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CN-121991150-A - Coupling method of double-target nucleic acid and application thereof

CN121991150ACN 121991150 ACN121991150 ACN 121991150ACN-121991150-A

Abstract

The invention relates to the technical field of biological medicine, in particular to a novel coupling method of double-target nucleic acid molecules and application thereof, and provides a strategy method for connecting two nucleic acid chains through cyclization reaction between tetrazine and alkene or alkyne. The cyclization reaction between the nucleic acid Strand of the tetrazine group (Strand 1) and the nucleic acid Strand of the alkene or alkyne group (Strand 2) yields a dihydropyridazine or pyridazine linker unit between the two nucleic acid strands, the linker mode being selected from among 5 'of Strand1 and 3' of Strand2, 5 'of Strand1 and 5' of Strand2, 3 'of Strand1 and 3' of Strand2, 3 'of Strand1 and 5' of Strand 2. Further, two nucleic acid strands connected by dihydropyridazine or pyridazine are respectively combined with complementary nucleic acid strands according to base complementary pairing to prepare the double-target oligonucleotide medicine.

Inventors

  • SUN GANG
  • SUN HONGWEI
  • ZHANG YUFEN
  • ZHU BIN
  • ZHENG ZHENSHENG
  • HUANG YUANSONG

Assignees

  • 苏州盛诺维生物科技有限公司

Dates

Publication Date
20260508
Application Date
20251215

Claims (10)

  1. 1. A coupling method of double-target nucleic acid and application thereof are characterized in that two nucleic acid chains are connected through a dihydropyridazine or pyridazine unit, and the connected nucleic acid structure comprises at least one of the modes shown in the formulas 1-8: ....formula 1; ... Formula 2; ....formula 3; ....formula 4; ... ....Formula 6; ....formula 7; ....formula 8; Wherein Strand1, strand2 are nucleic acid sequences including natural or chemically modified DNA, RNA strands, linker1, linker2 are alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polyglycol, polyester, polyamide, aminoacyl, monosaccharide or polysaccharide, amino acid or polypeptide, ester group, aryl, acyl, diacyl, etc., R and R ́ are alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polyglycol, aminoacyl, peptide, ester group, aryl, acyl, diacyl, etc., CR is cycloalkyl, cycloalkenyl, aryl cycloalkyl, aryl cycloalkenyl, etc.
  2. 2. The method for coupling double-target nucleic acids and application thereof according to claim 1, wherein a dihydropyridazine or pyridazine linking unit is constructed between the two nucleic acid chains by preparing a linker designed for 3 'or 5' introduction of the nucleic acid chains, comprising the structure shown in formula 9 and formula 10: ....formula 9; ....formula 10; Wherein Strand is a nucleic acid sequence comprising natural or chemically modified DNA, RNA strands, linker is alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polyglycol, polyester, polyamide, aminoacyl, monosaccharide or polysaccharide, amino acid or polypeptide, ester group, aromatic group, acyl, diacyl, etc., LH is a linker comprising but not limited to tetrazine, straight or branched alkene, straight or branched alkyne, cycloalkene, cycloalkyne, etc.
  3. 3. The method for coupling double-target nucleic acids according to claim 1 and 2, wherein a dihydropyridazine or pyridazine connecting unit is constructed between the two nucleic acid chains, and the 3 'or 5' introduced designed linker of the nucleic acid chain comprises tetrazine and alkene or alkyne, and comprises a structure shown in formula 11-14: ....formula 11; ....formula 12; ....formula 13; ....formula 14; wherein Strand is a nucleic acid sequence comprising a natural or chemically modified DNA, RNA Strand, linker is alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polyglycol, polyester, polyamide, aminoacyl, monosaccharide or polysaccharide, amino acid or polypeptide, ester group, aryl, acyl, diacyl, etc., R is alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polyglycol, aminoacyl, peptide, ester group, aryl, acyl, diacyl, etc., ALK is straight chain or branched chain, straight chain or branched alkyne, cycloalkene, including cyclooctene, norbornene, etc., cycloalkyne, etc.
  4. 4. A method for coupling a double-target nucleic acid according to any one of claims 1 to 3 and the use thereof, wherein the linker (linker) at the 3 ́ or 5 ́ end of the nucleic acid chain is selected from one or more of the following structures, or a combination of the following structures, alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polyglycol, polyester, polyamide, aminoacyl, monosaccharide or polysaccharide, amino acid or polypeptide, ester, aromatic, acyl, diacyl, etc., according to the physicochemical properties, bioactivity, etc.
  5. 5. The method for coupling double-target nucleic acid and application thereof according to any one of claims 1-4, wherein a dihydropyridazine or pyridazine connecting unit is constructed between the two nucleic acid chains, and the alkene introduced from the 3 'or 5' end of one nucleic acid chain is a cycloolefin structure such as cyclooctene, norbornene and the like.
  6. 6. The method for coupling double-target nucleic acids and the application thereof according to any one of claims 1 to 5, wherein a dihydropyridazine or pyridazine connecting unit is constructed between the two nucleic acid chains, and a1, 2,4, 5-tetrazine introduced from the 3 'or 5' end of one nucleic acid chain and a nucleic acid chain introduced with alkene or alkyne from the 3 'or 5' end undergo cyclization, so that a dihydropyridazine or pyridazine connecting unit is constructed between the two nucleic acid chains.
  7. 7. The method for coupling a double-target nucleic acid and the use thereof according to claim 1, wherein the tetrazine group of nucleic acid Strand (Strand 1) and the alkene or alkyne group of nucleic acid Strand (Strand 2) produce a dihydropyridazine or pyridazine linking unit, and the linking mode is selected from four types, namely, between 5 'of Strand1 and 3' of Strand2, between 5 'of Strand1 and 5' of Strand2, between 3 'of Strand1 and 3' of Strand2, and between 3 'of Strand1 and 5' of Strand 2.
  8. 8. The method for coupling double-target nucleic acids and the use thereof according to claim 1, wherein the double-target oligonucleotide is prepared by combining two nucleic acid strands connected by dihydropyridazine or pyridazine with their complementary nucleic acid strands according to base complementary pairing, respectively.
  9. 9. The method for coupling double-target nucleic acid and application thereof according to claim 8, wherein two nucleic acid strands connected by dihydropyridazine or pyridazine are taken as sense strands, and can be combined with two antisense strands which are complementary to bases to form a double-target siRNA molecule, and the combination direction of the antisense strands and the sense strands of the double-target siRNA has four modes, namely 5 ́ -3 ́ and 5 ́ -3 ́, 5 ́ -3 ́ and 3 ́ -5 ́,3 ́ -5 ́ and 5 ́ -3 ́,3 ́ -5 ́ and 3 ́ -5 ́.
  10. 10. The method of claim 1, wherein the oligonucleotide is selected from the group consisting of small interfering nucleotides, antisense oligonucleotides, micrornas, small activating RNAs, guide RNAs, transfer RNAs, template DNA, template RNAs and aptamers.

Description

Coupling method of double-target nucleic acid and application thereof Technical Field The invention relates to the technical field of biological medicine, in particular to a novel coupling method of double-target nucleic acid molecules and application thereof. Background The oligonucleotide drug is specifically combined with a target gene through base complementation pairing, expands a drug target point to upstream mRNA of pathogenic protein, has higher specificity, influences the expression of the target gene from the post-transcriptional level through regulating or knocking down the target gene, and has definite therapeutic potential in the fields of tumor, viral diseases, hereditary diseases and the like. The oligonucleotide drug has the advantages of rich targets, lasting drug effect, short research and development period, high research and development success rate and the like, and provides a solution for the treatment of a plurality of refractory diseases. Clinical practice shows that single-target gene silencing has obvious limitation that on one hand, the occurrence and development of most diseases involve multi-gene and multi-channel cooperative regulation and control, single-target intervention is difficult to block the disease process, and on the other hand, long-term single-target regulation and control or inhibition easily causes the mutation of target genes or the activation of compensatory channels, drug resistance is generated, and the treatment effect is reduced. In order to break through the bottleneck, double-target oligonucleotide drugs are generated, and the oligonucleotide drugs target two genes simultaneously, so that a synergistic treatment effect of 1+1>2 is realized, single-target dependence is reduced, and drug resistance risk is reduced. In recent years, with the maturation of sequence design technology and the optimization of delivery systems, research on double-target oligonucleotide drugs gradually changes from basic experiments to clinical, and becomes a new research and development focus in the nucleic acid field. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a novel coupling method of double-target nucleic acid molecules and application thereof, in particular to a coupling method of two nucleic acid chains connected through a dihydropyridazine or pyridazine unit, wherein the nucleic acid structure forming the connection comprises at least one of modes shown in the formulas 1-8: ....formula 1; ... Formula 2; ....formula 3; ....formula 4; ... ....Formula 6; ....formula 7; ....formula 8; Wherein Strand1, strand2 are nucleic acid sequences including natural or chemically modified DNA, RNA strands, linker1, linker2 are alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polyglycol, polyester, polyamide, aminoacyl, monosaccharide or polysaccharide, amino acid or polypeptide, ester group, aryl, acyl, diacyl, etc., R and R ́ are alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polyglycol, aminoacyl, peptide, ester group, aryl, acyl, diacyl, etc., CR is cycloalkyl, cycloalkenyl, aryl cycloalkyl, aryl cycloalkenyl, etc. Construction of a dihydropyridazine or pyridazine linker unit between two nucleic acid strands, the present invention is achieved by preparing a linker designed for 3 'or 5' introduction of a nucleic acid strand, comprising the structure shown in formula 9, formula 10: ....formula 9; ....formula 10; Wherein Strand is a nucleic acid sequence comprising natural or chemically modified DNA, RNA strands, linker is alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polyglycol, polyester, polyamide, aminoacyl, monosaccharide or polysaccharide, amino acid or polypeptide, ester group, aromatic group, acyl, diacyl, etc., LH is a linker comprising but not limited to tetrazine, straight or branched alkene, straight or branched alkyne, cycloalkene, cycloalkyne, etc. Preferably, the 3 'or 5' introduced designed linker of the nucleic acid chain comprises 1,2,4, 5-tetrazine (Tetrazine, tz) or alkene, alkyne, comprising the structure shown in formula 11-formula 14: ....formula 11; ....formula 12; ....formula 13; ....formula 14; wherein Strand is a nucleic acid sequence comprising a natural or chemically modified DNA, RNA Strand, linker is alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkoxy, alkoxyalkyl, polyglycol, polyester, polyamide, aminoacyl, monosaccharide or polysaccharide, amino acid or polypeptide, ester group, aryl, acyl, diacyl, etc., R is alkyl, alkylene, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, halogen, cyano, alkoxy, alkoxyalkyl, polyglycol, aminoacyl, peptide, ester group, aryl, acyl, diacyl, etc., ALK is straight chain or branched chain, straight chain or branched alkyne, cycloalkene, including cyclooctene, norbornene, etc., cycloalkyne, etc. Through solid phase synthesis, nucleotide phosphoramidite or phosp