Nucleic acid, composition and conjugate comprising the same, and preparation method and use thereof

1. A siRNA conjugate comprising siRNA and a conjugating group conjugated to the siRNA, and having a structure as shown by Formula (308):

wherein n1 is an integer of 1-2, and n3 is an integer of 0-[4]1, and n1+n3=2-3;

m1, m2, and m3 independently of one another are an integer of 2-10;

R 10 , R 11 , R 12 , R 13 , R 14 and R 15 independently of one another are H, or selected from the group consisting of C 1 -C 10 alkyl, C 1 -C 10 haloalkyl, and C 1 -C 10 alkoxy;

R 3 is a group having a structure as shown by Formula (A59):

wherein E1 is OH, SH or BH 2 ; Nu is siRNA;

R 2 is a linear alkylene of 1 to 20 carbon atoms in length, wherein one or more carbon atoms are optionally replaced with one or more groups selected from the group consisting of: C(O), NH, O, S, CH═N, S(O) 2 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocyclylene, and C 5 -C 10 heteroarylene, and wherein R 2 optionally has one or more substituents selected from the group consisting of: C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 1 -C 10 haloalkyl, —OC 1 -C 10 alkyl, —OC 1 -C 10 alkylphenyl, —C 1 -C 10 alkyl-OH, —OC 1 -C 10 haloalkyl, —SC 1 -C 10 alkyl, —SC 1 -C 10 alkylphenyl, —C 1 -C 10 alkyl-SH, —SC 1 -C 10 haloalkyl, halo, —OH, —SH, —NH 2 , —C 1 -C 10 alkyl-NH 2 , —N(C 1 -C 10 alkyl)(C 1 -C 10 alkyl), —NH(C 1 -C 10 alkyl), cyano, nitro, —CO 2 H, —C(O)O(C 1 -C 10 alkyl), —CON(C 1 -C 10 alkyl)(C 1 -C 10 alkyl), —CONH(C 1 -C 10 alkyl), —CONH 2 , —NHC(O)(C 1 -C 10 alkyl), —NHC(O)(phenyl), —N(C 1 -C 10 alkyl)C(O)(C 1 -C 10 alkyl), —N(C 1 -C 10 alkyl)C(O)(phenyl), —C(O)C 1 -C 10 alkyl, —C(O)C 1 -C 10 alkylphenyl, —C(O)C 1 -C 10 haloalkyl, —OC(O)C 1 -C 10 alkyl, —SO 2 (C 1 -C 10 alkyl), —SO 2 (phenyl), —SO 2 (C 1 -C 10 haloalkyl), —SO 2 NH 2 , —SO 2 NH(C 1 -C 10 alkyl), —SO 2 NH(phenyl), —NHSO 2 (C 1 -C 10 alkyl), —NHSO 2 (phenyl), and —NHSO 2 (C 1 -C 10 haloalkyl);

each L 1 is independently a linear alkylene of 1 to 70 carbon atoms in length, wherein one or more carbon atoms are optionally replaced with one or more groups selected from the group consisting of: C(O), NH, O, S, CH═N, S(O) 2 , C 2 -C 10 alkenylene, C 2 -C 10 alkynylene, C 6 -C 10 arylene, C 3 -C 18 heterocyclylene, and C 5 -C 10 heteroarylene, and wherein L 1 optionally has one or more substituents selected from the group consisting of: C 1 -C 10 alkyl, C 6 -C 10 aryl, C 5 -C 10 heteroaryl, C 1 -C 10 haloalkyl, —OC 1 -C 10 alkyl, —OC 1 -C 10 alkylphenyl, —C 1 -C 10 alkyl-OH, —OC 1 -C 10 haloalkyl, —SC 1 -C 10 alkyl, —SC 1 -C 10 alkylphenyl, —C 1 -C 10 alkyl-SH, —SC 1 -C 10 haloalkyl, halo, —OH, —SH, —NH 2 , —C 1 -C 10 alkyl-NH 2 , —N(C 1 -C 10 alkyl)(C 1 -C 10 alkyl), —NH(C 1 -C 10 alkyl), cyano, nitro, —CO 2 H, —C(O)O(C 1 -C 10 alkyl), —CON(C 1 -C 10 alkyl)(C 1 -C 10 alkyl), —CONH(C 1 -C 10 alkyl), —CONH 2 , —NHC(O)(C 1 -C 10 alkyl), —NHC(O)(phenyl), —N(C 1 -C 10 alkyl)C(O)(C 1 -C 10 alkyl), —N(C 1 -C 10 alkyl)C(O)(phenyl), —C(O)C 1 -C 10 alkyl, —C(O)C 1 -C 10 alkylphenyl, —C(O)C 1 -C 10 haloalkyl, —OC(O)C 1 -C 10 alkyl, —SO 2 (C 1 -C 10 alkyl), —SO 2 (phenyl), —SO 2 (C 1 -C 10 haloalkyl), —SO 2 NH 2 , —SO 2 NH(C 1 -C 10 alkyl), —SO 2 NH(phenyl), —NHSO 2 (C 1 -C 10 alkyl), —NHSO 2 (phenyl), and —NHSO 2 (C 1 -C 10 haloalkyl);

represents a site where a group is linked to the rest of the molecule;

M 1 represents a targeting group, wherein each of the targeting groups is independently a ligand that has affinity to asialoglycoprotein receptors (ASGP-R) on a surface of mammalian hepatocytes;

wherein the siRNA comprises a sense strand and an antisense strand, each nucleotide in the siRNA being independently a modified or unmodified nucleotide, wherein the sense strand comprises a nucleotide sequence I, and the antisense strand comprises a nucleotide sequence II;

the nucleotide sequence I and the nucleotide sequence II are at least partly reverse complementary to form a double stranded region; wherein the nucleotide sequence I comprises a nucleotide sequence A, which has the same length as the nucleotide sequence shown in SEQ ID NO: 1 with no more than 3 nucleotide differences; and the nucleotide sequence II comprises a nucleotide sequence B, which has the same length as the nucleotide sequence shown in SEQ ID NO: 2 with no more than 3 nucleotide differences:

(SEQ ID NO: 1)

5′-UGCUAUGCCUCAUCUUCUZ-3′;

(SEQ ID NO: 2)

5′-Z’AGAAGAUGAGGCAUAGCA-3′;

wherein,

Z is A; Z′ is U; and

the nucleotide sequence A comprises a nucleotide Z A at the position corresponding to Z; the nucleotide sequence B comprises a nucleotide Z′ B at the position corresponding to Z′; the nucleotide Z′ B is the first nucleotide at 5′ terminal of the antisense strand.

2. The siRNA conjugate according to claim 1 , wherein each Lis independently selected from the connection combinations of one or more of Formulae A1-A26:

wherein j1 is an integer of 1-20;

j2 is an integer of 1-20;

R′ is a C 1 -C 10 alkyl;

Ra is selected from the group consisting of Formulae A27-A45, and connection combination thereof:

Rb is a C 1 -C 10 alkyl.

3. The siRNA conjugate according to claim 1 , wherein L 1 is selected from the connection combinations of one or more of A1, A4, A5, A6, A8, A10, A11, A13, and thereof, or L 1 is selected from the connection combinations of at least two of A1, A4, A8, A10, and A11.

4. The siRNA conjugate according to claim 2 , wherein L 1 is 3 to 25 atoms in length, or the length of L 1 further is 4 to 15 atoms.

5. The siRNA conjugate according to claim 2 , wherein m1, m2 and m3 independently of one another are an integer of 2-5; or wherein m1=m2=m3.

6. The siRNA conjugate according to claim 1 , wherein each of the targeting groups is independently an asialoglycoprotein or saccharide.

7. The siRNA conjugate according to claim 1 , wherein at least one or each of the targeting groups is galactose or N-acetylgalactosamine.

8. The siRNA conjugate according to claim 1 , wherein the conjugate has a structure as shown by Formula (403), (404), (405), (406), (407), (408), (409), (410), (411), (412), (413), (414), (415), (416), (417), (418), (419), (420), (421), or (422):

9. The siRNA conjugate according to claim 1 , wherein the P atom in Formula A59 is linked to 3′ terminal of the sense strand of the siRNA.

10. A method for treating and/or preventing a pathological condition or disease caused by HBV infection, wherein the method comprises administering to a subject in need thereof an effective amount of the siRNA conjugate according to claim 1 .

11. A kit comprising the siRNA conjugate according to claim 1 .

12. The siRNA conjugate according to claim 1 , wherein R 2 is selected from B5, B6, B5′, or B6′:

wherein, represents the site where a group is covalently linked; and q 2 is an integer of 1-10.

13. The siRNA conjugate according to claim 1 , wherein the nucleotide sequence A is a nucleotide sequence shown in SEQ ID NO: 3; and the nucleotide sequence B is a nucleotide sequence shown in SEQ ID NO: 4:

(SEQ ID NO: 3)

5′-UGCUAUGCCUCAUCUUCUZ A -3′;

(SEQ ID NO: 4)

5′-Z′ B AGAAGAUGAGGCAUAGCA-3′;

wherein Z′ B is the first nucleotide at 5′ terminal of the antisense strand; Z A is selected from A, U, G or C, and Z′ B is a nucleotide complementary to Z A .

14. The siRNA conjugate according to claim 1 , wherein the nucleotide sequence I further comprises a nucleotide sequence III, and the nucleotide sequence II further comprises a nucleotide sequence IV; the nucleotide sequence III and the nucleotide sequence IV independently of one another are 1-4 nucleotides in length; the nucleotide sequence III is linked to 5′ terminal of the nucleotide sequence A; the nucleotide sequence IV is linked to 3′ terminal of the nucleotide sequence B; and the nucleotide sequence III and the nucleotide sequence IV have the same length and are substantially reverse complementary or completely reverse complementary; the “substantially reverse complementary” means that there is no more than 1 base mispairing between two nucleotide sequences; the “completely reverse complementary” means that there is no mispairing between two nucleotide sequences.

15. The siRNA conjugate according to claim 14 , wherein the nucleotide sequence III and the nucleotide sequence IV both are of 1 nucleotide in length, and the base of the nucleotide sequence III is G;

the nucleotide sequence III and the nucleotide sequence IV both are 2 nucleotides in length; in the direction from 5′ terminal to 3′ terminal, the base composition of the nucleotide sequence III is AG;

the nucleotide sequence III and the nucleotide sequence IV both are 3 nucleotides in length; in the direction from 5′ terminal to 3′ terminal, the base composition of the nucleotide sequence III is AAG; or

the nucleotide sequence III and the nucleotide sequence IV both are 4 nucleotides in length; in the direction from 5′ terminal to 3′ terminal, the base composition of the nucleotide sequence III is CAAG.

16. The siRNA conjugate according to claim 1 , wherein the nucleotide sequence II further comprises a nucleotide sequence V, which is 1-3 nucleotides in length and is linked to 3′ terminal of the antisense strand, thereby forming a 3′ overhang of the antisense strand.

17. The siRNA conjugate according to claim 1 , wherein the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 3, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 5:

(SEQ ID NO: 3)

5′-UGCUAUGCCUCAUCUUCUZ A -3′;

(SEQ ID NO: 5)

5′-Z′ B AGAAGAUGAGGCAUAGCAGC-3′;

or

the sense strand of the siRNA comprises the nucleotide sequence shown in SEQ ID NO: 3, and the antisense strand comprises the nucleotide sequence shown in SEQ ID NO: 6:

(SEQ ID NO: 3)

5′-UGCUAUGCCUCAUCUUCUZ A -3′;

(SEQ ID NO: 6)

5′-Z′ B AGAAGAUGAGGCAUAGCAUU-3′;

wherein the nucleotide Z′ B is the first nucleotide at 5′ terminal of the antisense strand; Z A is selected from A, U, G or C; and Z′ B is a nucleotide complementary to Z A .

18. The siRNA conjugate according to claim 1 , wherein the siRNA is siHBVS1 or siHBVS2:

siHBVS1

siHBVS1

Sense strand:

(SEQ ID NO: 1)

5′-UGCUAUGCCUCAUCUUCUZ-3′,

Antisense strand:

(SEQ ID NO: 7)

5′-Z′AGAAGAUGAGGCAUAGCAGC-3′,

siHBVS2

Sense strand:

(SEQ ID NO: 1)

5′-UGCUAUGCCUCAUCUUCUZ-3′,

Antisense strand:

(SEQ ID NO: 8)

5′-Z′AGAAGAUGAGGCAUAGCAUU-3′,

wherein Z is A, and Z′ is U.