CN116262917A - G-quadruplex-containing nucleic acids - Google Patents

Abstract

The present invention relates to a nucleic acid comprising a G-quadruplex. The invention discloses a nucleic acid molecule with a G-quadruplex structure for improving the stability of target nucleic acid, which is characterized by comprising the following components: n (N)_a (G_x N_y )_z G_b Or selected from SEQ ID NO: 67. 81-82, 93 and 97 wherein: g represents guanine; x is independently at each occurrence an integer selected from 2-13; n independently for each occurrence represents any base; y is independently at each occurrence an integer selected from 1-19; z is an integer selected from 3-15; a is an integer selected from 0-8; and b is selected from 0 and 2-An integer of 10, wherein when z=3, b is not 0; wherein when n=g and N_y When GT is included, y is not 9; when n=g and N_y When TGA is included, y is not 4, 15 or 16.

Description

G-quadruplex-containing nucleic acids

Technical Field

The invention relates to the field of biological medicine and biotechnology, in particular to a nucleic acid containing G-quadruplex.

Background

By exploiting the ability of nucleic acids to bind to proteins or other targets, a variety of nucleic acid tools or nucleic acid drugs can be developed, including mRNA drugs, antisense oligonucleotides (Antisense oligonucleotide, ASO), and nucleic acid aptamers (aptpers), regulatory RNAs, and the like. Wherein, ASO inhibits the gene expression by combining sequence specificity with target gene RNA, and is developed into a gene knockdown method and applied to the field of nucleic acid medicines. The aptamer can be combined with a target (protein, small molecule, cell and the like), plays a role similar to a chemical antibody, and is applied to the fields of biological detection and nucleic acid medicines. However, such nucleic acids have problems such as being easily degraded by nucleases (exonucleases, endonucleases), unstable affinity to targets, and difficulty in positioning and transportation.

To protect nucleic acids from degradation, various methods have been developed to combat nuclease degradation, including both chemical and non-chemical modification methods. The methods widely used at present are mainly chemical modification methods including phosphate skeleton modification, sugar modification, phosphate modification, base modification and the like, for enhancing the stability of nucleic acids. However, chemically modified nucleic acid drugs have potential safety issues such as eliciting an immune response by activating toll-like receptors, causing cytotoxicity (e.g., inducing apoptosis to cause proteinuria), affecting clotting functions, and causing thrombocytopenia, etc.

The currently reported methods for non-chemical modification are very few, short-chain oligonucleotides capable of forming extremely stable secondary structures were reported in 1989, and the Coulson task group in 1993 reported that a thermodynamically stable hairpin structure hairpin (GCGAAAGC) can protect oligonucleotides from 3-terminal exonuclease degradation and optimize the hairpin to give min-hairpin (GCGAAGC). But the protective effect of this method is very limited. In 2002, patent report (US 7022832B 2) that 5 'end or/and 3' end of oligonucleotide is designed into hairpin structure, which can protect 10% heat inactivated serum, and uses nanometer material to assist in knocking down gene in animal, but the protection effect is limited, and has not been widely used. In 2002, a patent report (US 6121434A) reports that continuous G bases (wherein the number of G is 0-10) can protect an oligonucleotide, but the method is usually combined with thio modification, so that the method is not widely applied at present.

G-quadruplex (G-quadruplex) is a higher structure formed by folding of DNA or RNA rich in tandem repeat guanine (G). G-tetrads (G-quartet) are building blocks of a quadruplex, where 4G's are hydrogen bonded by Hoogsteen to form a circular plane, and two or more layers of G-tetrads are stacked by pi-pi to form a G-quadruplex, which is a class of nucleic acids with a stable secondary structure. The Zaller topic group reported in 1991 that was described in K⁺ Telomeric DNA folded into a G-quadruplex structure cannot become a substrate for telomerase in the presence and can inhibit extension of telomere by telomerase. The stable structure formed by the G-quadruplex may enhance stability in serum by binding to proteins. However, the prior art has not provided nucleic acid pharmaceuticals with high stability and activity.

Accordingly, there is an unmet need for substances and methods that improve nucleic acid stability and/or activity.

Disclosure of Invention

The invention is based on the following findings: nucleic acid molecules of specific sequences having a G-quadruplex structure can be used to protect nucleic acids, to enhance their stability in serum, and to increase their activity.

In a first aspect, the present disclosure provides a nucleic acid molecule having a G-quadruplex structure for improving the stability of a nucleic acid of interest, characterized by comprising the formula: n (N)_a (G_x N_y )_z G_b Or selected from SEQ ID NO: 67. 81-82, 93 and 97;

wherein:

g represents guanine;

x is independently at each occurrence an integer selected from 2-13;

n independently for each occurrence represents any base;

y is independently at each occurrence an integer selected from 1 to 19, for example an integer from 1 to 13, preferably from 1 to 10, more preferably from 1 to 6;

z is an integer selected from 3 to 15, preferably 3 to 10, more preferably 3 to 6;

a is an integer selected from 0 to 8, preferably 0 to 6, more preferably 0 to 3; and

b is an integer selected from 0 and 2-10, preferably 2-9, wherein when z=3, b is not 0;

wherein when n=g and N_y When GT is included, y is not 9; when n=g and N_y Y is not 4, 15 or 16 when TGA is included;

wherein when said nucleic acid molecule having a G-quadruplex structure comprises the sequence of SEQ ID NO:67, the G-quadruplex structure of the nucleic acid molecule is formed by the nucleotide sequence of 2 molecules; and

wherein when said nucleic acid molecule having a G-quadruplex structure comprises the sequence of SEQ ID NO:81 or 82, the G-quadruplex structure of said nucleic acid molecule is formed by the nucleotide sequence of 4 molecules.

In some embodiments, the nucleic acid molecule having a G-quadruplex structure comprises the formula:

(G_x N_y )_z G_b ，

Wherein:

g represents guanine;

x is independently at each occurrence an integer selected from 2-13;

n independently for each occurrence represents any base;

y is independently at each occurrence an integer selected from 1 to 19, for example an integer from 1 to 13, preferably from 1 to 10, more preferably from 1 to 6;

z is an integer selected from 3 to 15, preferably 3 to 10, more preferably 3 to 6; and

b is an integer selected from 0 and 2-10, preferably 2-9, wherein when z=3, b is not 0; and

wherein when n=g and N_y When GT is included, y is not 9; when n=g and N_y When TGA is included, y is not 4 or 15.

In some preferred embodiments, the nucleic acid molecule having a G-quadruplex structure comprises the following formula or is represented by the following formula: (G)_x N_y )_z G_b Wherein:

g represents guanine;

x is independently at each occurrence an integer selected from 2 to 5, preferably 3 or 4;

n independently for each occurrence represents a T base or an A base or a C base, preferably a T base;

y is independently at each occurrence an integer selected from 1 to 4, preferably 1 to 3;

z is an integer of 3, 4 and 5, preferably an integer of 3-4, most preferably 3;

b is an integer selected from 1 to 5, preferably 0 to 6, more preferably 0 to 3.

In some more preferred embodiments, the nucleic acid molecule having a G-quadruplex structure comprises the following formula or is represented by the following formula:

(G_x1 )(N_y1 )(G_x2 )(N_y2 )(G_x3 )(N_y3 )(G_x4 ) Wherein:

g represents guanine;

x1 to x4 are independently integers from 2 to 10 and may be the same or different; preferably, x1-x4 are independently integers from 2 to 5, which may be the same or different; more preferably, x1=x2=x3=x4=3;

n is a T base or an A base or a C base, preferably a T base;

y1 to y3 are independently integers of 1 to 5 and may be the same or different; preferably, y1-y3 are integers from 1 to 3, which may be the same or different; more preferably, y1=2, y2=3, and y3=2.

In some embodiments, the nucleic acid molecule having a G-quadruplex structure comprises or consists of a nucleotide sequence selected from any one ofSEQ ID NOs 3, 41-66, 68-80, 83-92, 94-96 and 98-131. In some embodiments, the nucleic acid molecule having a G-quadruplex structure may not comprise or be the nucleotide sequence set forth in any of SEQ ID NO: 3, 42-48 and 110-131, or, when the nucleic acid molecule having a G-quadruplex structure comprises or is the nucleotide sequence set forth in any of SEQ ID NO: 3, 42-48 and 110-131, one or more (up to 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10) T bases are conjugated at the 3' end of the nucleic acid molecule.

In some embodiments, the nucleic acid of interest is DNA or RNA. In some embodiments, the nucleic acid of interest is modified or unmodified. In some embodiments, the nucleic acid molecule is DNA or RNA. In some embodiments, the nucleic acid molecule having a G-quadruplex structure is modified or unmodified, e.g., not thio-modified. In some embodiments, the G-quadruplex is an intramolecular, bimolecular, or tetramolecular G-quadruplex.

In some embodiments, the nucleic acid of interest is 8-5000 nt, e.g., 8-4000nt, 8-3000nt, 8-2000nt, 8-1000nt, 8-500 nt, 8-200 nt, 8-150 nt, 8-100 nt, 8-80 nt, 8-50 nt, 8-40 nt, 8-30 nt, 8-20 nt, 8-10 nt, 30-180 nt, 20-100 nt, 30-50 nt, etc.

In some embodiments, the nucleic acid of interest is a nucleic acid for gene knockdown, a nucleic acid for gene activation, a nucleic acid for gene modification, a nucleic acid for gene editing, a nucleic acid for gene regulation, a nucleic acid for protein expression, a nucleic acid for biological detection, or a nucleic acid drug.

In some embodiments, the nucleic acid of interest is an oligonucleotide, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, mRNA, or ncRNA (non-coding RNA).

In some embodiments, the oligonucleotide is an antisense oligonucleotide ASO or a nucleic acid aptamer.

In some embodiments, the ncRNA is miRNA (microrna), siRNA (small interfering RNA), saRNA (small activating RNA), piRNA (RNA that interacts with Piwi protein), lncRNA (long non-coding RNA), circRNA (circular RNA), fragments thereof, or other regulatory RNAs.

In some embodiments, one or more (up to 10) T bases may be conjugated to a nucleic acid molecule of the invention having a G-quadruplex structure without affecting its protective effect or activity.

In a second aspect, the present disclosure provides a protected nucleic acid of interest, characterized in that the nucleic acid of interest is conjugated at its 5' end, 3' end or both 5' and 3 ends by or inserted into a nucleic acid molecule of the first aspect having a G-quadruplex structure, and the stability of the nucleic acid of interest is increased.

In some embodiments, the conjugation is a chemical covalent linkage, preferably via a phosphodiester linkage.

In some embodiments, the protected nucleic acid of interest is constructed by: chemical synthesis, genetic engineering methods based on the principle of PCR, or biosynthesis methods.

In some embodiments, the nucleic acid of interest is DNA or RNA. In some embodiments, the protected nucleic acid of interest is modified or unmodified.

In some embodiments, the nucleic acid of interest is 8-5000 nt, e.g., 8-4000nt, 8-3000nt, 8-2000nt, 8-1000nt, 8-500 nt, 8-200 nt, 8-150 nt, 8-100 nt, 8-80 nt, 8-50 nt, 8-40 nt, 8-30 nt, 8-20 nt, 8-10 nt, 30-180 nt, 20-100 nt, 30-50 nt, etc. In some embodiments, the nucleic acid of interest is a nucleic acid for gene knockdown, a nucleic acid for gene activation, a nucleic acid for gene modification, a nucleic acid for gene editing, a nucleic acid for gene regulation, a nucleic acid for protein expression, a nucleic acid for protein regulation, a nucleic acid for biological detection, or a nucleic acid drug. In some embodiments, the nucleic acid of interest is an oligonucleotide, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, mRNA, or ncRNA (non-coding RNA). In some embodiments, the oligonucleotide is an antisense oligonucleotide ASO or a nucleic acid aptamer. In some embodiments, the ncRNA is miRNA (microrna), siRNA (small interfering RNA), saRNA (small activating RNA), piRNA (RNA that interacts with Piwi protein), lncRNA (long non-coding RNA), circRNA (circular RNA), fragments thereof, or other regulatory RNAs.

In a third aspect, the present disclosure provides a pharmaceutical composition characterized in that the pharmaceutical composition comprises the protected nucleic acid of interest of the second aspect and a pharmaceutically acceptable carrier.

In a fourth aspect, the present disclosure provides a method for increasing the stability and/or activity of a nucleic acid of interest, characterized in that a nucleic acid molecule having a G-quadruplex structure of the first aspect is conjugated to the 5' end, the 3' end or both the 5' and 3 end of the nucleic acid of interest, or inserted into the nucleic acid of interest.

In some embodiments, the conjugation is a chemical covalent linkage, preferably via a phosphodiester linkage.

In some embodiments, the protected nucleic acid of interest is constructed by: chemical synthesis, genetic engineering methods based on the principle of PCR, or biosynthesis methods.

In some embodiments, the nucleic acid of interest is DNA or RNA. In some embodiments, the protected nucleic acid of interest is modified or unmodified.

In some embodiments, the nucleic acid of interest is no less than 8 nt, e.g., 8-5000 nt, such as 8-4000nt, 8-3000nt, 8-2000nt, 8-1000nt, or 8-500 nt, preferably 8-200 nt, e.g., 8-150 nt, 8-100 nt, 8-80 nt, 8-50 nt, 8-40 nt, 8-30 nt, 8-20 nt, 8-10 nt, 30-180 nt, 20-100 nt, 30-50 nt, etc. In some embodiments, the nucleic acid of interest is a nucleic acid for gene knockdown, a nucleic acid for gene activation, a nucleic acid for gene modification, a nucleic acid for gene editing, a nucleic acid for gene regulation, a nucleic acid for protein expression, a nucleic acid for protein regulation, a nucleic acid for biological detection, or a nucleic acid drug. In some embodiments, the nucleic acid of interest is an oligonucleotide, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, mRNA, or ncRNA. In some embodiments, the oligonucleotide is an antisense oligonucleotide ASO or a nucleic acid aptamer. In some embodiments, the ncrnas are miRNA, siRNA, saRNA, piRNA, lncRNA, circRNA, fragments thereof, or other regulatory RNAs.

The following description and examples set forth embodiments of the invention in detail. It is to be understood that the invention is not limited to the specific embodiments described herein and that modifications may be made. Those skilled in the art will recognize that there are many variations and modifications of the present invention, which variations and modifications are included within its scope.

Drawings

The foregoing is merely an overview of the present invention, and the present invention is further described in detail below with reference to the accompanying drawings and detailed description.

FIG. 1 shows the stability of G1-containing oligonucleotide drugs in 50% serum;

FIG. 2 is a graph showing the comparison of the stability of a nucleic acid drug containing G1 with that of a nucleic acid drug protected by a currently-used nucleic acid protection method;

FIG. 3 shows the stability of a nucleic acid drug containing G1 at the 5 'or 3' end in 50% serum;

FIG. 4 shows the stability of nucleic acid drugs containing DNA G-quadruplexes;

FIG. 5 shows the effect of G1 engineered G-quadruplexes on nucleic acid drug stability;

FIG. 6 shows the stability of nucleic acid drugs of different lengths containing DNA G-quadruplexes in 50% serum (representative only provides the electrophoresis results of G1 (SEQ ID NO: 3);

FIG. 7 shows the stability of nucleic acid drugs containing different sequences of DNA G-quadruplexes in 50% serum (representative only of the electrophoresis results providing G1 (SEQ ID NO: 3);

FIG. 8 shows the stability of RNA drugs containing different sequences of the RNA G-quadruplex in 50% serum (only representative of the electrophoresis results providing G1 (SEQ ID NO: 3);

FIG. 9 shows the stability of a long-chain RNA drug containing a G-quadruplex in serum (only representative of the results of electrophoresis providing G1 (SEQ ID NO: 3);

FIG. 10 shows the stability of nucleic acid drug chimeras comprising RNA G-quadruplexes and nucleic acid drug chimeras comprising DNA G-quadruplexes in serum (only representative of the electrophoresis results provided for G1 (SEQ ID NO: 3);

FIG. 11 shows a graph of the knockdown effect of a G-quadruplex containing oligonucleotide drug on target RNA in cells (representative only of the electrophoresis results providing G1 (SEQ ID NO: 3); and

FIG. 12 shows a graph of the results of PCSK9 (a) knockdown and LDL-c (b) reduction in animals using a G-quadruplex-containing PCSK 9-targeting antisense oligonucleotide (electrophoresis results representative only of the provision of G1 (SEQ ID NO: 3)).

Detailed Description

Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One of ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods.

Methods of protecting nucleic acid drugs from degradation include both chemical modification methods and non-chemical modification methods, but have limitations. The inventors of the present application originally utilized G-quadruplexes with specific sequences to protect nucleic acid drugs, significantly enhancing their stability and/or activity in serum.

Compared with the prior art, the invention has the following beneficial effects:

1. the nucleic acid molecules and methods of the invention are capable of enhancing the stability of a nucleic acid in serum without any modification;

2. the nucleic acid molecules and the method can enhance the activity of antisense oligonucleotides, verify the capability of ASO to knock down RNA at the cellular level and the whole animal level, and show that the invention can be applied to the fields of gene knockdown and nucleic acid medicines;

3. Compared with the conventional chemical modification protection method and non-chemical modification protection method, the nucleic acid medicine has the advantage of high stability in 50% of serum.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the open-ended expressions "comprising" and "comprising" are to be interpreted to mean that structural components or method steps which are not mentioned can also be included, but it is to be noted that the open-ended expression also covers the case of only being composed of the components and method steps mentioned (i.e. the case of the closed-ended expression "composed of … …" is to be covered).

As used throughout, ranges are used as shorthand for describing each and every value that is within the range. Any numerical value, such as an integer value, within a range can be selected as the end of the range. For example, an integer in the range 2-13 represents an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13, and any subrange formed by them, for example, 2-10, 3-12, 4-9, etc.

All scientific and technical terms referred to in the present specification have the same meaning as commonly understood by one of ordinary skill in the art, and in case of conflict, the present specification will control. In order to make the description of the present invention easier to understand, some terms are explained below.

In the present invention, the terms "nucleic acid", "nucleic acid molecule", "oligonucleotide drug" and "nucleic acid drug" are used interchangeably to refer to any DNA, RNA or DNA/RNA chimeric and may be an oligonucleotide or polynucleotide and may be unmodified RNA or DNA or modified RNA or DNA. The term includes, but is not limited to, single-stranded and double-stranded DNA, DNA that is a mixture of single-stranded and double-stranded regions, single-stranded and double-stranded RNA, and RNA that is a mixture of single-stranded and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or double-stranded or a mixture of single-stranded and double-stranded regions.

In some embodiments, the nucleic acid may comprise one or more modified nucleotides, modified linkages, or the like. Examples of modified linkages or internucleotide linkages include phosphorothioates, phosphorodithioates, and the like. In some embodiments, the nucleotide comprises a phosphorus derivative. The phosphorus derivative (or modified phosphate group) that can be attached to the sugar or sugar analog moiety in the modified nucleotide of the present invention can be a monophosphate, diphosphate, triphosphate, alkyl phosphate, alkane phosphate, phosphorothioate, or the like. The preparation of the above-described phosphate analogs, and their incorporation into nucleotides, modified nucleotides and nucleic acids, are also known per se and need not be described herein.

For nucleic acid modification

(1) Nucleic acid constructs

In some embodiments, a nucleic acid of the invention (e.g., a DNA-targeting RNA) comprises one or more modifications (e.g., base modifications, backbone modifications, etc.) to provide new or enhanced features (e.g., improved stability) to the nucleic acid. As known in the art, nucleosides are base-sugar combinations. The base portion of a nucleoside is typically a heterocyclic base. Two of the most common classes of such heterocyclic bases are purine and pyrimidine. A nucleotide is a nucleoside that also includes a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranose, the phosphate group can be attached to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming nucleic acids, phosphate groups covalently link nucleosides adjacent to each other to form a linear polymeric compound. In turn, each end of the linear polymeric compound may be further linked to form a cyclic compound, however, linear compounds are generally suitable. In addition, linear compounds may have internal nucleotide base complementarity and thus may fold in a manner to produce full or partial double-stranded compounds. Within a nucleic acid, phosphate groups are commonly referred to as forming the internucleoside backbone of the nucleic acid. The normal bond or backbone of RNA and DNA is a 3 'to 5' phosphodiester bond.

(2) Modified backbone and modified internucleoside linkage

Examples of suitable nucleic acids containing modifications include nucleic acids containing modified backbones or non-natural internucleoside linkages. Nucleic acids (having modified backbones) include those that retain phosphorus atoms in the backbones and those that do not have phosphorus atoms in the backbones.

Suitable modified nucleic acids containing phosphorus atoms include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphates including 3' -alkylene phosphates, 5' -alkylene phosphates and chiral phosphates, phosphonates, phosphoramidates including 3' -phosphoramidates and aminoalkyl phosphoramidates, phosphorodiamidates, phosphorothioates, phosphorothioate alkyl phosphates, phosphorothioate alkyl phosphotriesters, seleno-phosphates and boro-phosphates having normal 3' -5' linkages, 2' -5' linked analogs of these, and those having reversed polarity, wherein one or more internucleotide linkages are 3' to 3', 5' to 5' or 2' to 2' linkages. Suitable nucleic acids having reverse polarity comprise a single 3' to 3' bond at the most 3' internucleotide linkage, i.e., a single inverted nucleoside residue that is abasic (nucleobase missing or substituted with hydroxyl). Also included are various salts (such as potassium or sodium, for example), mixed salts and free acid forms.

In some embodiments, the nucleic acids of the invention comprise one or more phosphorothioate linkages and/or heteroatomic internucleoside linkages, in particular-CH₂ -NH-O-CH₂ -、-CH₂ -N(CH₃ )-O-CH₂ - (known as methylene (methylimino) or MMI skeleton), -CH₂ -O-N(CH₃ )-CH₂ -、-CH₂ -N(CH₃ )-N(CH₃ )-CH₂ -and-O-N (CH)₃ )-CH₂ -CH₂ - (wherein natural phosphodiester internucleoside linkage is represented by-O-P (=O) (OH) -O-CH₂ -)。

Other backbone modifications also include, for example, nucleic acids of morpholino backbone structures. For example, in some embodiments, the nucleic acids of the invention comprise a 6-membered morpholino ring in place of a ribose ring. In some of these embodiments, phosphorodiamidite or other non-phosphodiester internucleoside linkages replace phosphodiester linkages.

Suitable modified polynucleotide backbones that do not include phosphorus atoms have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatoms or heterocyclic internucleoside linkages. These include those having the following: morpholino linkages (formed in part from the sugar moiety of the nucleoside); a siloxane backbone; sulfide, sulfoxide, and sulfone backbones; formylacetyl and thiocarboxyacetyl backbones; methylene formylacetyl and thioformylacetyl backbones; a riboacetyl (riboacetyl) backbone; a backbone comprising olefins; sulfamate backbone; methylene imino and methylene hydrazino backbones; sulfonate and sulfonamide backbones; an amide skeleton; with mixtures N, O, S and CH₂ Other skeletons of the constituent parts.

Another backbone modification includes a locked coreAn acid (LNA) in which the 2 '-hydroxy group is attached to the 4' carbon atom of the sugar ring to form a 2'-C, 4' -C-oxymethylene bond, thereby forming a bicyclic sugar moiety. The chain may be methylene (-CH)₂ (-) (a group bridging a 2 'oxygen atom and a 4' carbon atom), wherein n is 1 or 2 (Singh et al chem. Commun.,1998,4,455-456). LNA and LNA analogs exhibit very high duplex thermal stability with complementary DNA and RNA (T_m = +3 ℃ to +10 ℃), stability towards 3' -exonucleolytic degradation and good dissolution properties.

The synthesis and preparation of the LNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization and nucleic acid recognition properties have been described in the prior art (Koshkin et al, tetrahedron,1998,54,3607-3630).

(3) Modified sugar moieties

The nucleic acids of the invention may also include one or more substituted sugar moieties. Suitable polynucleotides comprise sugar substituents selected from the group consisting of: OH; f, performing the process; o-, S-or N-alkyl; o-, S-or N-alkenyl; o-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly suitable are O ((CH)₂ )_n O)_m CH₃ 、O(CH₂ )_n OCH₃ 、O(CH₂ )_n NH₂ 、O(CH₂ )_n CH₃ 、O(CH₂ )_n ONH₂ And O (CH)₂ )_n ON((CH₂ )_n CH₃ )₂ Wherein n and m are from 1 to about 10. Other suitable polynucleotides comprise sugar substituents selected from the group consisting of: c1 to C10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkylaryl, arylalkyl, O-alkylaryl or O-arylalkyl, SH, SCH₃ 、OCN、Cl、Br、CN、CF₃ 、OCF₃ 、SOCH₃ 、SO₂ CH₃ 、ONO₂ 、NO₂ 、N₃ 、NH₂ Heterocyclylalkyl, heterocyclylaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, drug metabolism for improving nucleic acidA group of kinetic nature or a group for improving the pharmacodynamic properties of nucleic acids, and other substituents having similar properties. Suitable modifications include 2 '-methoxyethoxy (2' -O-CH)₂ CH₂ OCH₃ Also known as 2'-O- (2-methoxyethyl) or 2' -MOE) (Martin et al, helv. Chim. Acta,1995,78,486-504), i.e., alkoxyalkoxy. Another suitable modification includes 2' -dimethylaminooxyethoxy, i.e., O (CH₂ )₂ ON(CH₃ )₂ A group, also known as 2' -DMAOE, as described in the examples below; and 2 '-dimethylaminoethoxyethoxy (also known in the art as 2' -O-dimethyl-amino-ethoxy-ethyl or 2 '-DMAEOE), i.e. 2' -O-CH₂ -O-CH₂ -N(CH₃ )₂ 。

Other suitable sugar substituents include methoxy (-O-CH)₃ ) Aminopropoxy (- -OCH)₂ CH₂ CH₂ NH₂ ) Allyl (-CH)₂ -CH＝CH₂ ) -O-allyl (- -O- -CH)₂ —CH＝CH₂ ) Fluorine (F). The 2' -sugar substituent may be in the arabinose (upper) or ribose (lower) position. Suitable 2 '-arabinose modifications are 2' -F. Similar modifications can also be made at other positions on the oligomeric compound, specifically at the 3 'position of the sugar and the 5' position of the 5 'terminal nucleotide on the 3' terminal nucleoside or in the 2'-5' linked nucleic acid. The oligomeric compounds may also have a glycomimetic such as a cyclobutyl moiety in place of the pentofuranosyl sugar.

(4) Base modification and substitution

Nucleic acids of the invention may also include nucleobase (often referred to in the art simply as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (a) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine Pyrimidine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-c=c-CH)₃ ) Uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-mercapto, 8-sulfanyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo (specifically 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Additional modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido (5, 4-b) (1, 4) benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido (5, 4-b) (1, 4) benzothiazin-2 (3H) -one), G-clamps such as substituted phenoxazine cytidine (e.g., 9- (2-aminoethoxy) -H-pyrimido (5, 4- (b) (1, 4) benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimido (4, 5-b) indol-2-one), pyrido-indole cytidine (H-pyrido (3 ',2':4, 5) pyrrolo (2, 3-d) pyrimidine-2-one).

Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced by other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Additional nucleobases include those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, kroschwitz, J.I. journal of John Wiley & Sons,1990, those disclosed by Angewandte Chemie, international Edition,1991,30,613, and those disclosed by Sanghvi, Y.S.,chapter 15, antisense Research and Applications, pages 289-302, rooke, S.T. and Lebleu, B.journal, CRC Press, 1993. Some of these nucleobases are useful for increasing the binding affinity of oligomeric compounds. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase the stability of nucleic acid duplex by 0.6-1.2 ℃ (Sanghvi et al, eds., antisense Research and Applications, CRC Press, boca Raton,1993, pages 276-278) and are suitable base substitutions, for example when combined with 2' -O-methoxyethyl sugar modifications.

(5) Conjugate(s)

Another possible modification of the nucleic acids of the invention involves chemically linking one or more moieties or conjugates to the polynucleotide that enhance the activity, cellular distribution or cellular uptake of the nucleic acid. These moieties or conjugates can include conjugate groups that are covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of the oligomers, and groups that enhance the pharmacokinetic properties of the oligomers. Suitable conjugate groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folic acid esters, phenanthridines, anthraquinones, acridines, fluorescein, rhodamine, coumarin, and dyes. Groups that enhance pharmacodynamic properties include groups that improve absorption, enhance resistance to degradation, and/or enhance sequence-specific hybridization with a target nucleic acid. Groups that enhance pharmacokinetic properties include groups that improve absorption, distribution, metabolism, or excretion of the nucleic acids of the invention.

Conjugate moieties include, but are not limited to: lipid moieties such as cholesterol moieties (Letsinger et al, proc.Natl. Acad.Sci.USA,1989,86,6553-6556), cholic acids (Manoharan et al, bioorg.Med. Chem. Let.,1994,4,1053-1060), thioethers such as hexyl-S-tritylthiol (Manoharan et al, ann.N.Y. Acad.Sci.,1992,660,306-309; manoharan et al, bioorg.Med. Chem. Let.,1993,3,2765-2770), thiocholesterol (Obohauser et al, nucl. Acids Res.,1992,20,533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaas et al, EMBO J.,1991,10,1111-1118; kabanov et al, FEBS Lett.,1990,259,327-330, svirchuk et al, biomie, 1993,75,49-54), phospholipids such as di-hexadecyl-racemic glycerol or 1, 2-di-O-hexadecyl-racemic glycerol-3-H-triethylammonium phosphate (Manoharan et al, tetrahedron lett.) 1995,36,3651-3654; shea et al, nucleic acids res.,1990,18,3777-3783), polyamines or polyethylene glycol chains (Manoharan et al, nucleic & nucleic oxides, 1995,14,969-973), or adamantaneacetic acid (Manoharan et al, tetrahedron lett.,1995,36,3651-3654), palmityl moieties (Mishra et al, biochem. Acta,1995,1264,229-237), or octadecylamine or hexylamino-carbonyl-oxy cholesterol moieties (crike et al, j. Pharmacol. Exp. Therer., 1996,277,923-937).

As used herein, "unmodified" or "natural" nucleotides include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleotides include nucleotides that are only rare or short-lived in natural nucleic acids, such as, for example, hypoxanthine, 6-methyladenine, 5-Me pyrimidine, especially 5-methylcytosine (also known as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-Hydroxymethylcytosine (HMC), glycosyl HMC and gentiobiosyl HMC, as well as synthetic nucleotides, such as, for example, 2-aminoadenine, 2- (methylamino) adenine, 2- (imidazolylalkyl) adenine, 2- (aminoalkylamino) adenine or other hetero-substituted alkyl adenine, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl) adenine and 2, 6-diaminopurine. "universal" bases known in the art, such as inosine, may be included.

The term "G-quadruplex" as used herein is a higher order structure formed by folding of DNA or RNA rich in tandem repeat guanine (G). The G-tetrads are structural units of a quadruplex, 4G are hydrogen-bonded by Hoogsteen to form a circular plane, and two or more layers of G-tetrads are stacked by pi-pi to form a G-quadruplex, which is a class of nucleic acids having a stable secondary structure (see Zahler, a.m., williamson, j.r., cech, t.r.). & Prescott, D. M. Inhibition of telomerase by G-quartet DMA structures.Nature350, 718-720 (1991), which are incorporated herein by reference for all purposes). The G-quadruplexes are further stabilized by the presence of cations, in particular potassium, located in the central channel between each pair of tetrads. The G-quadruplex may be formed of DNA, RNA, LNA and PNA, and may be intramolecular, bimolecular or tetra-molecular. Depending on the orientation of the strands or portions of strands forming the quadrants, the structure may be described as parallel or anti-parallel (see, e.g., parkinson G N, lee M P H, neidle S, crystall structure of parallel quadruplexes from human telomeric DNA.Nature 417 (6891): 876-880 (2002)；Wang Y, Patel D J, Solution structure of the human telomeric repeat d[AG₃ (T₂ AG₃ )₃ ]G-quadruplex. Structure 1 (4): 263-282 (1993); and Dai J. Carver M., yang D,. Polymorphism of human telonieric quadruples constructs. Biochimie. 90 (8): 1172-1183 (2008), which is incorporated herein by reference for all purposes).

In some embodiments, a nucleic acid molecule of the invention having a G-quadruplex structure comprises: n (N)_a (G_x N_y )_z G_b Or selected from SEQ ID NO: 67. 81-82, 93 and 97; wherein: g represents guanine; x is independently at each occurrence an integer selected from 2-13; n independently for each occurrence represents any base; y is independently at each occurrence an integer selected from 1 to 19, for example an integer from 1 to 13, preferably from 1 to 10, more preferably from 1 to 6; z is an integer selected from 3 to 15, preferably 3 to 10, more preferably 3 to 6; a is an integer selected from 0 to 8, preferably 0 to 6, more preferably 0 to 3; and b is an integer selected from 0 and 2-10, preferably 2-9, wherein when z=3, b is not 0; wherein when n=g and N_y When GT is included, y is not 9; when n=g and N_y Y is not 4, 15 or 16 when TGA is included; wherein when the nucleic acid molecule comprises SEQ ID NO:67, the G-quadruplex structure of the nucleic acid molecule is formed by the nucleotide sequence of 2 molecules; and wherein when the nucleic acid molecule comprises SEQ ID NO:81 or 82, the G-quadruplex structure of said nucleic acid molecule is formed by the nucleotide sequence of 4 molecules.

In some embodiments, x is independently selected for each occurrence from theintegers 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and 13 and any subrange they form, e.g., 2-10, 3-12, 4-9, etc. In some embodiments, y is independently selected for each occurrence from theintegers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 19 and any subrange they form, e.g., 2-10, 3-13, 1-15, 5-10, 2-13, and the like. In some embodiments, z is selected fromintegers 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 and any subrange they form, e.g., 3-9, 4-12, 5-10, 7-15, 6-14, etc. In some embodiments, a is selected from 0, 1, 2, 3, 4, 5, 6, 7, and 8 and any subrange they form, e.g., 2-8, 0-7, 3-6, 4-7, etc. In some embodiments, b is selected from 0, 2, 3, 4, 5, 6, 7, 8, 9, and 10 and any subrange they form, e.g., 2-8, 3-9, 4-6, 5-10, etc. In some embodiments, the G-quadruplexes of the present invention may be intramolecular, bimolecular, or tetramolecular G-quadruplexes. In some embodiments, the G-quadruplexes of the present invention may comprise cations, for example monovalent cations such as potassium, sodium, etc., and divalent cations such as magnesium, calcium, zinc, copper, etc.

The nucleic acid molecules according to the invention and the nucleic acids of interest protected by the nucleic acid molecules according to the invention, for example nucleic acid pharmaceuticals, can be used for various applications, for example for gene knockdown, gene activation, gene modification, gene editing, gene regulation, protein expression, protein regulation or biological detection or as nucleic acid pharmaceuticals. Thus, the invention contemplates the use of the nucleic acid molecules of the invention and nucleic acids of interest protected by the nucleic acid molecules of the invention in this regard, for example for the preparation of a medicament for the above-mentioned use, etc.

In the present invention, the terms "conjugate" and "linked" are used interchangeably to refer to linking through a chemical bond.

In this context, the stability of the nucleic acid according to the invention in serum refers to the stability of the nucleic acid in serum of various concentrations, for example 50% serum. In this context, an improved stability means that the stability of the nucleic acid of interest, for example in serum, is improved compared to the nucleic acid of interest which is not protected by the nucleic acid molecule of the invention or is protected by other means. As used herein, an increase in the activity of a nucleic acid of interest means that the biological activity of the nucleic acid of interest is increased after protection by a nucleic acid molecule of the invention as compared to a nucleic acid of interest that is not protected by a nucleic acid molecule of the invention or is otherwise protected. In some embodiments, the biological activity is selected from the group consisting of gene knockdown activity, gene activation activity, gene modification activity, gene editing activity, gene regulation activity, protein expression activity, protein regulation activity, or biological assay activity, or activity for use as a nucleic acid drug, and the like. In some embodiments, the activity of the nucleic acid of interest is equivalent or similar to the activity of a nucleic acid of interest that is not protected by a nucleic acid molecule of the invention or is otherwise protected, but the stability is improved.

The term "pharmaceutically acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable vectors are described in the latest version of Remington's Pharmaceutical Sciences (standard reference text in the art), which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solution, dextrose solution, and 5% human serum albumin. The use of such media and reagents is well known in the art. Except insofar as any conventional medium or agent is incompatible with the materials provided herein, use thereof in the compositions is contemplated.

The following specific examples are given to illustrate the present invention and are not to be construed as limiting the scope of the invention, and it will be understood by those skilled in the art that the present invention may be modified or substituted for others without departing from the spirit and scope of the invention.

Examples

Unless otherwise indicated, the materials used in the examples herein are commercially available, and the various specific experimental methods used to conduct the experiments are either routine in the art or are in accordance with the procedures and conditions suggested by the manufacturer and can be routinely determined by one of skill in the art as desired. In addition, all the serum used in the examples in this specification are Gibco extra-large bovine serum. The degradation system comprises: nucleic acid drug, 1 XPBS, 50% serum and water. The electrophoresis conditions used are: 180V,20min; the sample loading amount is as follows: 2. μg-5 μg. Unless otherwise indicated, the sequences used for protection in the examples of the present specification are all G-quadruplexes. Unless otherwise indicated, the G-quadruplexes in the examples of the present specification are added to both ends of the protected sequence. Unless otherwise indicated, the sequences used in the examples of the present specification are all deoxyribonucleotide sequences.

Example 1: assessment of stability of G1-containing oligonucleotide drug in 50% serum

The specific DNA sequence (G1) of the present invention was added to the 5 'and 3' ends of the deoxyribonucleotide (S1) to be protected, respectively, and incubated in 50% serum for various times: 24 h, 48 h and 72h, and then analyzing the result by 3% agarose gel electrophoresis, wherein the random sequence Rs is selected as a negative control, because the oligonucleotide itself has a short length and is easy to degrade, so that a random control sequence which is basically consistent with the length of the protected sequence is selected, and the electrophoresis result graph of the random control sequence is almost in a straight line, thereby being convenient for comparison. As a result, as shown in FIG. 1, the random sequence Rs without the addition of the specific sequence of the present invention was substantially completely degraded at 6 h as a Negative Control (NC), while G1-S1 remained in the intact DNA sequence which was not degraded at 72 hours, indicating that the oligonucleotide drug containing G1 had good stability in 50% serum. The inventors have also found that the comparative effect is more pronounced with S1 as negative control.

In example 1, sequence information of the sequence used is as follows:

S1：TTTGAATGTAGAGATGCGGTGGTTT (SEQ ID NO: 1)

Rs: ACCCGACCTCTTCTATCTGGACCCGACCGTCTCTTTTTTGAGCCCACACTCTACTCGAC (SEQ ID NO: 2)

G1：GGGTTGGGTTTGGGTTGGG (SEQ ID NO: 3)。

example 2: comparing the stability of a G1-containing nucleic acid drug with that protected by a currently used nucleic acid protection method

To evaluate the stability of the G1-containing nucleic acid drug, the inventors selected the reported nucleic acid protection methods (chemically modified and non-chemically modified) for comparison, 4 control groups were set, whereincontrol group 1 was chemically modified, including 2' OMe-Ps, 3' -dT, 5' -FAM, and Ps; thecontrol group 2 is non-chemically modified, including G-cap sequence modification (G-cap-1, G-cap-2, G-cap-3, G-cap-4, G-cap-5, G-cap-6), mini-hairpin sequence modification and hairpin sequence modification (H-10, DH-6, L-10); control group 3 (G-cap-7, G-cap-8, G-cap-9) is a sequence obtained by chemical modification (thio modification, expressed as a) of G-cap according to the content of patent (US 20020151512 A1);control group 4 is the sequence (P1-P20) protected by the protection method in the reported patent (US 20210139893A 1). The modifications described above were applied to the S1 nucleic acid drug or the sequences described above were added to both ends of the S1 nucleic acid drug, and incubated 24 h in 50% serum, respectively, and then the results were analyzed by 3% agarose gel electrophoresis. As a result, as shown in FIG. 2, the nucleic acid protected by the chemical modification and non-chemical modification method was substantially completely degraded after 24. 24 h incubation in 50% serum except the nucleic acid protected by the method of the present invention, and the G1-containing nucleic acid had a large amount of undegraded complete DNA sequence, which suggests that the G1-containing nucleic acid has the advantage of high stability in 50% serum.

In example 2, sequence information of the sequence used is as follows:

S1：TTTGAATGTAGAGATGCGGTGGTTT (SEQ ID NO: 1)

H10：TGAACACGCCATGTCGATTCTTTAGAATCGACA (SEQ ID NO: 4)

DH6：TGTTCATCTGAACACGCCATGTCGATTCTTTAGAATC (SEQ ID NO: 5)

L-10：GCGCTTATGAACACGCCATGTCGATTCTTAAGCGC (SEQ ID NO: 6)

G-cap-1： GGGGCTCCTGGAGCGGGGCACAC (SEQ ID NO: 7)

G-cap-2：GGGGTCGACACCCAATTCTGAAAATGGATAA (SEQ ID NO: 8)

G-cap-3：GGGGACACCCAATTCTGAAAATGGGG (SEQ ID NO: 9)

G-cap-4：GGGAGGTCCCTGTTCGGGCGCCAGGGG (SEQ ID NO: 10)

G-cap-5：GGGGACACCCAATTCTGAAAATGGGG (SEQ ID NO: 11)

G-cap-6：GGGGGAAGGAGGAGGATGAGGGGG (SEQ ID NO: 12)

G-cap-7：G*G*G*AGGT*CC*C*TGT*T*CGGGCGC*CAG*G*G*G (SEQ ID NO: 13)

G-cap-8：G*G*G*GACACCCAATTCTGAAAATG*G*G*G (SEQ ID NO: 14)

G-cap-9：G*G*GGGAAGGAGGAGGAT*GAGGG*G*G (SEQ ID NO: 15)

P1：TTGGGGTTAGGGTTAGGGTTAGGGATGAATGTAGAGATGCGGTGGTTGGGGTTAGGGTTAGGGTTAGGGA (SEQ ID NO: 16)

P2：TTGGGTGGGTGGGTGGGTTGAATGTAGAGATGCGGTGGTTGGGTGGGTGGGTGGGT (SEQ ID NO: 17)

P3：GGGGTGGGAGGAGGGTTGAATGTAGAGATGCGGTGGGGGGTGGGAGGAGGGT (SEQ ID NO: 18)

P4：TCCACGCACAGTTGAATGTAGAGATGCGGTGGTCCACGCACAGT (SEQ ID NO: 19)

P5：TTTCCACGCACAGTTGAATGTAGAGATGCGGTGGTTTCCACGCACAGT (SEQ ID NO: 20)

P6：GCTTTCCACGCACAGTTGAATGTAGAGATGCGGTGGGCTTTCCACGCACAGT (SEQ ID NO: 21)

P7：ACGCTTTCCACGCACAGTTGAATGTAGAGATGCGGTGGACGCTTTCCACGCACAGT (SEQ ID NO: 22)

P8：CTACGCTTTCCACGCACAGTTGAATGTAGAGATGCGGTGCTACGCTTTCCACGCACAGT (SEQ ID NO: 23)

P9：TTTCCACGTGAATGTAGAGATGCGGTGGTTTCCACG (SEQ ID NO: 24)

P10：CTACGCTGAATGTAGAGATGCGGTGGCTACGC (SEQ ID NO: 25)

P11：CTACGCTTTGAATGTAGAGATGCGGTGGCTACGCTT (SEQ ID NO: 26)

P12：CTACCGTTTCTGAATGTAGAGATGCGGTGGCTACCGTTTC (SEQ ID NO: 27)

P13：CTACGCTTTCCATGAATGTAGAGATGCGGTGGCTACGCTTTCCA (SEQ ID NO: 28)

P14：CTACGCTTTCCACGTGAATGTAGAGATGCGGTGGCTACGCTTTCCACG (SEQ ID NO: 29)

P15：CTACGCTTTCCACGCATGAATGTAGAGATGCGGTGGCTACGCTTTCCACGCA (SEQ ID NO: 30)

P16：CTACGCTTTCCACGCACATGAATGTAGAGATGCGGTGGCTACGCTTTCCACGCACA (SEQ ID NO: 31)

P17：CTTTCCACGCTGAATGTAGAGATGCGGTGGCTTTCCACGC (SEQ ID NO: 32)

P18：GCTTTCCACGCATGAATGTAGAGATGCGGTGGGCTTTCCACGCA (SEQ ID NO: 33)

P19：CGCTTTCCACGCACTGAATGTAGAGATGCGGTGGCGCTTTCCACGCAC (SEQ ID NO: 34)

P20：ACGCTTTCCACGCACATGAATGTAGAGATGCGGTGGACGCTTTCCACGCACA (SEQ ID NO: 35)

wherein represents Ps modification.

Example 3: assessment of stability of nucleic acid drugs containing G1 at 5 'or 3' end in 50% serum

To evaluate the stability of a nucleic acid containing G1 at the 5 'end or 3' end in 50% serum, the inventors selected to add G1 to the 5 'end, 3' end and both ends of nucleic acids of different lengths (S2, S3 and S4), wherein S2 is 25 nt in length, S3 is 8 nt in length and S4 is 13 nt in length. Each sequence was incubated in 50% serum for 24h, respectively, and then analyzed by 16% polyacrylamide gel. As a result, as shown in FIG. 3, the stabilizing effect was not ideal when G1 was contained only at the 5 'end, but there was some stability when G1 was contained only at the 3' end. In addition, the shorter the nucleic acid length, the better the stability of containing G1 only at the 3' end. When G1 is at both ends (5 '+3'), the stabilizing effect is best. The above results indicate that the nucleic acid having G1 at either the 5 '-end or the 3' -end has stability, and that the stability is the strongest when both ends (5 '+3') contain G1.

In addition, stability of the nucleic acid sequence when G1 was contained inside the nucleic acid sequence was evaluated. Two different sequences (G1-S and G1-D) were designed, which were incubated in 50% serum for different times, respectively, and then analyzed by 3% agarose gel electrophoresis. The results are shown in FIG. 3, where the intact DNA sequence remains undegraded at 6 h, indicating that the G1 has some stability when inside the nucleic acid sequence.

In example 3, sequence information of the sequence used is as follows:

S2：TTTGCCTTTAGGATTCTAGACATTT (SEQ ID NO: 36)

S3：AGACATTT (SEQ ID NO: 37)

S4：ATTCTAGACATTT (SEQ ID NO: 38)

G1-S: TTTGCCTTTAGGAGGGTTGGGTTTGGGTTGGGTTCTAGACATTT (SEQ ID NO: 39)

G1-D: TTTGCCTTTAGGAGGGTTGGGTTTGGGTTGGGTTTGCCTTTAGGATTCTAGACATTTGGGTTGGGTTTGGGTTGGGTTCTAGACATTT (SEQ ID NO: 40)。

example 4: evaluation of stability of nucleic acid drugs containing DNA G-quadruplexes

The inventors further constructed 9 different G-quadruplex sequences based on G1 (SEQ ID NO: 3), which were added at both ends of nucleic acid S1, respectively, and incubated 24 h in serum; in addition, 60 different G-quadruplex sequences were selected, added to the 3' end of S1, and incubated in serum for 3 h, with random sequences without G-quadruplex added as negative controls (DRs), and then analyzed by 3% agarose gel electrophoresis, respectively. The results are shown in FIG. 4, wherein the degradation degree of nucleic acid medicines with different sequences in serum is different, but the complete DNA sequences which are not degraded exist, so that the nucleic acid medicines containing DNA G-quadruplexes have certain stability in serum.

In example 4, sequence information of the sequence used is as follows:

G2: GGGTTTGGGTGGGTTTGGG (SEQ ID NO: 41)

G3: GGGTGGGTGGGTGGGT (SEQ ID NO: 42)

G4: GGTGGTGGTGGTTGTGGTGGTGGTGG (SEQ ID NO: 43)

G5: GTGGGGCATTGTGGGTGGGTGTGG (SEQ ID NO: 44)

G6: AGGGTTAGGGTTAGGGTTAGGG (SEQ ID NO: 45)

G7: GGTTGGTGTGGTTGG (SEQ ID NO: 46)

G8: GGGTAGGGCGGGGTTGGGG (SEQ ID NO: 47)

G9: GGTGGTGGTGGTTGTGGTGGGTGGGTGGG (SEQ ID NO: 48)

G10: GGTGGTGGTGGTTGTGGTGGTGGTGGGGTGGTGGTGGTTGTGGTGGTGGTGG (SEQ ID NO: 49)

1：GGTTAGGTTAGGTTAGG (SEQ ID NO: 50)

2：GGGGTTTTGGGGT TTTGGGGTTTTGGGG (SEQ ID NO: 51)

3：GGCTTAGGCTTAGGCTTAGG (SEQ ID NO: 52)

4：GGGTTCAGGGTTCAGGGTTCAGGG (SEQ ID NO: 53)

5：AGGGTTAGGGTTAGGGTTAGGGT (SEQ ID NO: 54)

6：GGGCTAGGGCTAGGGCTAGGG (SEQ ID NO: 55)

7：TGAGGGTGGGTAGGGTGGGTAA (SEQ ID NO: 56)

8：GGGAGGGCGCTGGGAGGAGGG (SEQ ID NO: 57)

9：GGGCGGGCGCGAGGGAGGGG (SEQ ID NO: 58)

10：ACAGGGGTGTGGGGACAGGGGTGTGGGG (SEQ ID NO: 59)

11：GCGGGCGGCTCGGGCGCGGG (SEQ ID NO: 60)

12：GGGAGGGTTGGGGTGGG (SEQ ID NO: 61)

13：GGGCAGGGAGGGAACTGGG (SEQ ID NO: 62)

14：GGGAGGGTTGGGGTGGGT (SEQ ID NO: 63)

15：GGGCAGGGAGGGAACTGGGT (SEQ ID NO: 64)

16：GGGCGGTGTGGGAAGAGGGAAGAGGGG (SEQ ID NO: 65)

17：GGTTGGTGTGGTTGGT (SEQ ID NO: 66)

18：GGGCTTTTGGGC (SEQ ID NO: 67)

19：TTTAAGGGTTAGGGTTAGGGTTAGGG (SEQ ID NO: 68)

20：TGAGGGTGGTGAGGGTGGGGAAGG (SEQ ID NO: 69)

21：TGAGGGTGGGTAGGGTGGGTAAT (SEQ ID NO: 70)

22：TGAGGGTGGGAGGGTGGGGAAGGA (SEQ ID NO: 71)

23：GGGCGCGGGAGGAATTGGGCGGG (SEQ ID NO: 72)

24：AGGGAGGGCGCTGGGAGGAGGG (SEQ ID NO: 73)

25：CGGGCGGGCGCGAGGGAGGGT (SEQ ID NO: 74)

26：CGGGCGGGCGCTAGGGAGGGT (SEQ ID NO: 75)

27：GGAGGAGGAGGA (SEQ ID NO: 76)

28：GGTTGGTGTGGTTGGTT (SEQ ID NO: 77)

29：GGTGGTGGTGGTTGTGGTGGTGGTGGT (SEQ ID NO: 78)

30：GGGCGGGCGGGCGGGC (SEQ ID NO: 79)

31：TGTGGGGGTGGACGGGCCGGGTAGA (SEQ ID NO: 80)

32：TTGGGTT (SEQ ID NO: 81)

33：TGGGAG (SEQ ID NO: 82)

34：GGGGTGGGAGGAGGGT (SEQ ID NO: 83)

35：GGTTGGTGTGGTTGGTTT (SEQ ID NO: 84)

36：ATGGGGTCGGGCGGGCCGGGTGTC (SEQ ID NO: 85)

37：GTGGGTAGGGCGGGTTGG (SEQ ID NO: 86)

38：GGGTTAGGGTTAGGGTAGGG (SEQ ID NO: 87)

39：CCTGGGGGAGTATTGCGGAGGAAGG (SEQ ID NO: 88)

40：AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGG (SEQ ID NO: 89)

41：AGGGCGGTGTGGGAAGAGGGAAGAGGGGGAGGCAG (SEQ ID NO: 90)

42：CGGTCGCTCCGTGTGGCTTGGGTTGGGTGTGGCAGTGAC (SEQ ID NO: 91)

43：GGGGGTGGGAGGGTAGGCCTTAGGTTTCTGA (SEQ ID NO: 92)

44：CGCCTGATTAGCGATACTCAGCGTTGGGGGGGGGGGG (SEQ ID NO: 93)

45：GGGCGTGGTGGGTGGGGTACTAATAATGTGCGTTTG (SEQ ID NO: 94)

46：AGCGGGCATATGGTGGTGGGTGGTATGGTC (SEQ ID NO: 95)

47：AACACATAGGTTTGGTTAGGTTGGTTGGTTGAATTA (SEQ ID NO: 96)

48：TTGCGCGTTAATTGGGGGGGTGGGTGGGTT (SEQ ID NO: 97)

49：GGTGGTGGGGGGGGTTGGTAGGGTGTCTTC (SEQ ID NO: 98)

50：GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA (SEQ ID NO: 99)

51：GGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGG (SEQ ID NO: 100)

52：GGGGCGCTTATGGGGAGGGTGGGGAGGGTGGGGAAGGTGGGGAGGAG (SEQ ID NO: 101)

53：CCCCCCGGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAGCCATG (SEQ ID NO: 102)

54：GGCGGGGGGGGGGGGGCGGGGGCGGGGGCGGGGGAGGGGCGCGGCGG (SEQ ID NO: 103)

55：CGCGGAGGGGCGGGCGCGGGAGGAAGGGGGCGGGAGCGGGGCTGTGG (SEQ ID NO: 104)

56：GAGGAGCGGGTAGGGGCGGGGCGGGGCGGGGGCGGTCCAGGGGTGGG (SEQ ID NO: 105)

57：CGCTCGGGCGCGCGGGGAGGGGAGAGGGGGCGGGAGCGCGCCCGCTC (SEQ ID NO: 106)

58：AGAGGAGGAGGAGGTCACGGAGGAGGAGGAGAAGGAGGAGGAGGAAA (SEQ ID NO: 107)

59：GGGGCGCTTATGGGGAGGGTGGGTAGGGTGGGTAAGGTGGGGAGGAG (SEQ ID NO: 108)

60：GGGGCGCTTATGGGGAGGGTGGGTAGGGTTGGGAAGGTGGGGAGGAG (SEQ ID NO: 109)

DRs：GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCC (SEQ ID NO: 166)。

example 5: assessment of stability of nucleic acid drugs containing G1 engineered G-rich sequences

The inventors selected to modify the G1 sequence differently, and add the modified sequences to both ends of the nucleic acid S1, respectively, to evaluate the stability of a nucleic acid drug containing a G1-modified G-rich sequence. The above sequences were incubated in 50% serum for 24 h, respectively, and then analyzed by 3% agarose gel electrophoresis. The results are shown in FIG. 5, wherein the nucleic acid drugs containing G-rich sequences based on G1 engineering are all stable.

In example 5, sequence information of the sequence used is as follows:

G1-1: GGGAAGGGAGGGAAGGG (SEQ ID NO: 110)

G1-2: GGGTTGGGTGGGTTGGG (SEQ ID NO: 111)

G1-3: GGGCCGGGCGGGCCGGG (SEQ ID NO: 112)

G1-4: GGGTTGGGTTGGGTTGGG (SEQ ID NO: 113)

G1-5: GGGTAGGGCGGGTTGGG (SEQ ID NO: 114)

G1-6: GGGTGGGTGGGTGGG (SEQ ID NO: 115)

G1-7: GGGTGGGTTGGGTGGG (SEQ ID NO: 116)

G1-8: GGGTGGGTTTGGGTGGG (SEQ ID NO: 117)

G1-9: GGGTTTGGGTTTGGGTTTGGG (SEQ ID NO: 118)

G1-10: GGGTTTTGGGTTTTGGGTTTTGGG (SEQ ID NO: 119)

G1-11: GGGTTTTTGGGTTTTTGGGTTTTTGGG (SEQ ID NO: 120)

G1-12: GGGGTTGGGGTTTGGGGTTGGGG (SEQ ID NO: 121)

G1-13: GGGGGTTGGGGGTTTGGGGGTTGGGGG (SEQ ID NO: 122)

G1-14: GGGGGGTTGGGGGGTTTGGGGGGTTGGGGGG (SEQ ID NO: 123)

G1-15: GGGGGGGTTGGGGGGGTTTGGGGGGGTTGGGGGGG (SEQ ID NO: 124)

G1-16: GGGGGGGGTTGGGGGGGGTTTGGGGGGGGTTGGGGGGGG (SEQ ID NO: 125)

G1-17: GGGGGGGGGTTGGGGGGGGGTTTGGGGGGGGGTTGGGGGGGGG (SEQ ID NO: 126)

G1-18: GGGGGGGGGGTTGGGGGGGGGGTTTGGGGGGGGGGTTGGGGGGGGGG (SEQ ID NO: 127)

G1-19: GGGTAGGGCGGGTTGGGTAGGG (SEQ ID NO: 128)

G1-20: GGGTAGGGCGGGTTGGGTAGGGCGGG (SEQ ID NO: 129)

G1-21: GGGTAGGGCGGGTTGGGTAGGGCGGGTTGGG (SEQ ID NO: 130)

G1-22: GGGTAGGGCGGGTTGGGGGGTAGGGCGGGTTGGG (SEQ ID NO: 131)。

example 6: assessment of stability of nucleic acid drugs of different lengths containing DNA G-quadruplexes in 50% serum

The stability of nucleic acid drugs of different lengths containing DNA G-quadruplexes (here G-quadruplexes (G1-G10 and G1-G1-22)) in 50% serum was evaluated. The DNA G-quadruplex was added to each of the two ends of the selected nucleic acids having different lengths. The selected lengths are 16-20nt, 25nt, 30nt, 35nt, 50nt, 80nt and 200nt, respectively, wherein the nucleic acids of 35nt and 50nt lengths are nucleic acid aptamers toCD 44 and c-Met, respectively. The above sequence was incubated 24 h in 50% serum with random sequence without addition of DNA G-quadruplex as negative Control (Rs/Control), and the results were analyzed by 3% agarose gel electrophoresis. The results are shown in FIG. 6, in which the degree of degradation of nucleic acids of different lengths in serum is different, but there is a complete DNA sequence which is not degraded, and the combination of the protection ability of 8nt nucleic acids in example 3 shows that the nucleic acid drugs of different lengths containing DNA G-quadruplexes have stability.

In example 6, sequence information of the sequence used is as follows:

16nt: AATGTAGAGATGCGGT (SEQ ID NO: 132)

17nt: AATGTAGAGATGCGGTG (SEQ ID NO: 133)

18nt: GAATGTAGAGATGCGGTG (SEQ ID NO: 134)

19nt: GAATGTAGAGATGCGGTGG (SEQ ID NO: 135)

20nt: TGAATGTAGAGATGCGGTGG (SEQ ID NO: 136)

25nt: TTTGAATGTAGAGATGCGGTGGTTT (SEQ ID NO: 137)

30nt: TTTAGATTATATGTCATACCTCCATTGGTT (SEQ ID NO: 138)

33nt: TTTGGACGGTGTTAAACGAAAGGGGACGACCTT (SEQ ID NO: 139)

35nt: GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCC (SEQ ID NO: 140)

50nt: ATCAGGCTGGATGGTAGCTCGGTCGGGGTGGGTGGGTTGGCAAGTCTGAT (SEQ ID NO: 141)

80nt: GCTGTGTGACTCCTGCAAAGTGTGGACAACTTCCCACGGAGGAATTCCCGTATCTAAAGGTGCAGCTGTATCTTGTCTCC (SEQ ID NO: 142)

200nt:

AACACCGGTTTGCCATGTGTTGCCATGTGTATGTGGGGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCCCCACATACTCTGATGATCCGAGACGGTCGGGTCCAGATATTCGTATCTGTCGAGTAGAGTGTGGGCTCGGATCATTCATGGCAACTCTGATGATCCTCTATCTATCTGTCGAG (SEQ ID NO: 143)。

example 7: assessment of stability of nucleic acid drugs containing different sequences of DNA G-quadruplexes in 50% serum

Nucleic acids S5-S13 of different sequences were selected and the stability of nucleic acid drugs containing different sequences of DNA G-quadruplexes (G1-G10 and G1-G1-22) were used here) in 50% serum was evaluated. The above sequences were incubated in 50% serum for various times with random sequences not protected by this method as negative controls (RS) and then analyzed by 3% agarose gel electrophoresis. As a result, as shown in FIG. 7, in which the degree of degradation of the nucleic acid drugs containing the different sequences of DNA G-quadruplexes was different in 50% serum, but the complete DNA sequences which were not degraded were all present, the protection ability against S1 in the binding example 1, demonstrated that the nucleic acid drugs containing the different sequences of DNA G-quadruplexes were stable in 50% serum.

In example 7, sequence information of the sequence used is as follows:

RS: GGATAGGGATTCTGTTGGTCGGCTGGTTGGTATCC (SEQ ID NO: 140)

S5: TGCCTTTAGGATTCTAGACA (SEQ ID NO: 144)

S6: ATGGAGGTATGACATATAAT (SEQ ID NO: 145)

S7: TACATTGCCTCTTCATT (SEQ ID NO: 146)

S8: TTCCGAATAAACTCCAGGC (SEQ ID NO: 147)

S9: AAGCAAAACAGGTCTAGAA (SEQ ID NO: 148)

S10: GTGTCTAGGAGATACACCT (SEQ ID NO: 149)

S11: TTCCGAATAAACTCCAGGCT (SEQ ID NO: 150)

S12: AAGCAAAACAGGTCTAGAAT (SEQ ID NO: 151)

S13: GTGTCTAGGAGATACACCTT (SEQ ID NO: 152)。

example 8: assessment of stability of RNA drugs containing different sequences of RNA G-quadruplexes in 50% serum

The stability of RNA drugs containing different sequences of RNA G-quadruplexes in 50% serum was evaluated. G-quadruplexes (G1-G10 and G1-G1-22 converted into RNA) are added at two ends of an RNA drug, directly transcribed into complete RNA (RNA-1 and RNA-2) in vitro, and then incubated in 50% serum for 3h, 6 h and 12 h respectively, wherein RNA without the G-quadruplexes is used as a control (Rs-RNA); the results were then analyzed by 3% agarose gel electrophoresis. As a result, as shown in FIG. 8, rs-RNA was degraded substantially all after 3 hours of incubation in serum, while RNA-1 and RNA-2 remained largely intact after 12 h of incubation in serum, indicating that RNA drugs containing different sequences of RNA G-quadruplexes were stable in 50% serum.

In example 8, sequence information of the sequence used is as follows:

RS-RNA:

UCUAUCUAUCUGUCGAGUAGAGUGUGGGCUCUUUUGGAAGAAACUGUGGCACUUCGGUGCCAG (SEQ ID NO: 153)

RNA1:

GGGUAGGGCGGGUUGGGUUUGCCUUUAGGAUUCUAGACAUUUGGGUAGGGCGGGUUGGG (SEQ ID NO: 154)

RNA2: GGGUUGGGUUUGGGUUGGGUUUGCCUUUAGGAUUCUAGACAUUUGGGUUGGGUUUGGGUUGGG (SEQ ID NO: 155)。

example 9: assessment of stability of long-chain RNA drugs containing RNA G-quadruplex sequences in serum

The stability of long-chain RNAs (including mRNA and lncRNA) containing RNA G-quadruplex (G1-G10 and G1-G1-22 converted to RNA) in serum was evaluated, wherein RNAs with lengths of 5000nt and 2000nt were selected as targets, long-chain RNAs containing RNA G-quadruplex were named L-RNAs, which were incubated in 10% serum for 10 min and 30min, respectively, and long-chain RNAs without RNA G-quadruplex were used as Negative Controls (NC); the results were then analyzed by 3% agarose gel electrophoresis. As a result, as shown in FIG. 8, NC was degraded substantially all after 10 min incubation in serum, while L-RNA remained largely intact after 10 min incubation in serum, indicating that the G-quadruplex long-chain RNA drug containing RNA was stable in serum.

In example 9, sequence information of the sequence used is as follows:

L-RNA:

GGGUUGGGUUUGGGUUGGGUUAUUUUUAAGAGCUGUGGAGUUCUUAAAUAUCAACCAUGGCACUUUCUCCUGACCCCUUCCCUAGGGGAUUUCAGGAUUGAGAAAUUUUUCCAUCGAGCCUUUUUAAAAUUGUAGGACUUGUUCCUGUGGGCUUCAGUGAUGGGAUAGUACACUUCACUCAGAGGCAUUUGCAUCUUUAAAUAAUUUCUUAAAAGCCUCUAAAGUGAUCAGUGCCUUGAUGCCAACUAAGGAAAUUUGUUUAGCAUUGAAUCUCUGAAGGCUCUAUGAAAGGAAUAGCAUGAUGUGCUGUUAGAAUCAGAUGUUACUGCUAAAAUUUACAUGUUGUGAUGUAAAUUGUGUAGAAAACCAUUAAAUCAUUCAAAAUAAUAAACUAUUUUUAUUAGAGAAUGUAUACUUUUAGAAAGCUGUCUCCUUAUUUAAAUAAAAUAGUGUUUGUCUGUAGUUCAGUGUUGGGGCAAUCUUGGGGGGGAUUCUUCUCUAAUCUUUCAGAAACUUUGUCUGCGAACACUCUUUAAUGGACCAGAUCAGGAUUUGAGCGGAAGAACGAAUGUAACUUUAAGGCAGGAAAGACAAAUUUUAUUCUUCAUAAAGUGAUGAGCAUAUAAUAAUUCCAGGCACAUGGCAAUAGAGGCCCUCUAAAUAAGGAAUAAAUAACCUCUUAGACAGGUGGGAGAUUAUGAUCAGAGUAAAAGGUAAUUACACAUUUUAUUUCCAGAAAGUCAGGGGUCUAUAAAUUGACAGUGAUUAGAGUAAUACUUUUUCACAUUUCCAAAGUUUGCAUGUUAACUUUAAAUGCUUACAAUCUUAGAGUGGUAGGCAAUGUUUUACACUAUUGACCUUAUAUAGGGAAGGGAGGGGGUGCCUGUGGGGUUUUAAAGAAUUUUCCUUUGCAGAGGCAUUUCAUCCUUCAUGAAGCCAUUCAGGAUUUUGAAUUGCAUAUGAGUGCUUGGCUCUUCCUUCUGUUCUAGUGAGUGUAUGAGACCUUGCAGUGAGUUUAUCAGCAUACUCAAAAUUUUUUUCCUGGAAUUUGGAGGGAUGGGAGGAGGGGGUGGGGCUUACUUGUUGUAGCUUUUUUUUUUUUUACAGACUUCACAGAGAAUGCAGUUGUCUUGACUUCAGGUCUGUCUGUUCUGUUGGCAAGUAAAUGCAGUACUGUUCUGAUCCCGCUGCUAUUAGAAUGCAUUGUGAAACGACUGGAGUAUGAUUAAAAGUUGUGUUCCCCAAUGCUUGGAGUAGUGAUUGUUGAAGGAAAAAAUCCAGCUGAGUGAUAAAGGCUGAGUGUUGAGGAAAUUUCUGCAGUUUUAAGCAGUCGUAUUUGUGAUUGAAGCUGAGUACAUUUUGCUGGUGUAUUUUUAGGUAAAAUGCUUUUUGUUCAUUUCUGGUGGUGGGAGGGGACUGAAGCCUUUAGUCUUUUCCAGAUGCAACCUUAAAAUCAGUGACAAGAAACAUUCCAAACAAGCAACAGUCUUCAAGAAAUUAAACUGGCAAGUGGAAAUGUUUAAACAGUUCAGUGAUCUUUAGUGCAUUGUUUAUGUGUGGGUUUCUCUCUCCCCUCCCUUGGUCUUAAUUCUUACAUGCAGGAACACUCAGCAGACACACGUAUGCGAAGGGCCAGAGAAGCCAGACCCAGUAAGAAAAAAUAGCCUAUUUACUUUAAAUAAACCAAACAUUCCAUUUUAAAUGUGGGGAUUGGGAACCACUAGUUCUUUCAGAUGGUAUUCUUCAGACUAUAGAAGGAGCUUCCAGUUGAAUUCACCAGUGGACAAAAUGAGGAAAACAGGUGAACAAGCUUUUUCUGUAUUUACAUACAAAGUCAGAUCAGUUAUGGGACAAUAGUAUUGAAUAGAUUUCAGCUUUAUGCUGGAGUAACUGGCAUGUGAGCAAACUGUGUUGGCGUGGGGGUGGAGGGGUGAGGUGGGCGCUAAGCCUUUUUUUAAGAUUUUUCAGGUACCCCUCACUAAAGGCACCGAAGGCUUAAAGUAGGACAACCAUGGAGCCGGGUUGGGUUUGGGUUGGG (SEQ ID NO: 156)

NC：

UUAUUUUUAAGAGCUGUGGAGUUCUUAAAUAUCAACCAUGGCACUUUCUCCUGACCCCUUCCCUAGGGGAUUUCAGGAUUGAGAAAUUUUUCCAUCGAGCCUUUUUAAAAUUGUAGGACUUGUUCCUGUGGGCUUCAGUGAUGGGAUAGUACACUUCACUCAGAGGCAUUUGCAUCUUUAAAUAAUUUCUUAAAAGCCUCUAAAGUGAUCAGUGCCUUGAUGCCAACUAAGGAAAUUUGUUUAGCAUUGAAUCUCUGAAGGCUCUAUGAAAGGAAUAGCAUGAUGUGCUGUUAGAAUCAGAUGUUACUGCUAAAAUUUACAUGUUGUGAUGUAAAUUGUGUAGAAAACCAUUAAAUCAUUCAAAAUAAUAAACUAUUUUUAUUAGAGAAUGUAUACUUUUAGAAAGCUGUCUCCUUAUUUAAAUAAAAUAGUGUUUGUCUGUAGUUCAGUGUUGGGGCAAUCUUGGGGGGGAUUCUUCUCUAAUCUUUCAGAAACUUUGUCUGCGAACACUCUUUAAUGGACCAGAUCAGGAUUUGAGCGGAAGAACGAAUGUAACUUUAAGGCAGGAAAGACAAAUUUUAUUCUUCAUAAAGUGAUGAGCAUAUAAUAAUUCCAGGCACAUGGCAAUAGAGGCCCUCUAAAUAAGGAAUAAAUAACCUCUUAGACAGGUGGGAGAUUAUGAUCAGAGUAAAAGGUAAUUACACAUUUUAUUUCCAGAAAGUCAGGGGUCUAUAAAUUGACAGUGAUUAGAGUAAUACUUUUUCACAUUUCCAAAGUUUGCAUGUUAACUUUAAAUGCUUACAAUCUUAGAGUGGUAGGCAAUGUUUUACACUAUUGACCUUAUAUAGGGAAGGGAGGGGGUGCCUGUGGGGUUUUAAAGAAUUUUCCUUUGCAGAGGCAUUUCAUCCUUCAUGAAGCCAUUCAGGAUUUUGAAUUGCAUAUGAGUGCUUGGCUCUUCCUUCUGUUCUAGUGAGUGUAUGAGACCUUGCAGUGAGUUUAUCAGCAUACUCAAAAUUUUUUUCCUGGAAUUUGGAGGGAUGGGAGGAGGGGGUGGGGCUUACUUGUUGUAGCUUUUUUUUUUUUUACAGACUUCACAGAGAAUGCAGUUGUCUUGACUUCAGGUCUGUCUGUUCUGUUGGCAAGUAAAUGCAGUACUGUUCUGAUCCCGCUGCUAUUAGAAUGCAUUGUGAAACGACUGGAGUAUGAUUAAAAGUUGUGUUCCCCAAUGCUUGGAGUAGUGAUUGUUGAAGGAAAAAAUCCAGCUGAGUGAUAAAGGCUGAGUGUUGAGGAAAUUUCUGCAGUUUUAAGCAGUCGUAUUUGUGAUUGAAGCUGAGUACAUUUUGCUGGUGUAUUUUUAGGUAAAAUGCUUUUUGUUCAUUUCUGGUGGUGGGAGGGGACUGAAGCCUUUAGUCUUUUCCAGAUGCAACCUUAAAAUCAGUGACAAGAAACAUUCCAAACAAGCAACAGUCUUCAAGAAAUUAAACUGGCAAGUGGAAAUGUUUAAACAGUUCAGUGAUCUUUAGUGCAUUGUUUAUGUGUGGGUUUCUCUCUCCCCUCCCUUGGUCUUAAUUCUUACAUGCAGGAACACUCAGCAGACACACGUAUGCGAAGGGCCAGAGAAGCCAGACCCAGUAAGAAAAAAUAGCCUAUUUACUUUAAAUAAACCAAACAUUCCAUUUUAAAUGUGGGGAUUGGGAACCACUAGUUCUUUCAGAUGGUAUUCUUCAGACUAUAGAAGGAGCUUCCAGUUGAAUUCACCAGUGGACAAAAUGAGGAAAACAGGUGAACAAGCUUUUUCUGUAUUUACAUACAAAGUCAGAUCAGUUAUGGGACAAUAGUAUUGAAUAGAUUUCAGCUUUAUGCUGGAGUAACUGGCAUGUGAGCAAACUGUGUUGGCGUGGGGGUGGAGGGGUGAGGUGGGCGCUAAGCCUUUUUUUAAGAUUUUUCAGGUACCCCUCACUAAAGGCACCGAAGGCUUAAAGUAGGACAACCAUGGAGCC (SEQ ID NO: 157)。

example 10: evaluation of nucleic acid drug chimeras containing RNA G-quadruplexes and stability of nucleic acid drug chimeras containing DNA G-quadruplexes in serum

The stability of nucleic acid drug chimeras containing DNA G-quadruplexes (G1-G10 and G1-G1-22) was evaluated: each DNA G-quadruplex sequence is added at two ends of RS1 (RNA) respectively to form G1-RS1 to G1-22-RS1, so as to form chimeras of DNA and RNA, and the chimeras are incubated in serum for 15min, wherein a nucleic acid chimera without DNA G-quadruplex is adopted as a control (Rs-D+R); the results were then analyzed by 3% agarose gel electrophoresis. Stability of nucleic acid drug chimeras containing RNA G-quadruplexes (represented by G1, RG 1) were evaluated: RG1 sequences are respectively added at two ends of S1 to form RG1-S1, so that chimeras of RNA and DNA are formed, and incubated in serum for 30min, 2h and 4h, wherein chimeras without RNA G-quadruplexes are adopted as a control (Rs-R+D); the results were then analyzed by 3% agarose gel electrophoresis. The results are shown in FIG. 10, wherein the G-quadruplex-containing nucleic acid drug chimeras have a certain stability in serum.

In example 10, sequence information of the sequence used is as follows:

G1-RS1：GGGTTGGGTTTGGGTTGGGUGAAUGUAGAGAUGCGGUGGGGGTTGGGTTTGGGTTGGG (SEQ ID NO: 158)

Rs-D+R:

TCTATCTATCTGTCGAGTAUGAAUGUAGAGAUGCGGUGAAACTGTGGCACTTCGGTGC (SEQ ID NO: 159)

RG1-S1：

GGGUUGGGUUUGGGUUGGGTGAATGTAGAGATGCGGTGGGGGUUGGGUUUGGGUUUGGG (SEQ ID NO: 160)

RS-R+D:

UCUAUCUAUCUGUCGAGUATGAATGTAGAGATGCGGTAGAAACUGUGGCACUUCGGUGA (SEQ ID NO: 161)。

example 11: assessing the knockdown effect of G-quadruplex containing oligonucleotide drugs on target RNA in cells

1. ASO (Su 1, su2, MA1, MA2, PC1 and PC 2) protected by Transeasy transfection of G-quadruplex (G1-G10 and G1-G1-22): a proper amount of ASO was diluted with 50. Mu.L of Opti-MEM, while 4.8. Mu.L of Transeasy was diluted with 50. Mu.L of Opti-MEM, and the mixture was gently stirred and stirred at room temperature for 5 minutes. The DNA dilutions were then added to the Transeasy dilutions to form a mixture, which was incubated for 20min at room temperature. Meanwhile, the cells of the 12-pore plate are replaced by a culture medium without double antibodies, the mixed liquid is dripped into the cells, the mixture is gently mixed, and the complete culture medium containing serum and double antibodies is replaced after 5-6 hours.

2. Extraction of total RNA of cells: since RNA is easily degraded and there is a large amount of exogenous RNase in the air and the experimental vessel, the RNase-free gun head and tube should be used in the operation. The glassware used in the experimental process needs to be baked for more than 4 hours at a high temperature of 160 ℃; the plastic vessel is treated with 0.5. 0.5M NaOH solution for 10 min, then cleaned with RNase-free water and autoclaved.

(1) The cells were harvested when the adherent cell density was about 80%, the medium was discarded, and the cells were rinsed once with pre-chilled 1 XPBS.

(2) 1mL of Trizol was added to the cells of the 6-well plate, and the cell amount of the 12-well plate was halved. And (5) blowing and uniformly mixing by using a pipette.

(3) 200. Mu.L of chloroform was added to each 1mL of Trizol, the tube was capped, and the mixture was thoroughly mixed by shaking vigorously, and allowed to stand at room temperature for 2min, followed by centrifugation at 12000g for 10min at 4 ℃.

(4) About 400. Mu.L of the aqueous layer was pipetted into a new EP tube, mixed with 500. Mu.L of isopropanol, placed in a low temperature refrigerator at-80℃for more than half an hour, and centrifuged at 12000g for 15min at 4 ℃.

(5) After centrifugation, a white precipitate was seen at the bottom of the tube, which was RNA. The supernatant was decanted, 1mL of 75% ethanol was added and the pellet was resuspended and then centrifuged at 8500g for 10min at 4 ℃.

(6) The ethanol was aspirated off, taking care not to aspirate the precipitate. The tube was allowed to stand at room temperature, and after the ethanol had evaporated, 50. Mu.L of RNase-free H was added₂ O, and incubated at 42℃for 2min to allow sufficient dissolution of RNA. A small amount of RNA was then removed to determine its concentration and the RNA integrity was checked by TBE electrophoresis.

3. Real-time fluorescent quantitative PCR:

(1) The cDNA was removed from the-20deg.C refrigerator, dissolved at room temperature, and 180. Mu.L of ddH was added₂ O was diluted 10-fold.

(2) The qPCR reaction system is as follows:

the PCR reaction conditions were: 95 ℃ for 2min;95℃5s,58℃5s,72℃15s (40 cycles of this procedure); 95 ℃ for 15s;60 s at 60 ℃; the dissolution curve is 60-95 ℃, and the fluorescence signal is absorbed once when the dissolution curve rises by 0.5 ℃; and 95 ℃ for 10s.

(3) PCR was performed using a ABI step one plus quantitative instrument and experimental data was analyzed according to the results using the ΔΔct algorithm as shown in fig. 11.

Wherein the ASO targets are respectively RNASurvivin、Malat1AndPCSK9three genes, respectively designing two corresponding ASOs for each gene, namely Su1, su2, MA1, MA2, PC1 and PC2, respectively testing the knockdown capability of the genes at the cellular level, wherein Su1, su2, MA1 and MA2 are tested in two cells, including A549 cells and Hela cells; PC1 and PC2 were tested on 97H cells. The untransfected group (Mock) was also set as a negative control. The results show that the oligonucleotide drug containing the G-quadruplex has good knocking-down ability in cells.

In example 11, sequence information of the sequence used is as follows:

Su1: GAATGTAGAGATGCGGTGG (SEQ ID NO: 135)

Su2: TGAGGGCGAATCAAATCCATC (SEQ ID NO: 162)

MA1: TGCCTTTAGGATTCTAGACA (SEQ ID NO: 144)

MA2: ATGGAGGTATGACATATAAT (SEQ ID NO: 145)

MA3: ACATTGCCTCTTCATT (SEQ ID NO: 163)

PC1: GTCTGTGGAAGCG (SEQ ID NO: 164)

PC2: GCCTGTCTGTGGAAGCGGGT (SEQ ID NO: 165)。

example 12: PCSK9 (a) knockdown and LDL (b) reduction in animals using G-quadruplex-containing antisense oligonucleotides targeting PCSK9

In order to evaluate the effectiveness of antisense oligonucleotides containing G-quadruplexes (G1-G10 and G1-22) in animals, the inventors designed ASO sequences targeting PCSK9, and designed fully thio-modified PS-ASO as a control experiment to compare the effectiveness of two different protection methods in animals. Prior to injection, the "ssDNA" required for the experiment was diluted with physiological saline. Mice were weighed in groups and numbered: PS-ASO (0.8. Mu. Mol/kg) and LT-001 (a sequence formed by adding G1 to both ends of the ASO sequence of PCSK 9) (0.8. Mu. Mol/kg) were combined in 2 groups. The injection mode adopts the high-pressure injection of the tail vein of the mouse, uses a 2ml empty needle tube and a 4.5-numbered needle to carry out injection, and injects diluted ssDNA physiological saline solution. The total amount of physiological saline solution injected into each mouse is 8% -10% (volume/body weight) of the body weight, the total volume is about 2ml, and the injection process is completed within 5-8 seconds. Tail cutting blood collection was performed everyday 3 days before injection, followed by 5 th and 30 th days.

The ELISA method is adopted to detect the contents of PCSK9 and LDL-c (low density lipoprotein cholesterol) in the plasma of mice respectively, and the effect of the targeting PCSK9 antisense oligonucleotide protected by the method for knocking down the PCSK9 in animals is examined. The results are shown in FIG. 12, where LT-001 injected mice had significantly lower values of PCSK9 and LDL-c compared to PS-ASO injected and more than PS-ASO; the ability to knock down PCSK9 and reduce LDL levels well in animals using G-quadruplex containing targeted PCSK9 antisense oligonucleotides is demonstrated.

Sequence information for PCSK 9-targeting antisense oligonucleotides described in the examples: GTCTGTGGAAGCG (SEQ ID NO: 164); PS-ASO is a modified form of the total phosphorothioate of this sequence.

The above description is only of the preferred embodiments of the present invention, and is not intended to limit the invention in any way, and some simple modifications, equivalent variations or modifications can be made by those skilled in the art using the teachings disclosed herein, which fall within the scope of the present invention.

<110> Chengdu Ling Tai Krypton Biotechnology Co., ltd

<120> G-quadruplex-containing nucleic acid

<130> CPCH2162766N

<160> 166

<170> PatentIn version 3.3

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<210> 16

<211> 70

<212> DNA

<213> artificial sequence

<400> 16

ttggggttag ggttagggtt agggatgaat gtagagatgc ggtggttggg gttagggtta 60

gggttaggga 70

<210> 17

<211> 56

<212> DNA

<213> artificial sequence

<400> 17

ttgggtgggt gggtgggttg aatgtagaga tgcggtggtt gggtgggtgg gtgggt 56

<210> 18

<211> 52

<212> DNA

<213> artificial sequence

<400> 18

ggggtgggag gagggttgaa tgtagagatg cggtgggggg tgggaggagggt 52

<210> 19

<211> 44

<212> DNA

<213> artificial sequence

<400> 19

tccacgcaca gttgaatgta gagatgcggt ggtccacgca cagt 44

<210> 20

<211> 48

<212> DNA

<213> artificial sequence

<400> 20

tttccacgca cagttgaatg tagagatgcg gtggtttcca cgcacagt 48

<210> 21

<211> 52

<212> DNA

<213> artificial sequence

<400> 21

gctttccacg cacagttgaa tgtagagatg cggtgggctt tccacgcacagt 52

<210> 22

<211> 56

<212> DNA

<213> artificial sequence

<400> 22

acgctttcca cgcacagttg aatgtagaga tgcggtggac gctttccacg cacagt 56

<210> 23

<211> 59

<212> DNA

<213> artificial sequence

<400> 23

ctacgctttc cacgcacagt tgaatgtaga gatgcggtgc tacgctttcc acgcacagt 59

<210> 24

<211> 36

<212> DNA

<213> artificial sequence

<400> 24

tttccacgtg aatgtagaga tgcggtggtt tccacg 36

<210> 25

<211> 32

<212> DNA

<213> artificial sequence

<400> 25

ctacgctgaa tgtagagatg cggtggctac gc 32

<210> 26

<211> 36

<212> DNA

<213> artificial sequence

<400> 26

ctacgctttg aatgtagaga tgcggtggct acgctt 36

<210> 27

<211> 40

<212> DNA

<213> artificial sequence

<400> 27

ctaccgtttc tgaatgtaga gatgcggtgg ctaccgtttc 40

<210> 28

<211> 44

<212> DNA

<213> artificial sequence

<400> 28

ctacgctttc catgaatgta gagatgcggt ggctacgctt tcca 44

<210> 29

<211> 48

<212> DNA

<213> artificial sequence

<400> 29

ctacgctttc cacgtgaatg tagagatgcg gtggctacgc tttccacg 48

<210> 30

<211> 52

<212> DNA

<213> artificial sequence

<400> 30

ctacgctttc cacgcatgaa tgtagagatg cggtggctac gctttccacgca 52

<210> 31

<211> 56

<212> DNA

<213> artificial sequence

<400> 31

ctacgctttc cacgcacatg aatgtagaga tgcggtggct acgctttcca cgcaca 56

<210> 32

<211> 40

<212> DNA

<213> artificial sequence

<400> 32

ctttccacgc tgaatgtaga gatgcggtgg ctttccacgc 40

<210> 33

<211> 44

<212> DNA

<213> artificial sequence

<400> 33

gctttccacg catgaatgta gagatgcggt gggctttcca cgca 44

<210> 34

<211> 48

<212> DNA

<213> artificial sequence

<400> 34

cgctttccac gcactgaatg tagagatgcg gtggcgcttt ccacgcac 48

<210> 35

<211> 52

<212> DNA

<213> artificial sequence

<400> 35

acgctttcca cgcacatgaa tgtagagatg cggtggacgc tttccacgcaca 52

<210> 36

<211> 25

<212> DNA

<213> artificial sequence

<400> 36

tttgccttta ggattctaga cattt 25

<210> 37

<211> 8

<212> DNA

<213> artificial sequence

<400> 37

agacattt 8

<210> 38

<211> 13

<212> DNA

<213> artificial sequence

<400> 38

attctagaca ttt 13

<210> 39

<211> 44

<212> DNA

<213> artificial sequence

<400> 39

tttgccttta ggagggttgg gtttgggttg ggttctagac attt 44

<210> 40

<211> 88

<212> DNA

<213> artificial sequence

<400> 40

tttgccttta ggagggttgg gtttgggttg ggtttgcctt taggattcta gacatttggg 60

ttgggtttgg gttgggttct agacattt 88

<210> 41

<211> 19

<212> DNA

<213> artificial sequence

<400> 41

gggtttgggt gggtttggg 19

<210> 42

<211> 16

<212> DNA

<213> artificial sequence

<400> 42

gggtgggtgg gtgggt 16

<210> 43

<211> 26

<212> DNA

<213> artificial sequence

<400> 43

ggtggtggtg gttgtggtgg tggtgg 26

<210> 44

<211> 24

<212> DNA

<213> artificial sequence

<400> 44

gtggggcatt gtgggtgggt gtgg 24

<210> 45

<211> 22

<212> DNA

<213> artificial sequence

<400> 45

agggttaggg ttagggttag gg 22

<210> 46

<211> 15

<212> DNA

<213> artificial sequence

<400> 46

ggttggtgtg gttgg 15

<210> 47

<211> 19

<212> DNA

<213> artificial sequence

<400> 47

gggtagggcg gggttgggg 19

<210> 48

<211> 29

<212> DNA

<213> artificial sequence

<400> 48

ggtggtggtg gttgtggtgg gtgggtggg 29

<210> 49

<211> 52

<212> DNA

<213> artificial sequence

<400> 49

ggtggtggtg gttgtggtgg tggtggggtg gtggtggttg tggtggtggt gg 52

<210> 50

<211> 17

<212> DNA

<213> artificial sequence

<400> 50

ggttaggtta ggttagg 17

<210> 51

<211> 28

<212> DNA

<213> artificial sequence

<400> 51

ggggttttgg ggttttgggg ttttgggg 28

<210> 52

<211> 20

<212> DNA

<213> artificial sequence

<400> 52

ggcttaggct taggcttagg 20

<210> 53

<211> 24

<212> DNA

<213> artificial sequence

<400> 53

gggttcaggg ttcagggttc aggg 24

<210> 54

<211> 23

<212> DNA

<213> artificial sequence

<400> 54

agggttaggg ttagggttag ggt 23

<210> 55

<211> 21

<212> DNA

<213> artificial sequence

<400> 55

gggctagggc tagggctaggg 21

<210> 56

<211> 22

<212> DNA

<213> artificial sequence

<400> 56

tgagggtggg tagggtgggtaa 22

<210> 57

<211> 21

<212> DNA

<213> artificial sequence

<400> 57

gggagggcgc tgggaggaggg 21

<210> 58

<211> 20

<212> DNA

<213> artificial sequence

<400> 58

gggcgggcgc gagggagggg 20

<210> 59

<211> 28

<212> DNA

<213> artificial sequence

<400> 59

acaggggtgt ggggacaggg gtgtgggg 28

<210> 60

<211> 20

<212> DNA

<213> artificial sequence

<400> 60

gcgggcggct cgggcgcggg 20

<210> 61

<211> 17

<212> DNA

<213> artificial sequence

<400> 61

gggagggttg gggtggg 17

<210> 62

<211> 19

<212> DNA

<213> artificial sequence

<400> 62

gggcagggag ggaactggg 19

<210> 63

<211> 18

<212> DNA

<213> artificial sequence

<400> 63

gggagggttg gggtgggt 18

<210> 64

<211> 20

<212> DNA

<213> artificial sequence

<400> 64

gggcagggag ggaactgggt 20

<210> 65

<211> 27

<212> DNA

<213> artificial sequence

<400> 65

gggcggtgtg ggaagaggga agagggg 27

<210> 66

<211> 16

<212> DNA

<213> artificial sequence

<400> 66

ggttggtgtg gttggt 16

<210> 67

<211> 12

<212> DNA

<213> artificial sequence

<400> 67

gggcttttgg gc 12

<210> 68

<211> 26

<212> DNA

<213> artificial sequence

<400> 68

tttaagggtt agggttaggg ttaggg 26

<210> 69

<211> 24

<212> DNA

<213> artificial sequence

<400> 69

tgagggtggt gagggtgggg aagg 24

<210> 70

<211> 23

<212> DNA

<213> artificial sequence

<400> 70

tgagggtggg tagggtgggtaat 23

<210> 71

<211> 24

<212> DNA

<213> artificial sequence

<400> 71

tgagggtggg agggtgggga agga 24

<210> 72

<211> 23

<212> DNA

<213> artificial sequence

<400> 72

gggcgcggga ggaattgggc ggg 23

<210> 73

<211> 22

<212> DNA

<213> artificial sequence

<400> 73

agggagggcg ctgggaggag gg 22

<210> 74

<211> 21

<212> DNA

<213> artificial sequence

<400> 74

cgggcgggcg cgagggagggt 21

<210> 75

<211> 21

<212> DNA

<213> artificial sequence

<400> 75

cgggcgggcg ctagggagggt 21

<210> 76

<211> 12

<212> DNA

<213> artificial sequence

<400> 76

ggaggaggag ga 12

<210> 77

<211> 17

<212> DNA

<213> artificial sequence

<400> 77

ggttggtgtg gttggtt 17

<210> 78

<211> 27

<212> DNA

<213> artificial sequence

<400> 78

ggtggtggtg gttgtggtgg tggtggt 27

<210> 79

<211> 16

<212> DNA

<213> artificial sequence

<400> 79

gggcgggcgg gcgggc 16

<210> 80

<211> 25

<212> DNA

<213> artificial sequence

<400> 80

tgtgggggtg gacgggccgg gtaga 25

<210> 81

<211> 7

<212> DNA

<213> artificial sequence

<400> 81

ttgggtt 7

<210> 82

<211> 6

<212> DNA

<213> artificial sequence

<400> 82

tgggag 6

<210> 83

<211> 16

<212> DNA

<213> artificial sequence

<400> 83

ggggtgggag gagggt 16

<210> 84

<211> 18

<212> DNA

<213> artificial sequence

<400> 84

ggttggtgtg gttggttt 18

<210> 85

<211> 24

<212> DNA

<213> artificial sequence

<400> 85

atggggtcgg gcgggccggg tgtc 24

<210> 86

<211> 18

<212> DNA

<213> artificial sequence

<400> 86

gtgggtaggg cgggttgg 18

<210> 87

<211> 20

<212> DNA

<213> artificial sequence

<400> 87

gggttagggt tagggtaggg 20

<210> 88

<211> 25

<212> DNA

<213> artificial sequence

<400> 88

cctgggggag tattgcggag gaagg 25

<210> 89

<211> 32

<212> DNA

<213> artificial sequence

<400> 89

agggcggtgt gggaagaggg aagaggggga gg 32

<210> 90

<211> 35

<212> DNA

<213> artificial sequence

<400> 90

agggcggtgt gggaagaggg aagaggggga ggcag 35

<210> 91

<211> 39

<212> DNA

<213> artificial sequence

<400> 91

cggtcgctcc gtgtggcttg ggttgggtgt ggcagtgac 39

<210> 92

<211> 31

<212> DNA

<213> artificial sequence

<400> 92

gggggtggga gggtaggcct taggtttctg a 31

<210> 93

<211> 37

<212> DNA

<213> artificial sequence

<400> 93

cgcctgatta gcgatactca gcgttggggg ggggggg 37

<210> 94

<211> 36

<212> DNA

<213> artificial sequence

<400> 94

gggcgtggtg ggtggggtac taataatgtg cgtttg 36

<210> 95

<211> 30

<212> DNA

<213> artificial sequence

<400> 95

agcgggcata tggtggtggg tggtatggtc 30

<210> 96

<211> 36

<212> DNA

<213> artificial sequence

<400> 96

aacacatagg tttggttagg ttggttggtt gaatta 36

<210> 97

<211> 30

<212> DNA

<213> artificial sequence

<400> 97

ttgcgcgtta attggggggg tgggtgggtt 30

<210> 98

<211> 30

<212> DNA

<213> artificial sequence

<400> 98

ggtggtgggg ggggttggta gggtgtcttc 30

<210> 99

<211> 36

<212> DNA

<213> artificial sequence

<400> 99

gatcgggtgt gggtggcgta aagggagcat cggaca 36

<210> 100

<211> 45

<212> DNA

<213> artificial sequence

<400> 100

gggttagggt tagggttagg gttagggtta gggttagggt taggg 45

<210> 101

<211> 47

<212> DNA

<213> artificial sequence

<400> 101

ggggcgctta tggggagggt ggggagggtg gggaaggtgg ggaggag 47

<210> 102

<211> 47

<212> DNA

<213> artificial sequence

<400> 102

ccccccgggg cgggccgggg gcggggtccc ggcggggcgg agccatg 47

<210> 103

<211> 47

<212> DNA

<213> artificial sequence

<400> 103

ggcggggggg ggggggcggg ggcgggggcg ggggaggggc gcggcgg 47

<210> 104

<211> 47

<212> DNA

<213> artificial sequence

<400> 104

cgcggagggg cgggcgcggg aggaaggggg cgggagcggg gctgtgg 47

<210> 105

<211> 47

<212> DNA

<213> artificial sequence

<400> 105

gaggagcggg taggggcggg gcggggcggg ggcggtccag gggtggg 47

<210> 106

<211> 47

<212> DNA

<213> artificial sequence

<400> 106

cgctcgggcg cgcggggagg ggagaggggg cgggagcgcg cccgctc 47

<210> 107

<211> 47

<212> DNA

<213> artificial sequence

<400> 107

agaggaggag gaggtcacgg aggaggagga gaaggaggag gaggaaa 47

<210> 108

<211> 47

<212> DNA

<213> artificial sequence

<400> 108

ggggcgctta tggggagggt gggtagggtg ggtaaggtgg ggaggag 47

<210> 109

<211> 47

<212> DNA

<213> artificial sequence

<400> 109

ggggcgctta tggggagggt gggtagggtt gggaaggtgg ggaggag 47

<210> 110

<211> 17

<212> DNA

<213> artificial sequence

<400> 110

gggaagggag ggaaggg 17

<210> 111

<211> 17

<212> DNA

<213> artificial sequence

<400> 111

gggttgggtg ggttggg 17

<210> 112

<211> 17

<212> DNA

<213> artificial sequence

<400> 112

gggccgggcg ggccggg 17

<210> 113

<211> 18

<212> DNA

<213> artificial sequence

<400> 113

gggttgggtt gggttggg 18

<210> 114

<211> 17

<212> DNA

<213> artificial sequence

<400> 114

gggtagggcg ggttggg 17

<210> 115

<211> 15

<212> DNA

<213> artificial sequence

<400> 115

gggtgggtgg gtggg 15

<210> 116

<211> 16

<212> DNA

<213> artificial sequence

<400> 116

gggtgggttg ggtggg 16

<210> 117

<211> 17

<212> DNA

<213> artificial sequence

<400> 117

gggtgggttt gggtggg 17

<210> 118

<211> 21

<212> DNA

<213> artificial sequence

<400> 118

gggtttgggt ttgggtttggg 21

<210> 119

<211> 24

<212> DNA

<213> artificial sequence

<400> 119

gggttttggg ttttgggttt tggg 24

<210> 120

<211> 27

<212> DNA

<213> artificial sequence

<400> 120

gggtttttgg gtttttgggt ttttggg 27

<210> 121

<211> 23

<212> DNA

<213> artificial sequence

<400> 121

ggggttgggg tttggggttg ggg 23

<210> 122

<211> 27

<212> DNA

<213> artificial sequence

<400> 122

gggggttggg ggtttggggg ttggggg 27

<210> 123

<211> 31

<212> DNA

<213> artificial sequence

<400> 123

ggggggttgg ggggtttggg gggttgggggg 31

<210> 124

<211> 35

<212> DNA

<213> artificial sequence

<400> 124

gggggggttg ggggggtttg ggggggttgg ggggg 35

<210> 125

<211> 39

<212> DNA

<213> artificial sequence

<400> 125

ggggggggtt ggggggggtt tggggggggt tgggggggg 39

<210> 126

<211> 43

<212> DNA

<213> artificial sequence

<400> 126

gggggggggt tggggggggg tttggggggg ggttgggggg ggg 43

<210> 127

<211> 47

<212> DNA

<213> artificial sequence

<400> 127

gggggggggg ttgggggggg ggtttggggg gggggttggg ggggggg 47

<210> 128

<211> 22

<212> DNA

<213> artificial sequence

<400> 128

gggtagggcg ggttgggtag gg 22

<210> 129

<211> 26

<212> DNA

<213> artificial sequence

<400> 129

gggtagggcg ggttgggtag ggcggg 26

<210> 130

<211> 31

<212> DNA

<213> artificial sequence

<400> 130

gggtagggcg ggttgggtag ggcgggttggg 31

<210> 131

<211> 34

<212> DNA

<213> artificial sequence

<400> 131

gggtagggcg ggttgggggg tagggcgggt tggg 34

<210> 132

<211> 16

<212> DNA

<213> artificial sequence

<400> 132

aatgtagaga tgcggt 16

<210> 133

<211> 17

<212> DNA

<213> artificial sequence

<400> 133

aatgtagaga tgcggtg 17

<210> 134

<211> 18

<212> DNA

<213> artificial sequence

<400> 134

gaatgtagag atgcggtg 18

<210> 135

<211> 19

<212> DNA

<213> artificial sequence

<400> 135

gaatgtagag atgcggtgg 19

<210> 136

<211> 20

<212> DNA

<213> artificial sequence

<400> 136

tgaatgtaga gatgcggtgg 20

<210> 137

<211> 25

<212> DNA

<213> artificial sequence

<400> 137

tttgaatgta gagatgcggt ggttt 25

<210> 138

<211> 30

<212> DNA

<213> artificial sequence

<400> 138

tttagattat atgtcatacc tccattggtt 30

<210> 139

<211> 33

<212> DNA

<213> artificial sequence

<400> 139

tttggacggt gttaaacgaa aggggacgac ctt 33

<210> 140

<211> 35

<212> DNA

<213> artificial sequence

<400> 140

ggatagggat tctgttggtc ggctggttgg tatcc 35

<210> 141

<211> 50

<212> DNA

<213> artificial sequence

<400> 141

atcaggctgg atggtagctc ggtcggggtg ggtgggttgg caagtctgat 50

<210> 142

<211> 80

<212> DNA

<213> artificial sequence

<400> 142

gctgtgtgac tcctgcaaag tgtggacaac ttcccacgga ggaattcccg tatctaaagg 60

tgcagctgta tcttgtctcc 80

<210> 143

<211> 200

<212> DNA

<213> artificial sequence

<400> 143

aacaccggtt tgccatgtgt tgccatgtgt atgtggggag acggtcgggt ccagatattc 60

gtatctgtcg agtagagtgt gggctcccca catactctga tgatccgaga cggtcgggtc 120

cagatattcg tatctgtcga gtagagtgtg ggctcggatc attcatggca actctgatga 180

tcctctatct atctgtcgag 200

<210> 144

<211> 20

<212> DNA

<213> artificial sequence

<400> 144

tgcctttagg attctagaca 20

<210> 145

<211> 20

<212> DNA

<213> artificial sequence

<400> 145

atggaggtat gacatataat 20

<210> 146

<211> 17

<212> DNA

<213> artificial sequence

<400> 146

tacattgcct cttcatt 17

<210> 147

<211> 19

<212> DNA

<213> artificial sequence

<400> 147

ttccgaataa actccaggc 19

<210> 148

<211> 19

<212> DNA

<213> artificial sequence

<400> 148

aagcaaaaca ggtctagaa 19

<210> 149

<211> 19

<212> DNA

<213> artificial sequence

<400> 149

gtgtctagga gatacacct 19

<210> 150

<211> 20

<212> DNA

<213> artificial sequence

<400> 150

ttccgaataa actccaggct 20

<210> 151

<211> 20

<212> DNA

<213> artificial sequence

<400> 151

aagcaaaaca ggtctagaat 20

<210> 152

<211> 20

<212> DNA

<213> artificial sequence

<400> 152

gtgtctagga gatacacctt 20

<210> 153

<211> 63

<212> RNA

<213> artificial sequence

<400> 153

ucuaucuauc ugucgaguag agugugggcu cuuuuggaag aaacuguggc acuucggugc 60

cag 63

<210> 154

<211> 59

<212> RNA

<213> artificial sequence

<400> 154

ggguagggcg gguuggguuu gccuuuagga uucuagacau uuggguaggg cggguuggg 59

<210> 155

<211> 63

<212> RNA

<213> artificial sequence

<400> 155

ggguuggguu uggguugggu uugccuuuag gauucuagac auuuggguug gguuuggguu 60

ggg 63

<210> 156

<211> 2038

<212> RNA

<213> artificial sequence

<400> 156

ggguuggguu uggguugggu uauuuuuaag agcuguggag uucuuaaaua ucaaccaugg 60

cacuuucucc ugaccccuuc ccuaggggau uucaggauug agaaauuuuu ccaucgagcc 120

uuuuuaaaau uguaggacuu guuccugugg gcuucaguga ugggauagua cacuucacuc 180

agaggcauuu gcaucuuuaa auaauuucuu aaaagccucu aaagugauca gugccuugau 240

gccaacuaag gaaauuuguu uagcauugaa ucucugaagg cucuaugaaa ggaauagcau 300

gaugugcugu uagaaucaga uguuacugcu aaaauuuaca uguugugaug uaaauugugu 360

agaaaaccau uaaaucauuc aaaauaauaa acuauuuuua uuagagaaug uauacuuuua 420

gaaagcuguc uccuuauuua aauaaaauag uguuugucug uaguucagug uuggggcaau 480

cuuggggggg auucuucucu aaucuuucag aaacuuuguc ugcgaacacu cuuuaaugga 540

ccagaucagg auuugagcgg aagaacgaau guaacuuuaa ggcaggaaag acaaauuuua 600

uucuucauaa agugaugagc auauaauaau uccaggcaca uggcaauaga ggcccucuaa 660

auaaggaaua aauaaccucu uagacaggug ggagauuaug aucagaguaa aagguaauua 720

cacauuuuau uuccagaaag ucaggggucu auaaauugac agugauuaga guaauacuuu 780

uucacauuuc caaaguuugc auguuaacuu uaaaugcuua caaucuuaga gugguaggca 840

auguuuuaca cuauugaccu uauauaggga agggaggggg ugccuguggg guuuuaaaga 900

auuuuccuuu gcagaggcau uucauccuuc augaagccau ucaggauuuu gaauugcaua 960

ugagugcuug gcucuuccuu cuguucuagu gaguguauga gaccuugcag ugaguuuauc 1020

agcauacuca aaauuuuuuu ccuggaauuu ggagggaugg gaggaggggg uggggcuuac 1080

uuguuguagc uuuuuuuuuu uuuacagacu ucacagagaa ugcaguuguc uugacuucag 1140

gucugucugu ucuguuggca aguaaaugca guacuguucu gaucccgcug cuauuagaau 1200

gcauugugaa acgacuggag uaugauuaaa aguuguguuc cccaaugcuu ggaguaguga 1260

uuguugaagg aaaaaaucca gcugagugau aaaggcugag uguugaggaa auuucugcag 1320

uuuuaagcag ucguauuugu gauugaagcu gaguacauuu ugcuggugua uuuuuaggua 1380

aaaugcuuuu uguucauuuc uggugguggg aggggacuga agccuuuagu cuuuuccaga 1440

ugcaaccuua aaaucaguga caagaaacau uccaaacaag caacagucuu caagaaauua 1500

aacuggcaag uggaaauguu uaaacaguuc agugaucuuu agugcauugu uuaugugugg 1560

guuucucucu ccccucccuu ggucuuaauu cuuacaugca ggaacacuca gcagacacac 1620

guaugcgaag ggccagagaa gccagaccca guaagaaaaa auagccuauu uacuuuaaau 1680

aaaccaaaca uuccauuuua aaugugggga uugggaacca cuaguucuuu cagaugguau 1740

ucuucagacu auagaaggag cuuccaguug aauucaccag uggacaaaau gaggaaaaca 1800

ggugaacaag cuuuuucugu auuuacauac aaagucagau caguuauggg acaauaguau 1860

ugaauagauu ucagcuuuau gcuggaguaa cuggcaugug agcaaacugu guuggcgugg 1920

ggguggaggg gugagguggg cgcuaagccu uuuuuuaaga uuuuucaggu accccucacu 1980

aaaggcaccg aaggcuuaaa guaggacaac cauggagccg gguuggguuu ggguuggg 2038

<210> 157

<211> 2000

<212> RNA

<213> artificial sequence

<400> 157

uuauuuuuaa gagcugugga guucuuaaau aucaaccaug gcacuuucuc cugaccccuu 60

cccuagggga uuucaggauu gagaaauuuu uccaucgagc cuuuuuaaaa uuguaggacu 120

uguuccugug ggcuucagug augggauagu acacuucacu cagaggcauu ugcaucuuua 180

aauaauuucu uaaaagccuc uaaagugauc agugccuuga ugccaacuaa ggaaauuugu 240

uuagcauuga aucucugaag gcucuaugaa aggaauagca ugaugugcug uuagaaucag 300

auguuacugc uaaaauuuac auguugugau guaaauugug uagaaaacca uuaaaucauu 360

caaaauaaua aacuauuuuu auuagagaau guauacuuuu agaaagcugu cuccuuauuu 420

aaauaaaaua guguuugucu guaguucagu guuggggcaa ucuugggggg gauucuucuc 480

uaaucuuuca gaaacuuugu cugcgaacac ucuuuaaugg accagaucag gauuugagcg 540

gaagaacgaa uguaacuuua aggcaggaaa gacaaauuuu auucuucaua aagugaugag 600

cauauaauaa uuccaggcac auggcaauag aggcccucua aauaaggaau aaauaaccuc 660

uuagacaggu gggagauuau gaucagagua aaagguaauu acacauuuua uuuccagaaa 720

gucagggguc uauaaauuga cagugauuag aguaauacuu uuucacauuu ccaaaguuug 780

cauguuaacu uuaaaugcuu acaaucuuag agugguaggc aauguuuuac acuauugacc 840

uuauauaggg aagggagggg gugccugugg gguuuuaaag aauuuuccuu ugcagaggca 900

uuucauccuu caugaagcca uucaggauuu ugaauugcau augagugcuu ggcucuuccu 960

ucuguucuag ugaguguaug agaccuugca gugaguuuau cagcauacuc aaaauuuuuu 1020

uccuggaauu uggagggaug ggaggagggg guggggcuua cuuguuguag cuuuuuuuuu 1080

uuuuacagac uucacagaga augcaguugu cuugacuuca ggucugucug uucuguuggc 1140

aaguaaaugc aguacuguuc ugaucccgcu gcuauuagaa ugcauuguga aacgacugga 1200

guaugauuaa aaguuguguu ccccaaugcu uggaguagug auuguugaag gaaaaaaucc 1260

agcugaguga uaaaggcuga guguugagga aauuucugca guuuuaagca gucguauuug 1320

ugauugaagc ugaguacauu uugcuggugu auuuuuaggu aaaaugcuuu uuguucauuu 1380

cugguggugg gaggggacug aagccuuuag ucuuuuccag augcaaccuu aaaaucagug 1440

acaagaaaca uuccaaacaa gcaacagucu ucaagaaauu aaacuggcaa guggaaaugu 1500

uuaaacaguu cagugaucuu uagugcauug uuuaugugug gguuucucuc uccccucccu 1560

uggucuuaau ucuuacaugc aggaacacuc agcagacaca cguaugcgaa gggccagaga 1620

agccagaccc aguaagaaaa aauagccuau uuacuuuaaa uaaaccaaac auuccauuuu 1680

aaaugugggg auugggaacc acuaguucuu ucagauggua uucuucagac uauagaagga 1740

gcuuccaguu gaauucacca guggacaaaa ugaggaaaac aggugaacaa gcuuuuucug 1800

uauuuacaua caaagucaga ucaguuaugg gacaauagua uugaauagau uucagcuuua 1860

ugcuggagua acuggcaugu gagcaaacug uguuggcgug gggguggagg ggugaggugg 1920

gcgcuaagcc uuuuuuuaag auuuuucagg uaccccucac uaaaggcacc gaaggcuuaa 1980

aguaggacaa ccauggagcc 2000

<210> 158

<211> 58

<212> DNA

<213> artificial sequence

<400> 158

gggttgggtt tgggttgggu gaauguagag augcgguggg ggttgggttt gggttggg 58

<210> 159

<211> 58

<212> DNA

<213> artificial sequence

<400> 159

tctatctatc tgtcgagtau gaauguagag augcggugaa actgtggcac ttcggtgc 58

<210> 160

<211> 59

<212> DNA

<213> artificial sequence

<400> 160

ggguuggguu uggguugggt gaatgtagag atgcggtggg gguuggguuu ggguuuggg 59

<210> 161

<211> 59

<212> DNA

<213> artificial sequence

<400> 161

ucuaucuauc ugucgaguat gaatgtagag atgcggtaga aacuguggca cuucgguga 59

<210> 162

<211> 21

<212> DNA

<213> artificial sequence

<400> 162

tgagggcgaa tcaaatccatc 21

<210> 163

<211> 16

<212> DNA

<213> artificial sequence

<400> 163

acattgcctc ttcatt 16

<210> 164

<211> 13

<212> DNA

<213> artificial sequence

<400> 164

gtctgtggaa gcg 13

<210> 165

<211> 20

<212> DNA

<213> artificial sequence

<400> 165

gcctgtctgt ggaagcgggt 20

<210> 166

<211> 57

<212> DNA

<213> artificial sequence

<400> 166

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggccc 57

Claims (10)

1. A nucleic acid molecule having a G-quadruplex structure for improving the stability of a nucleic acid of interest, comprising:

N_a (G_x N_y )_z G_b or (b)

Selected from SEQ ID NOs: 67. 81-82, 93 and 97;

wherein:

g represents guanine;

x is independently at each occurrence an integer selected from 2-13;

n independently for each occurrence represents any base;

y is independently at each occurrence an integer selected from 1 to 19, preferably 1 to 10, more preferably 1 to 6;

z is an integer selected from 3 to 15, preferably 3 to 10, more preferably 3 to 6;

a is an integer selected from 0 to 8, preferably 0 to 6, more preferably 0 to 3; and

b is an integer selected from 0 and 2-10, preferably 2-9, wherein when z=3, b is not 0;

wherein when n=g and N_y When GT is included, y is not 9; when n=g and N_y Y is not 4, 15 or 16 when TGA is included;

Wherein when said nucleic acid molecule having a G-quadruplex structure comprises the sequence of SEQ ID NO:67, the G-quadruplex structure of the nucleic acid molecule is formed by the nucleotide sequence of 2 molecules; and

wherein when said nucleic acid molecule having a G-quadruplex structure comprises the sequence of SEQ ID NO:81 or 82, the G-quadruplex structure of said nucleic acid molecule is formed by the nucleotide sequence of 4 molecules.

2. The nucleic acid molecule having a G-quadruplex structure according to claim 1, characterized by comprising the formula:

(G_x N_y )_z G_b ，

wherein:

g represents guanine;

x is independently at each occurrence an integer selected from 2-13;

n independently for each occurrence represents any base;

y is independently at each occurrence an integer selected from 1 to 19, preferably 1 to 10, more preferably 1 to 6;

z is an integer selected from 3 to 15, preferably 3 to 10, more preferably 3 to 6; and

b is an integer selected from 0 and 2-10, preferably 2-9, wherein when z=3, b is not 0; and

wherein when n=g and N_y When GT is included, y is not 9; when n=g and N_y When TGA is included, y is not 4 or 15.

3. The nucleic acid molecule of claim 1 or 2 having a G-quadruplex structure, characterized in that the nucleotide sequence thereof is selected from any one of SEQ ID NO. 3, 41-66, 68-80, 83-92, 94-96 and 98-131.

4. A protected nucleic acid of interest, characterized in that the nucleic acid of interest is conjugated at its 5', 3' or both 5' and 3 ends by a nucleic acid molecule having a G-quadruplex structure according to any of claims 1 to 3 or a nucleic acid molecule having a G-quadruplex structure according to any of claims 1 to 3 is inserted into the nucleic acid of interest and the stability of the nucleic acid of interest is increased.

5. The protected nucleic acid of interest according to claim 4, characterized in that the length of the nucleic acid of interest is ≡8/nt, preferably 8-5000 nt; preferably, the target nucleic acid is a nucleic acid for gene knockdown, a nucleic acid for gene activation, a nucleic acid for gene modification, a nucleic acid for gene editing, a nucleic acid for gene regulation, a nucleic acid for protein expression, a nucleic acid for protein regulation, a nucleic acid for biological detection, or a nucleic acid drug; more preferably, the nucleic acid of interest is an oligonucleotide, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, mRNA or ncRNA.

6. The protected nucleic acid of interest according to claim 5, characterized in that the oligonucleotide is an antisense oligonucleotide ASO or a nucleic acid aptamer.

7. The protected nucleic acid of interest according to claim 5, characterized in that the ncrnas are miRNA, siRNA, saRNA, piRNA, lncRNA, circRNA, fragments thereof or other regulatory RNAs.

8. A pharmaceutical composition, characterized in that it comprises a protected nucleic acid of interest according to any one of claims 4-7 and a pharmaceutically acceptable carrier.

9. A method for increasing the stability and/or activity of a nucleic acid of interest, characterized in that a nucleic acid molecule having a G-quadruplex structure according to any of claims 1-3 is conjugated to the 5' end, 3' end or both the 5' and 3 end of the nucleic acid of interest, or inserted into the nucleic acid of interest.

10. The method according to claim 9, characterized in that the nucleic acid of interest has a length of ≡8. 8 nt, preferably 8-5000 nt; preferably, the target nucleic acid is a nucleic acid for gene knockdown, a nucleic acid for gene activation, a nucleic acid for gene modification, a nucleic acid for gene editing, a nucleic acid for gene regulation, a nucleic acid for protein expression, a nucleic acid for protein regulation, a nucleic acid for biological detection, or a nucleic acid drug; more preferably, the nucleic acid of interest is an oligonucleotide (e.g., antisense oligonucleotide ASO or a nucleic acid aptamer), single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, mRNA or ncRNA (e.g., miRNA, siRNA, saRNA, piRNA, lncRNA, circRNA, fragments thereof or other regulatory RNAs).

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