CN109161572B - Single-stranded circular nucleic acid, and preparation method and application thereof - Google Patents

Abstract

Translated fromChinese

本发明提供了一种单链环状核酸及其制备方法和应用，涉及分子生物学技术领域，本发明提供的环状核酸的制备方法，包括具有末端发夹结构的线性核酸在与成环辅助链杂交后，经连接酶连接使两端闭合，得到单链环状核酸。该方法显著降低了副产物的生成。运用这种“末端发夹”连接方法，使线性核酸在连接酶连接下，在常规体系中的单环成环率可达近100％。并且，大多数的核酸单链在连接体系中均会形成发夹结构，所以本发明提供的制备方法的通用性极高。此外，该方法操作简单，耗时较短，无需专门的实验人员即可进行操作，在节约人力及时间成本的基础上，还具有普适性广的优点。

The present invention provides a single-stranded circular nucleic acid and a preparation method and application thereof, and relates to the technical field of molecular biology. After the strands are hybridized, the two ends are closed by ligase ligase to obtain a single-stranded circular nucleic acid. This method significantly reduces the formation of by-products. Using this "terminal hairpin" ligation method, the ligation rate of linear nucleic acids in a conventional system can reach nearly 100%. In addition, most nucleic acid single strands will form a hairpin structure in the connection system, so the preparation method provided by the present invention has extremely high versatility. In addition, the method is simple to operate, takes a short time, and can be operated without special experimental personnel, and has the advantage of being widely applicable on the basis of saving manpower and time cost.

Description

Single-stranded circular nucleic acid, and preparation method and application thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a single-stranded circular nucleic acid and a preparation method and application thereof.

Background

Nucleic acid is not only a carrier of genetic information, but also a material for constructing a nano structure, and particularly in recent years, the design of nucleic acid machines is a research hotspot, and different nucleic acid machines can be in the shapes of 'net cages', 'chain locks', and the like, which are possibly used for drug carriers, substance transportation, liquid calculation, and the like. The single-stranded circular nucleic acid is a circular structure formed by connecting single-stranded DNA, RNA or other modified nucleic acid chains end to end, and plays an important role in constructing nano materials due to unique kinetic and topological properties of the single-stranded circular nucleic acid. In addition, circular nucleic acids can be used for constructing nanomaterials, rolling circle amplification, food traceability, research on nucleic acid topological structures and the like. Therefore, whether the circularization of single-stranded linear nucleic acids is successful or whether single circular nucleic acid products can be obtained after the circularization of linear nucleic acids has been currently studied.

Currently, most of the single-stranded circular nucleic acids are synthesized by joining them end to form closed loops (see pathway (r) in FIG. 1) by ligase and a loop-forming auxiliary strand (a short strand complementary to the 3 '-and 5' -ends of a single-stranded DNA or RNA). However, a major problem encountered in the preparation of single-chain cyclic nucleic acids is the difficulty in suppressing by-products generated by intermolecular linkage at high substrate concentrations. For example, in the reaction system, a plurality of template single-chains are connected with each other under the action of a ring-forming auxiliary chain (as in pathway II of FIG. 1) to form a polymer, that is, under the condition of high concentration of a connecting substrate, the distance between molecules in the system is shortened, so that the collision probability between molecules is increased, the collision probability between molecules at two ends of the linear nucleic acid is unchanged, the difference of the collision probability between molecules is reduced, the intermolecular connection efficiency is increased, and the yield of the polymer is increased sharply. Recently, experiments prove that the byproducts in the interlinking process can be effectively reduced under the condition of low buffer concentration, and the low buffer reduces the reaction efficiency, greatly reduces the intermolecular connection efficiency, and ensures that intramolecular connection is in an advantage, but the method has the defects of complex operation, long time consumption and the like.

Therefore, it is important to develop a method for preparing a circular nucleic acid, which can efficiently link a linear nucleic acid to a circular nucleic acid, and has high linking efficiency, few by-products, simple operation, and short time.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first objective of the present invention is to provide a method for preparing single-stranded circular nucleic acid, so as to alleviate the technical problems of low efficiency of single-stranded circular nucleic acid ligation, more byproducts, tedious operation and long time consumption in the prior art.

The second object of the present invention is a single-stranded circular nucleic acid produced by the above-mentioned method.

The third purpose of the invention is to provide the application of the single-stranded circular nucleic acid in one or more of nano material construction, rolling circle amplification, food traceability or nucleic acid topological structure research.

The invention provides a preparation method of a single-stranded circular nucleic acid, which comprises the following steps:

connecting with ligase to close two ends of the linear nucleic acid hybridized with the looping auxiliary chain to obtain the circular nucleic acid;

wherein the linear nucleic acid has a hairpin structure at the junction of the linear nucleic acid and the looped auxiliary strand;

the looping auxiliary strand is complementary to the 3 'end and the 5' end of the linear nucleic acid.

Further, the linear nucleic acid was subjected to secondary structure simulation, and a stable hairpin structure was selected as the junction of the linear nucleic acid and the loop-forming auxiliary strand.

Further, the length of the linear nucleic acid is 40 to 200nt, preferably 44 to 200nt, and more preferably 54 to 200 nt.

Further, T of the hairpin structure_mThe value is 10 to 60 ℃ higher than the ligation reaction temperature, preferably 20 to 40 ℃ higher than the ligation reaction temperature.

Further, mixing the linear nucleic acid, the cyclization auxiliary chain, the ligase and the connection buffer solution into the same connection system for connection reaction;

preferably, the time of the ligation reaction is 1 to 5 hours;

preferably, the buffer of the ligation reaction is 0.1-1 times that of the standard buffer.

Further, the ligase is a ligase that can ligate nicking moieties in double-stranded DNA, double-stranded RNA, or DNA-RNA hybrid strands;

preferably, the ligase is any one of T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase or e.

Further, the concentration of the linear nucleic acid is 1 to 100. mu.M, preferably 5 to 50. mu.M.

Further, the molar ratio of the linear nucleic acid to the looping auxiliary strand is 1:1-5, preferably 1: 1-3.

The invention also provides the circular nucleic acid prepared by the preparation method.

In addition, the invention also provides application of the circular nucleic acid in one or more of construction of nano materials, rolling circle amplification, food traceability or nucleic acid topological structure research.

The preparation method of the circular nucleic acid comprises the steps of connecting through ligase to close two ends of the linear nucleic acid with the hairpin structure after hybridization with the looping auxiliary chain, so as to obtain the circular nucleic acid. The joint of the linear nucleic acid and the looping auxiliary chain is designed into a hairpin structure, so that a small part of hairpin structure is firstly opened to be connected with the looping auxiliary chain for looping in a connecting system, molecules which can be connected in the connecting system are always kept at a level with lower concentration, the advantages of the interconnection in the molecules are in a more obvious position, and the generation of byproducts is greatly reduced. By using the connection method of the terminal hairpin, the single-loop cyclization rate of the linear nucleic acid can reach nearly 100% in a conventional system under the connection of ligase. In addition, most of the nucleic acid single strands form hairpin structures in the ligation system, so that the preparation method provided by the invention has extremely high versatility. In addition, the method is simple to operate, short in time consumption, capable of being operated without special experimenters, and wide in universality on the basis of saving labor and time cost.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic diagram of the ligation of a hairpin-terminated linear nucleic acid into a loop according to the invention;

FIG. 2A is L64 provided inembodiment 1 of the present invention_3-4,24-4Mfold simulation diagram of (1);

FIG. 2B is L64 provided inembodiment 1 of the present invention_16-4,37-4Mfold simulation diagram of (1);

FIG. 2C is a graph showing the results of comparing the effect of hairpin location on the amount of loop-forming target product attached as provided in example 1 of the present invention;

FIG. 3A is L74 provided inembodiment 2 of the present invention_{3-4,24-4,65-2}Mfold mimetic maps of linear nucleic acids of (a);

FIG. 3B is L64 provided inembodiment 2 of the present invention_{3-4,24-4,55-2}Mfold mimetic maps of linear nucleic acids of (a);

FIG. 3C is L54 provided in example 2 of the present invention_{3-4,24-4,45-2}Mfold mimetic maps of linear nucleic acids of (a);

FIG. 3D is L44 provided inembodiment 2 of the present invention_{14-6,24-4,55-2}Mfold mimetic maps of linear nucleic acids of (a);

FIG. 3E shows L34 provided in example 2 of the present invention_14-6,35-2Mfold mimetic maps of linear nucleic acids of (a);

FIG. 3F is a graph showing the results of ligation comparison of single strands having terminal hairpin structures of different lengths provided in example 2 of the present invention;

FIG. 4A is L64 provided inembodiment 3 of the present invention_{2-4,23-4,51-2}Mfold mimetic maps of linear nucleic acids of (a);

FIG. 4B is L64 provided inembodiment 3 of the present invention_3-4,24-4Mfold mimetic maps of linear nucleic acids of (a);

FIG. 4C is L64 provided inembodiment 3 of the present invention_4-4,25-4Mfold mimetic maps of linear nucleic acids of (a);

FIG. 4D is L64 provided inembodiment 3 of the present invention_5-4,26-4Mfold mimetic maps of linear nucleic acids of (a);

FIG. 4E shows L64 provided inembodiment 3 of the present invention_6-4,27-4Mfold mimetic maps of linear nucleic acids of (a);

FIG. 4F is a graph showing the comparison result of different connection locations provided inembodiment 3 of the present invention;

FIG. 5A is L64 provided inembodiment 4 of the present invention_0-4,24-4Mfold mimetic maps of linear nucleic acids of (a);

FIG. 5B is L64 provided inembodiment 4 of the present invention_0-6,24-4Mfold mimetic maps of linear nucleic acids of (a);

FIG. 5C is L64 provided inembodiment 4 of the present invention_0-7,24-4Mfold mimetic maps of linear nucleic acids of (a);

FIG. 5D is L64 provided inembodiment 4 of the present invention_0-7,20-4Mfold mimetic maps of linear nucleic acids of (a);

FIG. 5E is a graph showing the comparison of the loop formation of hair clips of different stability at the ends of a chain according to example 4 of the present invention;

FIG. 6A is L64 provided inembodiment 5 of the present invention_3-4,24-4A statistical plot of ligation conversion and circular nucleic acid yield of (a);

FIG. 6B is L64 provided inembodiment 5 of the present invention_16-4,37-4A statistical plot of ligation conversion and circular nucleic acid yield of (a);

FIG. 7A is L64 provided inembodiment 6 of the present invention_3-4,24-4High concentration ring formation electrophoretogram;

FIG. 7B is L64 provided inembodiment 6 of the present invention_16-4,37-4High concentration ring formation electrophoretogram;

FIG. 8A is a drawing showing L in example 7 of the present invention_DNAMfold mimetic maps of linear nucleic acids of (a);

FIG. 8B is a diagram showing a linear nucleic acid L having a terminal hairpin provided in example 7 of the present invention_DNALooping electrophoretogram.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a preparation method of single-stranded circular nucleic acid, which comprises the following steps:

connecting with ligase to close two ends of the linear nucleic acid hybridized with the looping auxiliary chain to obtain circular nucleic acid;

wherein the linear nucleic acid has a hairpin structure at the hybridization of the linear nucleic acid and the loop-forming auxiliary strand;

the looping auxiliary strand is complementary to the 3 'end and the 5' end of the linear nucleic acid.

In the invention, the single-stranded circular nucleic acid is obtained by connecting the same strand of linear nucleic acid end to end and closing the same into a ring.

In the present invention, the linear nucleic acid is a single-stranded linear nucleic acid, and the nucleic acid may be DNA, RNA, or other modified nucleic acid; accordingly, the circular nucleic acid prepared is a single-stranded circular nucleic acid, which can be DNA, RNA, or other modified nucleic acid.

In the present invention, the nucleic acid molecule is folded back on itself, some bases are close to each other, and the bases in the folded region are complementarily paired, and the folded part forms a hairpin structure. When the hairpin structure of the linear nucleic acid is positioned at the hybridization part of the linear nucleic acid and the loop-forming auxiliary chain, in the process of the connection reaction, the linear nucleic acid in the connection system needs to open the hairpin structure of the linear nucleic acid first to be matched with the loop-forming auxiliary chain for forming a loop, so that the aim of always keeping the molecules which can be connected in the connection system at a low concentration level is fulfilled, the advantages of intramolecular connection are more obvious, and the generation of byproducts is greatly reduced.

Meanwhile, in the preparation method of the circular nucleic acid provided by the invention, most of the nucleic acid single strands form hairpin structures in a connection system, and the selection of the connection position does not influence the sequence of the finally obtained circular nucleic acid, so the preparation method provided by the invention has extremely high universality. In addition, the method is simple to operate, short in time consumption, capable of being operated without special experimenters, and wide in universality on the basis of saving labor and time cost.

In a preferred embodiment, the linear nucleic acid is subjected to secondary structure modeling, and a stable hairpin structure is selected as the point of hybridization of the linear nucleic acid to the looped auxiliary strand.

The secondary structure simulation may be performed by computer software, for example, but not limited to, the secondary structure simulation may be performed by using software such as Mfold, IDT, and the like.

The hairpin with certain stability is selected as the junction, so that the hairpin structure is not easy to open, and the purpose of keeping molecules which can be connected in a connection system at a low concentration level all the time is achieved, thereby ensuring that the advantages of intramolecular connection are in a more obvious position and greatly reducing the generation of byproducts.

In a preferred embodiment, the linear nucleic acid is 40-200nt in length, for example, and may be, but is not limited to, 40nt, 50nt, 60nt, 80nt, 100nt, 120nt, 150nt, 180nt, or 200 nt. Preferably 44 to 200nt, more preferably 54 to 200 nt.

When the length of the linear nucleic acid is 40 to 200nt, a high loop formation rate can be ensured.

Further adjustment and optimization of the length of the linear nucleic acid of the present invention enables higher cyclization ratio.

In a preferred embodiment, T is a hairpin structure_mThe value is 10-60 ℃ higher than the ligation reaction temperature, and may be, for example, but not limited to, 10 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, or 60 ℃, preferably 20-40 ℃ higher than the ligation reaction temperature.

T in hairpin structure_mWhen the value is 20-40 ℃ higher than the temperature of the connection reaction, higher ring formation rate can be ensured.

In a preferred embodiment, the effective ligation concentration is reduced by increasing the distance between intramolecular and intermolecular ligations.

According to the relationship between the geps free energy and the equilibrium constant:

ΔG＝-RTlnK (1)

it is known that, as the- Δ G of the terminal hairpin structure increases, a high yield (> 85%) of the substrate concentration increases exponentially, and it was preliminarily determined from the experimental results that when the- Δ G of the terminal hairpin structure is in the range of 1 to 4, the amount of the linked by-product is greatly suppressed, and the amount of the obtained monocyclic cyclic nucleic acid is greatly increased.

In a preferred embodiment, the linear nucleic acid, the loop forming auxiliary chain, the ligase and the ligation buffer are mixed into the same ligation system to perform ligation reaction;

preferably, the temperature of the ligation reaction is 20-40 ℃, for example, but not limited to, 20 ℃, 22 ℃, 25 ℃, 28 ℃, 30 ℃, 32 ℃, 35 ℃, or 37 ℃ or 40 ℃, preferably 25-37 ℃;

preferably, the time of the ligation reaction is 1 to 5 hours, for example, but not limited to, 1 hour, 2 hours, 3 hours, 4 hours or 5 hours, preferably 2 to 4 hours.

Preferably, the buffer of the ligase reaction is 0.1 to 1 times of the standard buffer, and may be, for example, but not limited to, 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times, 0.8 times, 0.9 times, or 1 times.

The conditions of the ligation reaction in the invention are further adjusted and optimized, so that the ring formation rate can be higher.

In a preferred embodiment, the ligase is a ligase that can ligate nicking moieties in double-stranded DNA, double-stranded RNA, or DNA-RNA hybrids;

preferably, the ligase is any one of T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase or e.

In a preferred embodiment, the concentration of the linear nucleic acid is 1-100. mu.M, and may be, for example, but not limited to, 1. mu.M, 10. mu.M, 20. mu.M, 30. mu.M, 40. mu.M, 50. mu.M, 60. mu.M, 70. mu.M, 80. mu.M, 90. mu.M or 100. mu.M, preferably 5-50. mu.M.

When the concentration of the linear nucleic acid is 1 to 100. mu.M, a higher cyclization ratio can be ensured.

Further adjustment and optimization of the concentration of the linear nucleic acid of the present invention enables higher cyclization ratio.

In a preferred embodiment, the molar ratio of linear nucleic acid to looping auxiliary strand is 1:1 to 5, and may be, for example, but not limited to, 1:1, 1:2, 1:3, 1:4 or 1:5, preferably 1:1 to 3.

When the molar ratio of the linear nucleic acid to the auxiliary strand for loop formation is 1:1-5, a higher loop formation rate can be ensured.

The loop formation rate can be made higher by further adjusting and optimizing the molar ratio of the linear nucleic acid to the loop forming auxiliary chain.

The invention also provides the single-stranded circular nucleic acid prepared by the preparation method.

In addition, the invention also provides application of the single-stranded circular nucleic acid in one or more of construction of nano materials, rolling circle amplification, food traceability or nucleic acid topological structure research.

To facilitate a clearer understanding of the contents of the present invention, reference will now be made in detail to the following specific embodiments.

The experimental procedures in the following examples are conventional unless otherwise specified.

Unless otherwise indicated, the drugs and reagents used in the examples of the present invention are available from normal and readily available sources:

the linear nucleic acid and the looping auxiliary chain are both purchased from Suzhou Jinzhi Biotechnology GmbH and artificially synthesized; t4 DNA ligase, T4 RNA ligase was purchased from Saimer Feishel, USA (Thermo Scientific); coli DNA ligase, purchased from New England Biolabs; other compounds were purchased from Sigma Aldrich (Sigma-Aldrich) usa.

Example 1

1. Raw materials

Linear nucleic acid 1(5 '→ 3', L64_3-4,24-4)：

ACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCCGG (5' -phosphorylated, 64nt in length, SEQ ID NO: 1);

linear nucleic acid 2(5 '→ 3', L64)_16-4,37-4)：

CCATTTTCGCCGGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCC (5' -phosphorylated, 64nt in length, SEQ ID NO: 2);

looping auxiliary chain 1(5 '→ 3'):

AGCGGTCCGGCG (length 12nt, SEQ ID NO: 3);

looping auxiliary chain 2(5 '→ 3'):

AAATGGGGAGAA (length 12nt, SEQ ID NO: 4).

The source is as follows: artificially synthesized (Suzhou Jinzhi Biotechnology, Inc.).

Two linear nucleic acids are joined into circular nucleic acids whose sequences are identical except that the positions of the junctions selected when joining the rings are different. Linearnucleic acid 1 selects the junction next to the hairpin, and linearnucleic acid 2 selects the junction further away from the hairpin.

2. Test of

1) Connected into a ring

Mixing the linear nucleic acid and the corresponding looping auxiliary strand, ligase and ligation buffer in a system in which the concentration of the linear nucleic acid is 5 μ M and the molar ratio of linear nucleic acid to looping auxiliary strand is 1:2, 10U T4 DNA Ligase (purchased from Thermo Scientific), 1XT 4 DNA Ligase Buffer (Ligase Buffer) in a total volume of 20. mu.L; ligation was carried out at 25 ℃ for 1 hour. The sequences of the circular nucleic acids formed after ligation of two linear nucleic acids are identical except for the different ligation positions selected at the time of ligation.

2) Confirmation of enzyme cleavage

Taking a part of the connected system, adding exoenzyme Exonuclease I, and removing the single strand in the system. Enzyme digestion system: the ligation system was 5. mu.L, 1 XExonuclease I Exonuclease buffer, 5UExonuclease I, and the digestion was carried out at 37 ℃ for 1 hour, with a total volume of 10. mu.L.

3) Electrophoretic detection

As shown in fig. 2A, 2B and 2C. Wherein, FIG. 2A is L64_3-4,24-4The MFold simulation chart in FIG. 2B is L64_16-4,37-4Under the simulation condition of Mfold simulation diagram of (1) [ Mg²⁺]10mM, 25 ℃. Reaction system and conditions in fig. 2C: [ Linear nucleic acid]5 μ M, [ Loop-forming auxiliary chain]10 μ M,10U T4 DNA ligase, 1xt 4 DNA ligase buffer, 25 ℃,1 hour.Lane 1 is L64_3-4,24-4The loop-forming auxiliary chain and the ligase are not added;lane 2 is L64_3-4,24-4A band after addition of the loop forming auxiliary strand and T4 DNA ligase;lane 3 is L64_3-4,24-4Adding exonuclease into the circular band after the single chain is removed;lane 4 is L64_16-4,37-4The loop-forming auxiliary chain and the ligase are not added;lane 5 is L64_16-4,37-4A band after addition of the loop forming auxiliary strand and T4 DNA ligase;lane 6 is L64_16-4,37-4After ligation to form a loop, exonuclease is added to remove the single strand.

FIG. 2C shows a comparison of the results after ligation and cleavage of the hairpin at the ends and in the middle of the strand (10% urea-denatured acrylamide gel electrophoresis). The results show that when the hairpin structure is at the end of the strand, the yield of the generated circular nucleic acid can reach 93%, and can reach 100% after the enzyme digestion. However, when the ligation site is at a position without a hairpin, the yield of the resulting circular nucleic acid is only 45%, and 64% after cleavage. From the results, it can be seen that when the place with the terminal hairpin structure is selected for ligation, the ring formation rate can be significantly increased, resulting in a ring formation yield from 45% to 93%.

Example 2

1. Raw materials

Linear nucleic acid 1(5 '→ 3', L74_{3-4,24-4,65-2})：

ACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGG (5' -phosphorylated, 74nt in length, SEQ ID NO: 5);

linear nucleic acid 2(5 '→ 3', L64)_{3-4,24-4,55-2})：

ACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCGCCACGTTCGCCGG (5' -phosphorylated, 64nt in length, SEQ ID NO: 6);

linear nucleic acid 3(5 '→ 3', L54_{3-4,24-4,45-2})：

ACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCGTTCGCCGG (5' -phosphorylated, 54nt in length, SEQ ID NO: 7);

linear nucleic acid 4(5 '→ 3', L44)_{14-6,24-4,55-2})：

ACCGCTACACTTGCCAGCGCCCTAGCGCCGCTTTCGTTCGCCGG (5' -phosphorylated, 44nt in length, SEQ ID NO: 8);

linear nucleic acid 5(5 '→ 3', L34_14-6,35-2)：

ACCGCTACACTTGCCAGCGCGCCACGTTCGCCGG (5' -phosphorylated, 34nt in length, SEQ ID NO: 9);

looping auxiliary chain (5 '→ 3'):

AGCGGTCCGGCG (length 12nt, SEQ ID NO: 10).

The source is as follows: artificially synthesized (Suzhou Jinzhi Biotechnology, Inc.).

2. Test of

1) Connected into a ring

Mixing the linear nucleic acid and the corresponding looping auxiliary strand, ligase and ligation buffer in a system in which the concentration of the linear nucleic acid is 5 μ M and the molar ratio of linear nucleic acid to looping auxiliary strand is 1:2, 10U e.coli DNA ligase, 1 × e.coli DNA ligase buffer,total volume 40 μ L; ligation was carried out at 16 ℃ for 2 hours. Single strands of different lengths with terminal hairpins were selected for ligation.

2) Confirmation of enzyme cleavage

Taking a part of the connected system, adding exoenzyme Exonuclease I, and removing the single strand in the system. Enzyme digestion system: the ligation system was 5. mu.L, 1 XExonuclease I Exonuclease buffer, 5UExonuclease I, and the digestion was carried out at 37 ℃ for 1 hour, with a total volume of 10. mu.L.

3) Electrophoretic detection

As shown in fig. 3A, 3B, 3C, 3D, 3E, and 3F. Wherein, FIG. 3A is L74_{3-4,24-4,65-2}The MFold simulation of the linear nucleic acid of (1), FIG. 3B is L64_{3-4,24-4,55-2}The MFold simulation of the linear nucleic acid of (1), FIG. 3C is L54_{3-4,24-4,45-2}The MFold simulation of the linear nucleic acid of (1), FIG. 3D is L44_{14-6,24-4,55-2}The MFold simulation of the linear nucleic acid of (1), FIG. 3E is L34_14-6,35-2Under the conditions of Mfold simulation of the linear nucleic acid of (1), the simulation is that [ Mg²⁺]10mM, 25 ℃. FIG. 3F reaction system and conditions: [ Linear nucleic acid]5 μ M, [ Loop-forming auxiliary chain]Coli DNA ligase 10U, 1 × e.coli DNA ligase buffer, 16 ℃, 2 hours.Lanes 1, 4, 7, 10, 13 are linear nucleic acids with no added loop forming auxiliary strand and ligase;lanes 2, 5, 8, 11, 14 are bands of linear nucleic acids with added loop forming auxiliary strand and e.coli DNA ligase;lanes 3, 6, 9, 12 and 15 are bands obtained by adding exonuclease to remove single strands after the linear nucleic acids are ligated into loops.

FIG. 3F is a comparison of the results of looping linear nucleic acids of different lengths using the "terminal hairpin" method (10% urea denaturing acrylamide gel electrophoresis). The results showed that the cyclization ratio of the single strand with the terminal hairpin was 94% or more when the length of the linear nucleic acid was 54nt or more, and that the cyclization ratio was reduced to about 60% when the length of the linear nucleic acid was reduced to about 44 nt. And when the length of the linear nucleic acid was reduced to 34nt, the loop formation rate was only 14%.

Example 3

1. Raw materials

Linear nucleic acid 1(5 '→ 3', L64_{2-4,23-4,51-2})：

CCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCCGGA (5' -phosphorylated, 64nt in length, SEQ ID NO: 11);

linear nucleic acid 2(5 '→ 3', L64)_3-4,24-4)：

ACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCCGG (5' -phosphorylated, 64nt in length, SEQ ID NO: 12);

linear nucleic acid 3(5 '→ 3', L64_4-4,25-4)：

GACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCCG (5' -phosphorylated, 64nt in length, SEQ ID NO: 13);

linear nucleic acid 4(5 '→ 3', L64)_5-4,26-4)：

GGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCC (5' -phosphorylated, 64nt in length, SEQ ID NO: 14);

linear nucleic acid 5(5 '→ 3', L64_6-4,27-4)：

CGGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGC (5' -phosphorylated, 64nt in length, SEQ ID NO: 15);

looping auxiliary chain 1(5 '→ 3'):

TAGCGGTCCGGC (12 nt in length, SEQ ID NO: 16);

looping auxiliary chain 2(5 '→ 3'):

AGCGGTCCGGCG (length 12nt, SEQ ID NO: 17);

looping auxiliary chain 3(5 '→ 3'):

GCGGTCCGGCGA (12 nt in length, SEQ ID NO: 18);

looping auxiliary chain 4(5 '→ 3'):

CGGTCCGGCGAA (12 nt in length, SEQ ID NO: 19);

looping auxiliary chain 5(5 '→ 3'):

GGTCCGGCGAAA (length 12nt, SEQ ID NO: 20).

The source is as follows: artificially synthesized (Suzhou Jinzhi Biotechnology, Inc.).

2. Test of

1) Connected into a ring

Mixing the linear nucleic acid and the corresponding looping auxiliary strand, ligase and ligation buffer in a system in which the concentration of the linear nucleic acid is 5 μ M and the molar ratio of linear nucleic acid to looping auxiliary strand is 1:2, 5U T4 DNA ligase, 1XT 4 DNA ligase buffer, total volume 10u L; ligation was carried out at 25 ℃ for 4 hours. The sequences of two linear nucleic acids after ligation are identical except for the chosen ligation positions at the time of ligation.

2) Confirmation of enzyme cleavage

Taking a part of the connected system, adding exoenzyme Exonuclease I, and removing the single strand in the system. Enzyme digestion system: the ligation system was 5. mu.L, 1 XExonuclease I Exonuclease buffer, 5U Exonuclease I, and the total volume was 10. mu.L at 37 ℃ for 1 hour.

4) Electrophoretic detection

As shown in fig. 4A, 4B, 4C, 4D, 4E, and 4F. Wherein, FIG. 4A is L64_{2-4,23-4,51-2}The MFold simulation of the linear nucleic acid of (1), FIG. 4B is L64_3-4,24-4The MFold simulation of the linear nucleic acid of (1), FIG. 4C is L64_4-4,25-4The MFold model of linear nucleic acid (1), FIG. 4D is L64_5-4,26-4The MFold simulation of the linear nucleic acid of (1), FIG. 4E is L64_6-4,27-4Under the conditions of Mfold simulation of the linear nucleic acid of (1), the simulation is that [ Mg²⁺]10mM, 25 ℃. FIG. 4F reaction system and conditions: [ Linear nucleic acid]5 μ M, [ Loop-forming auxiliary chain]10 μ M,10U T4 DNA ligase, 1xt 4 DNA ligase buffer, 25 ℃, 12 hours.Lanes 1, 3, 5, 7, 9 are linear nucleic acids with no added loop forming auxiliary strand and ligase;lanes 2, 4, 6, 8, and 10 show bands of linear nucleic acids with added loop forming helper strands and T4 DNA ligase.

FIG. 4F is a comparison of the looping results for linear nucleic acids with hairpins at different positions on the strand ends (10% urea denaturing acrylamide gel electrophoresis). The results showed that the yield of the cyclic nucleic acid decreased with increasing base at the junction, and that the yield of the cyclic nucleic acid was about 90% when the hairpin was at a base length of 1-3nt from the junction, and about 70% when the junction was at a distance of 4, 5nt in the hairpin.

Example 4

1. Raw materials

Linear nucleic acid 1(5 '→ 3', L64_0-4,24-4)：

CGCTACACTTGCCAGCGCCCACTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCCGG (5' -phosphorylated, 64nt in length, SEQ ID NO: 21);

linear nucleic acid 2(5 '→ 3', L64)_0-6,24-4)：

GGCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCCGG (5' -phosphorylated, 64nt in length, SEQ ID NO: 22);

linear nucleic acid 3(5 '→ 3', L64_0-7,24-4)：

GGCGCGCCACTTGCGCGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCCGG (5' -phosphorylated, 64nt in length, SEQ ID NO: 23);

linear nucleic acid 4(5 '→ 3', L64)_0-7,20-4)：

GGCGCGCGAAGCGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCTCCCCATTTTCGCCGG (5' -phosphorylated, 64nt in length, SEQ ID NO: 24);

looping auxiliary chain 1(5 '→ 3'):

GTAGCGCCGGCG (12 nt in length, SEQ ID NO: 25);

looping auxiliary chain 2(5 '→ 3'):

AGCGCCCCGGCG (12 nt in length, SEQ ID NO: 26);

looping auxiliary chain 3(5 '→ 3'):

CGCGCCCCGGCG (length 12nt, SEQ ID NO: 27).

The source is as follows: artificially synthesized (Suzhou Jinzhi Biotechnology, Inc.).

2. Test of

1) Connected into a ring

Mixing the linear nucleic acid and the corresponding looping auxiliary strand, ligase and ligation buffer in a system in which the concentration of the linear nucleic acid is 5 μ M and the molar ratio of linear nucleic acid to looping auxiliary strand is 1:2,1000U T3 DNA ligase (from New England Biolabs), 1XT 3 DNA ligase buffer, in a total volume of 20. mu.L; ligation was carried out at 25 ℃ for 12 hours. Single strands of different lengths with terminal hairpins were selected for ligation.

2) Electrophoretic detection

As shown in fig. 5A, 5B, 5C, 5D, and 4E. Wherein, FIG. 5A is L64_0-4,24-4Mfold simulation of the linear nucleic acid of (1), T of the terminal hairpin thereof_mThe value is 57 ℃ and L64 in FIG. 5B_0-6,24-4Mfold mimic of the linear nucleic acid of (1), T of the terminal hairpin thereof_mThe value is 73 ℃ and L64 in FIG. 5C_0-7,24-4Mfold mimic of the linear nucleic acid of (1), T of the terminal hairpin thereof_mThe value is 85 ℃ and L64 in FIG. 5D_0-7,20-4Mfold mimic of the linear nucleic acid of (1), T of the terminal hairpin thereof_mThe value was 90 ℃ under the simulated conditions of [ Mg ]²⁺]10mM, 25 ℃. FIG. 5E reaction system and conditions: [ Linear nucleic acid]5 μ M, [ Loop-forming auxiliary chain]₀10 μ M,1000U T3 DNA ligase, 1xt 3 DNA ligase buffer, 25 ℃, 12 hours.Lanes 1, 3, 5, and 7 are linear nucleic acids with no added loop-forming auxiliary strand and ligase;lanes 2, 4, 6 and 8 show the bands of linear nucleic acids with the addition of the circular auxiliary strand and T3 DNA ligase.

FIG. 5E is a comparison of the looping results for linear nucleic acids with hairpins of different stability at the ends (12% urea denaturing acrylamide gel electrophoresis). The results show that T is a terminal hairpin_mAt about 50-70 deg.C, the amount of cyclic nucleic acid after ligation is about 90%, when T is_mAbove 80 deg.C, the ring forming rate is reduced to about 64%.

Example 5

1. Raw materials

The ligation rate and the circularity were determined using the two linear nucleic acids in example 1.

2. Test of

1) Connected into a ring

Mixing the linear nucleic acid and the corresponding looping auxiliary strand, ligase and ligation buffer in a system in which the concentration of the linear nucleic acid is 5 μ M and the molar ratio of linear nucleic acid to looping auxiliary strand is 1:2, 10U T4 DNA ligase, 1XT 4 DNA ligase buffer,total volume 30 uL; the connection time is 2min, 5min, 10min, 20min, 40min, 1h, 2h and 3h respectively at 25 ℃.

2) Electrophoretic detection

FIGS. 6A and 6B are a comparison of the looping results of the hairpin at the terminal linear nucleic acid and the hairpin at the middle linear nucleic acid over different periods of time (12% urea denaturing acrylamide gel electrophoresis). Conversion ═ amount of (circular nucleic acid + polymeric by-product)/total linear nucleic acid, conversion indicates ligation rate of linear nucleic acid; the loop formation rate is the amount of circular nucleic acid/total substrate, and indicates the loop formation effect of linear nucleic acid. The results showed that the ligation rate of the linear nucleic acid with the hairpin in the middle (no hairpin at the end) was 10 times or more higher than that of the linear nucleic acid with the hairpin at the end. When no hairpin is present at the end, the probability of intermolecular collision increases, and the ligation rate becomes too high, so that the amount of by-products increases rapidly. The yield of the circular nucleic acid can reach more than 90% after the linear nucleic acid with the hairpin at the tail end is connected into a ring, and the yield of the circular nucleic acid of the linear nucleic acid with the hairpin in the middle is only 45%.

Example 6

1. Raw materials

High concentration ligation comparisons were performed using the two linear nucleic acids of example 1.

2. Test of

1) Connected into a ring

Mixing the linear nucleic acid and the corresponding looping auxiliary strand, ligase and ligation buffer in a system in which the concentration of the linear nucleic acid is 10. mu.M, 20. mu.M, 40. mu.M, 60. mu.M, 100. mu.M, and the molar ratio of the linear nucleic acid to the looping auxiliary strand is 1:2, 10-50U T4 DNA ligase, 1XT 4 DNA ligase buffer and 0.1XT 4 DNA ligase buffer, the total volume is 10 μ L; the ligation time was 12 hours at 25 ℃.

3) Electrophoretic detection

As shown in fig. 7A and 7B, L64 in fig. 7A_3-4,24-4FIG. 7B is L64_16-4,37-4High concentration of (2) into a ring electrophoretogram. Lanes 2-6, 9 ligation were performed using 1XT 4 ligase buffer;lanes 7 and 10 were ligated with 0.1XT 4 ligase buffer.

As can be seen from the results, the yield of the circular nucleic acid was 80% or more at a final concentration of 100. mu.M for the linear nucleic acid having the terminal hairpin (linear nucleic acid 1), while the loop formation rate of the linear nucleic acid having the hairpin at the middle portion (linear nucleic acid 2) was only 27%.

Example 7

1. Raw materials

Linear nucleic acid (5 '→ 3', L)_RNA)：

CUGCUGUUGUUCCCUUAUAAUUUCCUCCUCCAUAGUUUCCUUCUGAGUUGUGUU (5' -phosphorylated, 54nt in length, SEQ ID NO: 28);

looping auxiliary chain (5 '→ 3'):

AACAGCAGAACACAAC (length 16nt, SEQ ID NO: 29);

the source is as follows: artificially synthesized (Suzhou Jinzhi Biotechnology, Inc.).

2. Test of

1) Connected into a ring

Mixing the linear nucleic acid and the corresponding looping auxiliary chain, ligase and ligation buffer in a system in which the concentration of the linear nucleic acid is 1 μ M and the molar ratio of linear nucleic acid to looping auxiliary chain is 1:2, 5U T4 RNA ligase, 1XT 4 RNA ligase buffer,total volume 20 uL; ligation was carried out at 37 ℃ for 12 hours.

2) Electrophoretic detection

As shown in fig. 8A and 8B. Wherein, FIG. 8A is L_RNAUnder the simulation condition of Mfold simulation diagram of (1) [ Mg²⁺]10mM, 37 ℃. Reaction system and conditions in fig. 8B: [ Linear nucleic acid]1 μ M, [ Loop-forming auxiliary chain]2 μ M,5U T4 RNA ligase, 1xt 4 RNA ligase buffer, 37 ℃, 12 hours.Lane 1 is L_RNAThe loop-forming auxiliary chain and the ligase are not added;lane 2 is L_RNAA band after addition of the loop forming auxiliary strand and T4 RNA ligase;

FIG. 8B shows the looping result of the RNA strand with the hairpin structure at the end (12% urea-denatured acrylamide gel electrophoresis). The results show that when the hairpin structure is located at the end of the RNA strand, the ligation loop formation rate can reach more than 90% (lane 2), indicating that the terminal hairpin structure also has a significant promoting effect on the ligation loop formation of the RNA strand.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> China oceanic university

<120> single-stranded circular nucleic acid, and preparation method and application thereof

<160> 29

<170> PatentIn version 3.5

<210> 1

<211> 64

<212> DNA

<213> Artificial sequence

<400> 1

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttctc cccattttcg 60

ccgg 64

<210> 2

<211> 64

<212> DNA

<213> Artificial sequence

<400> 2

ccattttcgc cggaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 60

ctcc 64

<210> 3

<211> 12

<212> DNA

<213> Artificial sequence

<400> 3

agcggtccgg cg 12

<210> 4

<211> 12

<212> DNA

<213> Artificial sequence

<400> 4

aaatggggag aa 12

<210> 5

<211> 74

<212> DNA

<213> Artificial sequence

<400> 5

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 60

gccacgttcg ccgg 74

<210> 6

<211> 64

<212> DNA

<213> Artificial sequence

<400> 6

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttctc gccacgttcg 60

ccgg 64

<210> 7

<211> 54

<212> DNA

<213> Artificial sequence

<400> 7

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcgttcg ccgg 54

<210> 8

<211> 44

<212> DNA

<213> Artificial sequence

<400> 8

accgctacac ttgccagcgc cctagcgccg ctttcgttcg ccgg 44

<210> 9

<211> 34

<212> DNA

<213> Artificial sequence

<400> 9

accgctacac ttgccagcgc gccacgttcg ccgg 34

<210> 10

<211> 12

<212> DNA

<213> Artificial sequence

<400> 10

agcggtccgg cg 12

<210> 11

<211> 64

<212> DNA

<213> Artificial sequence

<400> 11

ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttctcc ccattttcgc 60

cgga 64

<210> 12

<211> 64

<212> DNA

<213> Artificial sequence

<400> 12

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttctc cccattttcg 60

ccgg 64

<210> 13

<211> 64

<212> DNA

<213> Artificial sequence

<400> 13

gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttct ccccattttc 60

gccg 64

<210> 14

<211> 64

<212> DNA

<213> Artificial sequence

<400> 14

ggaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc tccccatttt 60

cgcc 64

<210> 15

<211> 64

<212> DNA

<213> Artificial sequence

<400> 15

cggaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt ctccccattt 60

tcgc 64

<210> 16

<211> 12

<212> DNA

<213> Artificial sequence

<400> 16

tagcggtccg gc 12

<210> 17

<211> 12

<212> DNA

<213> Artificial sequence

<400> 17

agcggtccgg cg 12

<210> 18

<211> 12

<212> DNA

<213> Artificial sequence

<400> 18

gcggtccggc ga 12

<210> 19

<211> 12

<212> DNA

<213> Artificial sequence

<400> 19

cggtccggcg aa 12

<210> 20

<211> 12

<212> DNA

<213> Artificial sequence

<400> 20

ggtccggcga aa 12

<210> 21

<211> 64

<212> DNA

<213> Artificial sequence

<400> 21

cgctacactt gccagcgccc actagcgccc gctcctttcg ctttcttctc cccattttcg 60

ccgg 64

<210> 22

<211> 64

<212> DNA

<213> Artificial sequence

<400> 22

ggcgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttctc cccattttcg 60

ccgg 64

<210> 23

<211> 64

<212> DNA

<213> Artificial sequence

<400> 23

ggcgcgccac ttgcgcgcgc cctagcgccc gctcctttcg ctttcttctc cccattttcg 60

ccgg 64

<210> 24

<211> 60

<212> DNA

<213> Artificial sequence

<400> 24

ggcgcgcgaa gcgcgcccta gcgcccgctc ctttcgcttt cttctcccca ttttcgccgg 60

<210> 25

<211> 12

<212> DNA

<213> Artificial sequence

<400> 25

gtagcgccgg cg 12

<210> 26

<211> 12

<212> DNA

<213> Artificial sequence

<400> 26

agcgccccgg cg 12

<210> 27

<211> 12

<212> DNA

<213> Artificial sequence

<400> 27

cgcgccccgg cg 12

<210> 28

<211> 54

<212> RNA

<213> Artificial sequence

<400> 28

cugcuguugu ucccuuauaa uuuccuccuc cauaguuucc uucugaguug uguu 54

<210> 29

<211> 16

<212> DNA

<213> Artificial sequence

<400> 29

aacagcagaa cacaac 16

Claims (10)

Translated fromChinese

1.一种单链环状核酸的制备方法，其特征在于，所述制备方法包括：1. a preparation method of single-stranded circular nucleic acid, is characterized in that, described preparation method comprises:经连接酶连接，使与成环辅助链杂交后的线性核酸两端闭合，得到所述单链环状核酸；After ligation by ligase, the two ends of the linear nucleic acid hybridized with the circular auxiliary chain are closed to obtain the single-stranded circular nucleic acid;其中，所述线性核酸具有发夹结构，所述发夹结构位于所述线性核酸与所述成环辅助链的杂交处；Wherein, the linear nucleic acid has a hairpin structure, and the hairpin structure is located at the hybridization of the linear nucleic acid and the looping auxiliary strand;所述成环辅助链与所述线性核酸的3’端和5’端互补；the looping auxiliary strand is complementary to the 3' and 5' ends of the linear nucleic acid;所述发夹距离连接处1-3nt；The hairpin is 1-3nt from the junction;所述线性核酸的长度为54-200nt；The length of the linear nucleic acid is 54-200nt;所述发夹结构的Tm值高于连接反应温度20-40℃；The Tm value of the hairpin structure is 20-40°C higher than the connection reaction temperature;将所述线性核酸、成环辅助链、连接酶和连接缓冲液混合为同一连接体系，进行连接反应。The linear nucleic acid, the loop-forming auxiliary chain, the ligase and the ligation buffer are mixed into the same ligation system to carry out the ligation reaction.2.根据权利要求1所述的制备方法，其特征在于，将所述线性核酸进行二级结构模拟，选择稳定的发夹结构作为所述线性核酸的杂交处。2 . The preparation method according to claim 1 , wherein the linear nucleic acid is subjected to secondary structure simulation, and a stable hairpin structure is selected as the hybridization site of the linear nucleic acid. 3 .3.根据权利要求1或2所述的制备方法，其特征在于，所述连接反应的时间为1-5小时。3. The preparation method according to claim 1 or 2, wherein the time of the ligation reaction is 1-5 hours.4.根据权利要求1或2所述的制备方法，其特征在于，所述连接反应的缓冲液为标准缓冲液的0.1-1倍。4. The preparation method according to claim 1 or 2, wherein the buffer for the ligation reaction is 0.1-1 times that of the standard buffer.5.根据权利要求1所述的制备方法，其特征在于，所述连接酶为可连接双链DNA、双链RNA或DNA-RNA杂交链中的切刻部分的连接酶。5 . The preparation method according to claim 1 , wherein the ligase is a ligase capable of connecting double-stranded DNA, double-stranded RNA or a nicked portion in a DNA-RNA hybrid strand. 6 .6.根据权利要求5所述的制备方法，其特征在于，所述连接酶为T4 DNA连接酶、T4 RNA连接酶、T3 DNA连接酶、T7 DNA连接酶或E.coli DNA连接酶中的任意一种。6. preparation method according to claim 5 is characterized in that, described ligase is any in T4 DNA ligase, T4 RNA ligase, T3 DNA ligase, T7 DNA ligase or E.coli DNA ligase A sort of.7.根据权利要求1所述的制备方法，其特征在于，所述线性核酸的浓度为1-100μM。7 . The preparation method according to claim 1 , wherein the concentration of the linear nucleic acid is 1-100 μM. 8 .8.根据权利要求7所述的制备方法，其特征在于，所述线性核酸的浓度为5-50μM。8 . The preparation method according to claim 7 , wherein the concentration of the linear nucleic acid is 5-50 μM. 9 .9.根据权利要求1所述的制备方法，其特征在于，所述线性核酸与所述成环辅助链的摩尔比为1:1-5。9 . The preparation method according to claim 1 , wherein the molar ratio of the linear nucleic acid to the looping auxiliary chain is 1:1-5. 10 .10.根据权利要求9所述的制备方法，其特征在于，所述线性核酸与所述成环辅助链的摩尔比为1:1-3。10 . The preparation method according to claim 9 , wherein the molar ratio of the linear nucleic acid to the ring-forming auxiliary chain is 1:1-3. 11 .

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