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Specific modification for inhibiting RNA interference off-target effect

A small interference and chemical modification technology, applied in the field of nucleic acids, can solve the problems of misinterpreting the inhibitory activity of siRNA, the toxic and side effects of siRNA drugs, and affecting the application of RNA interference technology, so as to reduce off-target effects and improve specificity.

Inactive Publication Date: 2012-10-10
BIOMICS BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the study of gene function, this off-target effect of the sense strand of siRNA may lead to misinterpretation of the inhibitory activity of siRNA; in small nucleic acid pharmaceuticals, it may cause a series of consequences such as toxic side effects of siRNA drugs, which will seriously affect Application of RNA Interference Technology

Method used

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  • Specific modification for inhibiting RNA interference off-target effect
  • Specific modification for inhibiting RNA interference off-target effect
  • Specific modification for inhibiting RNA interference off-target effect

Examples

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preparation example Construction

[0064] Generally speaking, the synthetic method of oligonucleotide molecule comprises the following four steps: (1) synthesis of oligoribonucleotide; (2) deprotection; (3) purification and separation; (4) desalting.

[0065] For example, the chemical synthesis steps of siRNA having the nucleotide sequence shown in SEQ ID NO: 1 are as follows:

[0066] (1) Synthesis of siRNA constituent strands: set the sense strand or antisense strand oligonucleotide sequence of synthetic 1 millimolar amount of siRNA on the automatic DNA / RNA synthesizer (for example, Applied Biosystems EXPEDITE8909), set each The coupling time of each cycle is 10-15 minutes. The starting material is 5'-O-dimethoxy-thymidine support connected to the solid phase. The first cycle connects a base to the solid phase support. , and then in the nth (2≤n≤35) cycle, a base is connected to the base connected in the n-1th cycle, and this cycle is repeated until the synthesis of the entire nucleic acid sequence is complet...

Embodiment 1

[0097] Example 1. siRNA target sites and siRNA sequences

[0098] Invitrogen Beijing Branch was commissioned to synthesize the siRNA target sequence fragments in Table 1 for the construction of fusion reporter gene expression vectors.

[0099] Design siRNA for the target site sequence in Table 1, and entrust Guangzhou Ruibo Biotechnology Co., Ltd. to synthesize the chemically modified and unmodified small interfering RNA (siRNA) listed in Table 2-11.

[0100] Table 1. siRNA target site sequences

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120]

[0121]

[0122]

[0123]

[0124]

Embodiment 2

[0125] Example 2. Detection of siRNA inhibitory activity on target sites

[0126] 1) Construction of recombinant firefly luciferase reporter gene plasmid with siRNA isolation target site

[0127] Refer to the following literature to construct a recombinant firefly luciferase reporter gene plasmid containing siRNA isolation target sites. The reporter gene plasmid vector can be obtained from the author of the paper. (Du Q, Thonberg H, Zhang HY, Wahlestedt C&Liang Z. Validating siRNA Using a Reporter Made from Synthetic DNA Oligonucleotides. Biochem Biophys Res Commun. 325:243-9, 2004; Du Q, Thonberg H, Wang J, Wahlestedt C&Liang Z.A Systematic Analysis of the Silencing Effects of an Active siRNA at All Single-nucleotide Mismatched Target Sites. Nucleic Acids Res. 33:1671-7, 2005; Li ZS, Qiao RP, Du Q, Yang ZJ, Zhang LR, Zhang PZ, Liang Z&Zhang LH. Studies on Aminoisonucleoside Modified siRNAs: Stability and Silencing Activity. Bioconjugate Chem.18:1017-24, 2007; Dahlgren C, Zha...

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Abstract

The invention provides a chemically modified double-strand small interfering RNA (siRNA) molecule and its preparation and application methods. Chemically modified nucleotide is introduced into the 14th site or / and 16th site beginning from 5' terminal of the siRNA molecular sense strand, thereby reducing the off-target effect caused by the modified siRNA molecular sense strand and enhancing specificity of the modified siRNA molecule for silence of target gene expression.

Description

technical field [0001] The invention relates to the technical field of nucleic acids, in particular to a chemically modified double-stranded small interfering RNA (siRNA) molecule capable of reducing the off-target effect of the sense strand and a preparation method thereof, and the application of the modified siRNA molecule in the preparation of pharmaceutical compositions . Background technique [0002] RNA interference (RNA interference, RNAi) is a phenomenon in which double-stranded RNA molecules (double-stranded RNA, dsRNA) reduce the expression of homologous genes at the mRNA level. RNA interference technology, also known as gene knockdown (knock-down) or gene silencing (gene silencing), is a post-transcriptional gene expression regulation method, so it is also called post-transcriptional gene silencing (post-transcriptional gene silencing) , PTGS). The earliest reports on RNA interference appeared around 1990. Two different research groups simultaneously reported th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/10A61K48/00
CPCC12N2320/53C12N2310/336C12N2310/333C12N2310/335C12N2310/334C12N2310/344C12N15/111C12N15/113C12N2310/3341
Inventor 杜权梁子才
Owner BIOMICS BIOTECH
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