means that the nucleic acid combination contains at least 2 DNA nucleic acid molecules with different sequences and / or different modifications. As described herein, "composition" can be used interchangeably with "combination", "system" and "system", which does not limit the components to be in one product or package. In some embodiments, the "composition" described herein may refer to a product or package in which the corresponding components are in one product or package. In some embodiments, the "composition" may also refer to a product or package in which the corresponding components are not in one product or package. The prior art cited in this specification is hereby incorporated by reference in its entirety and for all purposes. The third DNA fragment The "third DNA fragment" described herein refers to a DNA fragment used to synthesize the single-stranded circular DNA of the present invention. The third DNA fragment can be connected into a single-stranded circular DNA by the method of the present invention. Since the third DNA fragment is short, those skilled in the art can make the third DNA fragment carry a precisely located DNA modification at any site by conventional methods in the art, so that the final synthesized single-stranded circular DNA carries a DNA modification at a precise site. The third DNA fragment herein can be synthesized by any known method in the art. In some embodiments, the third DNA fragment is a 5' phosphorylated third DNA fragment. There are many types of DNA modifications that can be used in the present invention. All DNA modifications existing in the art can be used for the purpose of the present invention. DNA modifications that can be used in the present invention include, but are not limited to, base modifications such as m 6 A, Ψ, m 1 A.m 5 A.ms2 i 6 A.i 6 A.m 3 C.m 5 C. ac 4 C.m 7 G, m2,2G, m 2 G、m 1 G, Q, m 5 U、mcm 5 U、ncm 5 U、ncm 5 Um、D、mcm 5 s 2 U、Inosine(I)、hm 5 C.s 4 U.s. 2 U, azobenzene, Cm, Um, Gm, t 6 A, yW, ms 2 t 6 A or its derivatives; ribose modifications include, for example, LNA, 2'-OMe, 3'-OMeU, vmoe, 2'-F or 2'-OBn (2'-O-benzyl group) or their derivatives; phosphodiester bond modifications include, for example, phosphorothioate (PS), nucleotide triphosphate (NTPαS) or its derivatives, etc. In some embodiments, the length of the third DNA fragment is between 16-100nt. Preferentially, in some embodiments, the length of the third DNA fragment is 20nt, 25nt, 30nt, 35nt, 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, 80nt, 85nt or any integer therebetween. More preferably, in some embodiments, the length of the third DNA fragment is 40nt, 45nt, 50nt, 55nt, 60nt, 65nt, 70nt, 75nt, 80nt, 85nt or any integer therebetween. In some embodiments, the third DNA fragment comprises a 5' spacer, a functional sequence region and a 3' spacer. Preferably, in some embodiments, the third DNA fragment is a 5' spacer (5'spacer), a functional sequence region and a 3' spacer (3'spacer) from the 5' end to the 3' end. In some embodiments, the length of the 5' spacer and / or the 3' spacer is 8-20nt or any integer therebetween. Preferably, in some embodiments, the length of the 5' spacer and / or the 3' spacer is 10-18nt or any integer therebetween. More preferably, in some embodiments, the length of the 5' spacer and / or the 3' spacer is 13-16nt or any integer therebetween. In some embodiments, the functional sequence region is a specific sequence that interacts with miRNA, mRNA, pre-mRNA, lncRNA, circRNA, DNA, protein, etc. Preferably, in some embodiments, the functional sequence region is a specific sequence that interacts with miRNA. In order to realize the different functions of single-stranded circular DNA, such as serving as a miRNA sponge, regulating transcription, localizing proteins, and expressing target genes, those skilled in the art can adjust the sequence and sequence length of the functional sequence region based on the knowledge in the art to realize the above functions. The third DNA fragment can be connected according to the method of the present invention to form a long single-stranded DNA. The long single-stranded DNA can be used as an intermediate of the single-stranded circular DNA synthesized according to the method of the present invention. The third DNA fragment can be synthesized using any means known in the art, such as solid phase synthesis. The first DNA fragment The "first DNA fragment" described herein is a single-stranded DNA nucleic acid fragment, which can be used to prepare single-stranded circular DNA according to the method of the present invention. In some embodiments, the first DNA fragment comprises a sequence complementary to the 5' spacer region of the third DNA fragment and a sequence complementary to the 3' spacer region of the third DNA fragment. Preferably, in some embodiments, the first DNA fragment comprises a sequence completely complementary to the 5' spacer region of the third DNA fragment and a sequence completely complementary to the 3' spacer region of the third DNA fragment. In some embodiments, the length of the first DNA fragment is 16-40 nt. Preferably, in some embodiments, the length of the first DNA fragment is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nt or any integer therebetween. The second DNA fragment The "second DNA fragment" described herein is a single-stranded DNA nucleic acid fragment, which can be used to prepare single-stranded circular DNA according to the method of the present invention. In some embodiments, the second DNA fragment comprises a sequence that is completely or partially complementary to the sequence at the 5' end of the long single-stranded DNA formed by the third DNA fragment, and a sequence that is completely or partially complementary to the sequence at the 3' end of the long single-stranded DNA formed by the third DNA fragment. In some embodiments, the second DNA fragment comprises a sequence that is complementary to a 4-35nt sequence at the 5' end of the long single-stranded DNA, and a sequence that is complementary to a 4-35nt sequence at the 3' end of the long single-stranded DNA. The sequence at the 5' end of the long single-stranded DNA described herein refers to a nucleic acid sequence extending a certain length from the first base at the 5' end of the long single-stranded DNA to the 3' end. In some embodiments, the certain length may be 300nt, 280nt, 260nt, 240nt, 220nt, 200nt, 180nt, 160nt, 140nt, 120nt, 100nt, 80nt, 60nt, 40nt, 20nt, preferably less than 200nt. The sequence at the 3' end of the long single-stranded DNA described herein refers to a nucleic acid sequence extending from the first base at the 3' end of the long single-stranded DNA to the 5' end for a certain length. In some embodiments, the certain length may be 300nt, 280nt, 260nt, 240nt, 220nt, 200nt, 180nt, 160nt, 140nt, 120nt, 100nt, 80nt, 60nt, 40nt, 20nt, preferably less than 200nt. In some embodiments, the length of the second DNA fragment is 20-80 nt, such as 20 nt, 30 nt, 40 nt, 50 nt, 60 nt, 70 nt, 80 nt or any integer therebetween. Preferably, in some embodiments, the length of the second DNA fragment is 64 nt. In some embodiments, the 5' and 3' end sequences of the second DNA fragment comprise sequences that are completely or partially complementary to the 5' end sequence of the aforementioned long single-stranded DNA. In some embodiments, the non-5'-non-3' sequence of the second DNA fragment comprises a sequence that is completely or partially complementary to the 3' sequence of the aforementioned long single-stranded DNA. The 5' end sequence of the second DNA fragment described herein refers to a nucleic acid sequence extending a certain length from the first base of the 5' end of the DNA fragment to the 3' end. In some embodiments, the certain length can be 300nt, 280nt, 260nt, 240nt, 220nt, 200nt, 180nt, 160nt, 140nt, 120nt, 100nt, 80nt, 60nt, 40nt, 20nt, preferably less than 200nt. The 3' end sequence of the second DNA fragment described herein refers to a nucleic acid sequence extending from the first base of the 3' end of the DNA fragment to the 5' end for a certain length. In some embodiments, the certain length may be 300nt, 280nt, 260nt, 240nt, 220nt, 200nt, 180nt, 160nt, 140nt, 120nt, 100nt, 80nt, 60nt, 40nt, 20nt, preferably less than 200nt. The non-5'-non-3' sequence of the second DNA fragment described herein refers to a nucleic acid sequence of any length located outside the 5'-end and 3'-end sequences of the DNA fragment. Nucleic acid combination The present invention provides a nucleic acid combination, which can be used to prepare single-stranded circular DNA according to the method of the present invention. The single-stranded circular DNA can have a precise positioning chemical modification at any site, or can be a single-stranded circular DNA with a large length (such as greater than 200 nt). In some embodiments, the third DNA fragment is a 5' phosphorylated third DNA fragment. In some embodiments, the nucleic acid combination comprises at least 2 third DNA fragments, at least 2 first DNA fragments, and optionally, at least 1 second DNA fragment. Preferably, the number of the third DNA fragments is 2-20 or any integer therebetween, preferably, the number of the third DNA fragments is 4-10, more preferably 4-8, such as 4, 5, 6, 7, 8. In some embodiments, the 5' spacer sequences of different third DNA fragments are different. In some embodiments, the 3' spacer sequences of different third DNA fragments are different. In some embodiments, the functional sequence regions of different third DNA fragments are identical. In some embodiments, the functional sequence regions of different third DNA fragments are not identical. In some embodiments, the functional sequence regions of different third DNA fragments are all different. The molar amounts of the different third DNA fragments can be in any ratio. Preferably, in some embodiments, the molar amounts of the different third DNA fragments are the same. The molar amounts of the different first DNA fragments can be in any ratio. Preferably, in some embodiments, the molar amounts of the different first DNA fragments are the same. The molar amount of the total third DNA fragments (i.e., the sum of the molar amounts of the different third DNA fragments) and the molar amount of the total first DNA fragments (i.e., the sum of the molar amounts of the different first DNA fragments) can be in any ratio. Preferably, in some embodiments, the molar amount of the total third DNA fragments is the same as the molar amount of the total first DNA fragments. In some embodiments, the nucleic acid combination herein may also include enzymes, buffers and other reagents for preparing single-stranded circular DNA according to the methods herein, and optionally constitute a kit with the enzymes, buffers and other reagents. Method for preparing single-stranded circular DNA The present invention provides a method for preparing single-stranded circular DNA. The single-stranded circular DNA can carry a precisely located DNA modification at any site. The preparation method comprises the following steps: Step (1): connecting the third DNA fragment to obtain a long single-stranded DNA; Step (2): Connect two long single-stranded DNAs to obtain single-stranded circular DNA. Preferably, in some embodiments, the third DNA fragment and the first DNA fragment in step (1) are in equimolar amounts. In some embodiments, the third DNA fragment is 5'-phosphorylated before step (1), ie, a phosphate group is added to the 5' end of the third DNA fragment. i. Step (1) In some embodiments, step (1) comprises the following steps: Step (1.1): mixing the third DNA fragment and the first DNA fragment and then annealing them; Step (1.2): connecting the product of step (1.1); Optionally, in some embodiments, step (1) further comprises step (1.3): purifying the product of step (1.2). In some embodiments, step (1.2) uses T4 DNA ligase for ligation. In some embodiments, step (1.3) comprises an ultrafiltration step, optionally using an ultrafiltration tube or purification using size-sorting magnetic beads. ii. Step (2) In some embodiments, step (2) comprises the following steps: Step (2.1): mixing the product of step (1) with the second DNA fragment and then annealing; Step (2.2): connecting the product of step (2.1); Optionally, in some embodiments, step (2.3): purifying the product of step (2.2). In some embodiments, step (2.2) uses a circularizing ligase for ligation. In some embodiments, step (2.3) comprises the following steps: Step (2.3.1): adding DNase I for digestion; and / or Step (2.3.2): Purification, optionally, in some embodiments, is performed using ethanol and using an ultrafiltration method. The method for preparing single-stranded circular DNA of the present invention can be used to prepare single-stranded circular DNA of any length. In some embodiments, the length of the single-stranded circular DNA is 180nt-1000nt. Preferably, in some embodiments, the length of the single-stranded circular DNA is 240nt-1000nt or any integer therebetween. An example of a method for preparing a single-stranded circular DNA with precise site chemical modification of the present invention is as follows, which comprises the following steps: Short chain synthesis: multiple third DNA fragments, multiple first DNA fragments and second DNA fragments are synthesized by solid phase synthesis. The multiple third DNA fragments can be recorded as, for example, circ-n i 、circ-n i+1 、circ-n i+2 、circ-n i+3 , the multiple first DNA fragments can be recorded as DNAm i 、DNAm i+1 、DNAm i+2 、DNAm i+2 , the second DNA fragment is denoted as cycle-n i . The plurality of third DNA fragments are sequentially composed of a 5' spacer, a functional sequence region and a 3' spacer from the 5' end to the 3' end. Among them, the first DNA fragment DNAm i The 5' end sequence of the DNA fragment circ-n i+1 The first DNA fragment m i The 3' end sequence of the DNA fragment circ-n i The 3' spacer region is completely complementary to the sequence. 1. Annealing step: Annealing the plurality of third DNA fragments (circ-n i 、circ-n i+1… ) and the first DNA fragment (DNAm i , DNAm i+1… ) are mixed in the same reaction system in equimolar equivalents, annealed, and form a double-stranded assembly precursor; wherein there are connectors between adjacent third DNA fragments, and there are connectors between adjacent first DNA fragments; and the connectors between adjacent third DNA fragments and the connectors between adjacent first DNA fragments are staggered. 2. Ligation step: The double-stranded assembly after annealing in step 1 is subjected to enzyme ligation treatment (T4 DNA Ligase 2), and each connection port is connected to obtain a double-stranded assembly formed by the complementarity of the continuous long single-stranded DNA and the fragmented DNA nucleic acid chain. 3. In a crude purification step, the long single-stranded DNA system obtained in step 2 is subjected to ultrafiltration for concentration and desalting. In some embodiments, the operation is performed using an ultrafiltration tube. In some embodiments, the operation is performed using volume separation magnetic beads. 4. Annealing step: Cycle-n the product from step 3 and the second DNA fragment i The equimolar amounts are mixed in the same reaction system and annealed to form an assembly precursor containing a double cross structure. The second DNA fragment cycle-n i It can be completely complementary to the 4-35nt part of the 5' end and 3' end of the long single-stranded DNA formed by connecting the above-mentioned multiple third DNA fragments, so that cycle-n i It forms a double crossover structure with two long single-stranded DNAs. 5. Connection step: The assembly in step 4 is treated with circligase to connect all interfaces near the double cross structures to obtain a double-stranded assembly formed by the complementarity of the circular long single-stranded DNA and the fragmented DNA nucleic acid chain. 6. Purification steps: A. Enzyme digestion step 1: Use DNase exonuclease to treat the product of step 5 to completely remove the complementary DNA strand. B. Enrichment: Ethanol precipitation followed by ultrafiltration to remove salts to enrich the target single-stranded circular DNA. In some embodiments, according to the method for preparing single-stranded circular DNA of the present invention, the length of the single-stranded circular DNA is greater than 180 nt, preferably 240 nt-1000 nt. Example The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially. The experimental techniques and experimental methods used in this example are all conventional technical methods unless otherwise specified. For example, the experimental methods in the following examples that do not specify specific conditions are usually carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The materials, reagents, etc. used in the examples can be obtained through regular commercial channels unless otherwise specified. Experimental Materials The experimental reagent information used in this example is shown in Table 1, the 5' phosphorylation-modified third DNA fragment used is shown in Table 2, and the sequences of the first DNA fragment and the second DNA fragment used are shown in Table 3. Table 1 Experimental reagent information Table 2 The third DNA fragment Table 3 The first DNA fragment and the second DNA fragment The DNA fragment sequences in the examples were synthesized by mermade12 solid phase synthesizer. All experimental water was enzyme-free water. Other chemical reagents were of premium grade or higher. Example 1 Preparation, characterization and functional verification of unmodified 254 nt single-stranded circular DNA In the third DNA fragment of the raw material, circ-1 and circ-4 each contain a complementary sequence of miRNA-21, circ-2 can contain a complementary sequence of miRNA-221, and circ-3 contains a complementary sequence of miRNA-195. Therefore, the prepared target circDNA contains 4 complementary sequences of miRNA-21, 2 complementary sequences of miRNA-221 and 2 complementary sequences of miRNA-195, which can fully exert the effect of resisting multiple miRNAs. The multiple targeting sites of a single drug also increase the local concentration of the drug, thereby improving the binding effect with the targeted miRNA. Preparation of circDNA: Step 1: Take circ-1, circ-2, circ-3, circ-4, DNA-1, DNA-2 and DNA-3 in an equivalent amount at a concentration of 10 μmol / L in 1×TAE-12.5mmol / L magnesium acetate buffer. The mixture was incubated at 95°C for 5 minutes and then cooled to room temperature for annealing. Step 2: Treat the above assembly mixture system with T4 DNA Ligase 2 and keep it at 37°C for 10 h. Desalt it by ultracentrifugation in RNase free ddH2O at room temperature using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. Step 3: The above solution was concentrated to a fixed concentration and mixed with an equivalent amount of cycle-1 at a concentration of 1 umol / L in 1×TAE-1 2.5 mmol / L magnesium acetate buffer, incubated at 95°C for 5 minutes, and then cooled and annealed at room temperature. Step 4: Then treat with circligase and keep at 45°C for 2 hours to obtain single-stranded circular DNA. Step 5: Use Exonuclease III to digest the linear DNA fragments in the system. Finally, use ethanol precipitation to enrich the target product. Characterization tests of circDNA: Polyacrylamide gel electrophoresis characterization: 10% (19:1) polyacrylamide gel and sample solution mixed with formamide at a ratio of 1:1 were prepared separately. The sample was loaded at 50°C in 1×TBE buffer and run at a voltage of 200V. The synthesis characterization is shown in Figure 2, and the purification characterization and ring structure identification are shown in Figure 3. Functional validation of circDNA as a miRNA sponge MCF-7 cells were selected and cultured to the logarithmic growth phase, and the single-stranded circular DNA obtained in Example 1 was transfected into the cells through Messenger Max for co-culture, and the cell activity and the expression level of the circular DNA targeted gene in the cells were detected. The specific steps are as follows: Step 1: MCF-7 adherent cells were routinely cultured in DMEM culture medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, final concentration of penicillin 100 units / mL, streptomycin 100 μg / mL), and cultured in a 5% CO2 incubator at 37°C. Step 2: The cells were cultured in a 96-well plate, 8,000 cells were plated per well, and 100 μL of DMEM medium containing 10% fetal bovine serum was added. After culturing for 12 h, the circular DNA obtained in Example 1 and the dual luciferase reporter gene plasmid were co-transfected into the cells using Messenger Max transfection reagent, and the final concentration was 20 nmol / L. The blank control group was added with an equal volume of PBS. Step 3: After 24 hours of cell culture, the cells were treated with a dual luciferase reporter gene detection kit and analyzed using a microplate reader to detect the expression level of the targeted gene by comparing the intensities of the two chemiluminescence signals ( FIG. 4 ). Step 4: Only single-stranded circular DNA was transfected in step 2, and cell activity was detected. After 24h, 48h, 72h and 120h of cell culture, 10ul of MTS reagent was added to each 96-well well, and the cells were returned to the incubator for incubation for 1h, and the absorbance of the samples at 450nm and 690nm was detected using a microplate reader (Figure 4). Functional test results show that for the three miRNAs, compared with the miRNA complementary short chain (positive reference), single-stranded circular DNA showed better anti-miRNA effects at lower concentrations. Specifically, it improves the expression of the miRNA-targeted luciferase reporter gene. In the cell activity test experiment, compared with the control group without drugs, the addition of single-stranded circular DNA and miRNA complementary short chain (positive reference) can inhibit the rate of cell proliferation, among which the single-stranded circular DNA has a more obvious effect. On a time scale of 5 days, the inhibition efficiency can reach 50%. Example 2 Preparation and Characterization of Unmodified 240nt Single-Stranded Circular DNA The raw DNA fragments d23-1 and d23-2 each include a functional sequence that can cause exon skipping in the exon 23 of the mouse DMD gene during the splicing process of the pre-mRNA. Therefore, the prepared target circDNA can fully exert the function of exon skipping. The multiple targeting sites of a single drug also increase the local concentration of the drug, thereby improving the binding effect with the targeted pre-mRNA. Preparation of circDNA: Step 1: Take d23-1, d23-2 and d23-C1 in equivalent amounts at a concentration of 10 μmol / L and mix them in a 1×TAE-12.5mmol / L magnesium acetate buffer. Incubate the mixture at 95°C for 5 minutes and then cool and anneal at room temperature. Long single-stranded DNA was prepared by the method shown in step 2 of Example 1. Step 3: The above solution was concentrated to a fixed concentration and mixed with an equivalent amount of cycle-d23 at a concentration of 1 μmol / L in a 1×TAE-12.5mmol / L magnesium acetate buffer, incubated at 95°C for 5 minutes, and then cooled and annealed at room temperature. A 240 nt single-stranded circular DNA was prepared by the method described in Steps 4-5 of Example 1. The raw DNA fragments d23-1 and d23-2 each include a functional sequence that can cause exon skipping in the exon 23 of the mouse DMD gene during the splicing process of the pre-mRNA. Therefore, the prepared target circDNA can fully exert the function of exon skipping. The multiple targeting sites of a single drug also increase the local concentration of the drug, thereby improving the binding effect with the targeted pre-mRNA. Characterization tests of circDNA: Polyacrylamide gel electrophoresis characterization: 10% (19:1) polyacrylamide gel and sample solution mixed with formamide at a ratio of 1:1 were prepared separately. The sample was loaded at 50°C in 1×TBE buffer and run at a voltage of 200V. The synthesis and purification characterization is shown in Figure 5. The single target band proves the successful synthesis of the target product. Example 3 Preparation, characterization and functional verification of unmodified 264 nt single-stranded circular DNA The functional region of the raw DNA fragment ASO-1 contains a sequence that can target PCSK9 mRNA and a sequence that can target nanoLUC mRNA expression, ASO-2 contains another sequence that can target PCSK9 mRNA, and ASO-3 contains another sequence that can target nanoLUC mRNA. Therefore, the prepared target circDNA contains 2 different functional sequences targeting PCSK9 mRNA, each functional sequence has 2 repeats, and a total of 4 functional sequences targeting PCSK9 mRNA. Similarly, the circDNA also contains 2 different functional sequences targeting nanoLUC mRNA, each functional sequence has two repeats, and a total of 4 functional sequences targeting nanoLUC mRNA. Therefore, the circDNA targets PCSK9 and nanoLUC mRNA at the same time, and can target different regions of the same mRNA at the same time, which can fully exert the function of inhibiting gene expression. The multiple targeting sites of a single drug also increase the local concentration of the drug, thereby improving the binding effect with the targeted mRNA. Preparation and characterization of circDNA: Step 1: ASO-1, ASO-2, ASO-3, ASO-C1 and ASO-C2 were mixed in 1×TAE-12.5mmol / L magnesium acetate buffer at an equivalent concentration of 10umol / L. The mixture was incubated at 95°C for 5 minutes and then cooled to room temperature for annealing. Long single-stranded DNA was prepared by the method shown in step 2 of Example 1. Step 3: The above solution was concentrated to a fixed concentration and mixed with an equivalent amount of cycle-ASO at a concentration of 1 umol / L in 1×TAE-12.5 mmol / L magnesium acetate buffer, incubated at 95°C for 5 minutes, and then cold annealed at room temperature. The 264 nt single-stranded circular DNA was prepared by the method shown in steps 4-5 in Example 1. Characterization and functional testing of circDNA: A. Synthesis characterization (polyacrylamide gel electrophoresis characterization): Prepare 10% (19:1) polyacrylamide gel and sample solution mixed with formamide at 1:1. Load the sample at 50°C in 1×TBE buffer and run the gel at 200V. The synthesis and purification characterization is shown in Figure 5. The single target band proves the successful synthesis of the target product. B. Functional characterization (polyacrylamide gel electrophoresis characterization): Step 1: Mix circDNA or ASO short chain (positive reference) and targeted nanoLUC mRNA in 1×TAE buffer according to equivalent amount, incubate at 70°C for 5 minutes, and then cool down to 30°C at a rate of 0.5°C / min. Step 2: Treat the mixed system with RNase H and incubate at 37°C for 30 min. Step 3: Prepare 10% (19:1) polyacrylamide gel and sample solution mixed with formamide at a ratio of 1:1, load the sample at 50°C in 1×TBE buffer, and run the gel at a voltage of 200V. The characterization results are shown in Figure 6A. circDNA carries two sequences complementary to nanoLUC mRNA, which can successfully recruit RNase H to cleave nanoLUC mRNA to form three RNA fragments of different lengths. ASO short chain can only cause nanoLUC mRNA to be cleaved to form two RNA fragments. At the same time, the length of the generated RNA fragment is exactly the same as expected. This proves that circDNA can recruit RNase H to degrade RNA molecules after complementary pairing with RNA. Experimental verification of intracellular inhibition of mRNA levels: A. Functional validation of circDNA inhibiting PCSK9 mRNA levels by ASO Hep-G2 cells were selected and cultured to the logarithmic growth phase, and the single-stranded circular DNA obtained in Example 3 was transfected into the cells by Messenger Max for co-culture, and the inhibitory effect of the single-stranded circular DNA on PCSK9 mRNA was detected. The specific steps are as follows: Step 1: Hep-G2 adherent cells were routinely cultured in DMEM culture medium supplemented with 10% fetal bovine serum and cultured in a 5% CO2 incubator at 37°C. Step 2: The cells were cultured in a 12-well plate, 200,000 cells were plated per well, 1 mL of DMEM medium containing 10% fetal bovine serum was added, and after culturing for 12 h, the circular DNA obtained in Example 1 and Example 2 was transfected into the cells using Messenger Max transfection reagent, respectively, and the final concentration was 20 nmol / L. The blank control group was added with an equal volume of PBS. Step 3: After culturing the cells for 24 hours, treat the cells with an RNA extraction kit to extract total RNA from the cells. Step 4: Use a reverse transcription-qPCR kit to process total RNA and characterize the expression level of PCSK9. The experimental results (Figure 6C) show that compared with the control without drug addition, circDNA can significantly inhibit the mRNA level of PCSK9, and has a concentration-dependent property. And at a lower concentration, it can achieve the same inhibitory effect as the positive reference PC-1 and PC-2 (commercial ASO products that efficiently inhibit PCSK9 expression), and the effect is significantly better than PCS-1 and PCS-2 (unmodified short DNA chains, positive references). B. Functional verification of circDNA inhibiting the mRNA level of co-transfected nanoLUC by ASO Hep-G2 cells were selected and cultured to the logarithmic growth phase, and the single-stranded circular DNA obtained in Example 3 and the nanoLUC gene expression vector were co-transfected into the cells through Messenger Max, and the inhibitory effect of the single-stranded circular DNA on the expression of nanoLUC mRNA was detected. The specific steps are as follows: Step 1: Hep-G2 adherent cells were routinely cultured in DMEM culture medium supplemented with 10% fetal bovine serum and cultured in a 5% CO2 incubator at 37°C. Step 2: The cells were cultured in a 96-well plate, 8,000 cells were plated per well, 100 μL of DMEM medium containing 10% fetal bovine serum was added, and after culturing for 12 h, the circular DNA obtained in Example 3 was transfected into the cells using Messenger Max transfection reagent, and the final concentration was 20 nmol / L. The blank control group was added with an equal volume of PBS. Step 3: After 24 hours of cell culture, the cells were treated with the Nano Glo luciferase reporter gene detection system, and the chemiluminescence intensity was detected using a microplate reader to characterize the inhibitory effect of single-stranded circular DNA on the expression level of the target gene by comparison. The experimental results (Figure 6B) show that compared with the control without drug addition, circDNA can significantly inhibit the expression level of nanoLUC in a concentration-dependent manner. And at a lower concentration, it can achieve an inhibitory effect comparable to that of the positive reference LUC-2 and LUC-4 (unmodified DNA short chains). Example 4 Preparation of 254 nt single-stranded circular DNA with precise site 5mC modification Step 1: DNA fragments of circ-1, circ-2, circ-3 and circ-4 with 5mC modification and DNA fragments of DNA-1, DNA-2 and DNA-3 as in Example 1 were mixed in 1×TAE-1 2.5mmol / L magnesium acetate buffer at an equivalent concentration of 10umol / L. The mixture was incubated at 70°C for 5 minutes and then cooled at a rate of 0.5°C / min to 4°C. The 254 nt single-stranded circular DNA with precise site Ψ modification was prepared by the method shown in steps 2-5 of Example 1 ( FIG. 7 ). References [1]Bajan S, Hutvagner G.RNA-Based Therapeutics: From Antisense Oligonucleotides to miRNAs[J]. Cells, 2020, 9(1). [2]Rinaldi C, Wood MJ A. Antisense oligonucleotides: the next frontier for treatment of neurological disorders[J]. Nature Reviews Neurology, 2018, 14(1):9-21. [3]Statello L, Guo CJ, Chen LL, et al. Gene regulation by long non-coding RNAs and its biological functions[J]. Nature Reviews Molecular Cell Biology, 2021, 22(2):96-118. [4]Adachi T, Nakamura Y. Aptamers: A Review of Their Chemical Properties and Modifications for Therapeutic Application[J]. Molecules, 2019, 24(23). [5]Micura R, C. Fundamental studies of functional nucleic acids: aptamers, riboswitches, ribozymes and DNAzymes [J]. Chemical Society Reviews, 2020, 49(20): 7331-7353. [6]Müller S,Wedler A,Breuer J,et al.Synthetic circular miR-21 RNA decoys enhance tumor suppressor expression and impair tumor growth in mice[J].NAR Cancer,2020,2(3):zcaa014. [7]Lavenniah A,Luu T D A,Li Y P,et al.Engineered Circular RNA Sponges Act as miRNA Inhibitors to Attenuate Pressure Overload-Induced Cardiac Hypertrophy[J].Molecular Therapy,2020,28(6):1506-1517. [8]Liu X,Abraham J M,Cheng Y,et al.Synthetic Circular RNA Functions as a miR-21 Sponge to Suppress Gastric Carcinoma Cell Proliferation[J].Mol Ther Nucleic Acids,2018,13:312-321. [9]Brazier J.Chemical Synthesis of Oligonucelotide Sequences:Phosphoramidite Chemistry[J].Methods in molecular biology,2023,2633:185–193.

[0010] Marie Flamme,Luke K McKenzie,Ivo Sarac,et al.Chemical methods for the modification of RNA[J].Methods,2019,161:64-82.

Claims

1. A nucleic acid combination comprising at least 2 third DNA fragments, at least 2 first DNA fragments, and optionally, at least 1 second DNA fragment; wherein The third DNA fragment comprises a 5' spacer region, a functional sequence region and a 3' spacer region; the third DNA fragment is used for connection to form a long single-stranded DNA; The first DNA fragment comprises a sequence complementary to the 5' spacer region of the third DNA fragment and a sequence complementary to the 3' spacer region of another third DNA fragment; The second DNA fragment comprises a sequence that is completely or partially complementary to the sequence at the 5' end of the long single-stranded DNA, and a sequence that is completely or partially complementary to the sequence at the 3' end of the long single-stranded DNA. 2 . The nucleic acid combination according to claim 1 , wherein the third DNA fragment comprises a 5′ spacer region, a functional sequence region and a 3′ spacer region from the 5′ end to the 3′ end. 3 . The nucleic acid combination according to claim 1 , wherein the first DNA fragment comprises a sequence that is completely or partially complementary to the 5′ spacer of the third DNA fragment and a sequence that is completely or partially complementary to the 3′ spacer of another third DNA fragment.

4. The nucleic acid combination according to claim 1, wherein the 5' spacer sequence of each third DNA fragment is different, the 3' spacer sequence of each third DNA fragment is different, and / or the 5' spacer sequence and 3' spacer sequence of the same third DNA fragment are different. 5 . The nucleic acid combination according to claim 1 , wherein the second DNA fragment comprises a sequence complementary to a 4-35 nt sequence at the 5′ end of the long single-stranded DNA, and a sequence complementary to a 4-35 nt sequence at the 3′ end of the long single-stranded DNA.

6. The nucleic acid combination according to claim 1, wherein the functional sequence region is a sequence that interacts with miRNA, mRNA, pre-mRNA, lncRNA, circRNA, DNA or protein. The nucleic acid combination according to claim 1 , wherein the length of the 5′ spacer and / or the 3′ spacer is 8-20 nt.

8. The nucleic acid combination according to claim 1, wherein the second DNA fragment is 20-80 nt in length, preferably 64 nt.

9. The nucleic acid combination according to claim 1, wherein the third DNA fragment is 16-100 nt in length.

10. The nucleic acid combination according to claim 1, wherein the first DNA fragment is 16-40 nt in length. The nucleic acid combination according to claim 1 , wherein the number of the third DNA fragments is at most 20, preferably 4-10.

12. A method for preparing single-stranded circular DNA, comprising the following steps: Step (1): connecting the third DNA fragment according to any one of claims 1 to 11 to obtain a long single-stranded DNA; Step (2): Connect two long single-stranded DNAs to obtain single-stranded circular DNA.

13. The method according to claim 12, wherein the step (1) comprises the following steps: Step (1.1): mixing the third DNA fragment and the first DNA fragment according to any one of claims 1 to 11 and then annealing them; Step (1.2): connecting the product of step (1.1); Optionally, step (1.3): purifying the product of step (1.2).

14. The method according to claim 13, wherein step (1.2) uses T4 DNA ligase for ligation.

15. The method according to claim 13, wherein step (1.3) comprises an ultrafiltration step, optionally, purification is performed using an ultrafiltration tube operation or using size-sorting magnetic beads.

16. The method according to claim 13, wherein the step (2) comprises the following steps: Step (2.1): mixing the product of step (1) with the second DNA fragment according to any one of claims 1 to 11 and then annealing; Step (2.2): connecting the product of step (2.1); Optionally, step (2.3): purifying the product of step (2.2).

17. The method according to claim 16, wherein step (2.2) uses a circularizing ligase for ligation.

18. The method according to claim 16, wherein step (2.3) comprises the following steps: Step (2.3.1): adding DNA exonuclease for digestion; and / or Step (2.3.2): Purification is optionally carried out using ethanol and using an ultrafiltration method.

19. The method according to claim 12, wherein the length of the single-stranded circular DNA is 180 nt-1000 nt, preferably 240 nt-1000 nt.

20. Use of the nucleic acid combination according to claim 1 in preparing single-stranded circular DNA, optionally, the third DNA fragment comprises a DNA modification.

21. The single-stranded circular DNA according to the preceding claim, for use in treating, preventing or diagnosing a disease or in preparing a drug for treating or preventing a disease or a diagnostic or detection reagent.

22. The single-stranded circular DNA according to claim 21, which regulates the target nucleic acid in the subject; preferably regulates the function of pre-mRNA, mRNA, miRNA, LncRNA, circRNA and tRNA in the subject.

23. The single-stranded circular DNA according to claim 22, which can act as a miRNA sponge to adsorb targeted miRNA or antisense nucleotides to induce degradation of targeted mRNA, and preferably the single-stranded circular DNA is a miRNA inhibitor and an antisense nucleotide drug.

24. Use of a second DNA fragment for preparing single-stranded circular DNA, the second DNA fragment comprising a sequence complementary to the sequence at the 5' end of the long single-stranded DNA according to the preceding claim, and a sequence complementary to the sequence at the 3' end of the long single-stranded DNA according to the preceding claim.

25. The use according to claim 24, wherein the 5' and 3' end sequences of the second DNA fragment comprise sequences that are completely or partially complementary to the 5' end sequence of the long single-stranded DNA according to the preceding claim.

26. The use according to claim 24, wherein the non-5'-non-3' sequence of the second DNA fragment comprises a sequence that is completely or partially complementary to the 3'-end sequence of the long single-stranded DNA according to the preceding claim.

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