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Nucleic acid inducing RNA interference modified for preventing off-target, and use thereof

一种RNA干扰、脱靶效应的技术，应用在用于阻止脱靶效应而变形的RNA干扰诱导核酸及其用途领域，能够解决减少命中目标抑制效率等问题，达到缓解不准确性及副作用、防止脱靶效应的效果

Active Publication Date: 2016-08-03
ENCODEGEN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For this reason, although it can reduce off-target effects, it cannot completely prevent them from happening, and in addition, it also presents the problem of reducing the efficiency of on-target suppression

Method used

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  • Nucleic acid inducing RNA interference modified for preventing off-target, and use thereof
  • Nucleic acid inducing RNA interference modified for preventing off-target, and use thereof
  • Nucleic acid inducing RNA interference modified for preventing off-target, and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Comparison of target gene expression inhibition effect and off-target effect of siRNA molecules replaced by deoxyribonucleotide spacer (dSpacer)

[0069] Such as figure 1 For the example shown in the diagram, the inventors speculate that applying a base-pairing-incapable spacer deformation to the 5' seed region of all RNAi-inducing nucleic acids bound to the AGO protein could completely prevent off-target effects while improving target gene repression effect (on-target effect), the inventors' model based on the microRNA recognition mechanism, the transitional nuclear model (NatStructMolBiol.2012Feb12; 19(3):321-7), focuses on the 5' seed region, especially the so-called The present RNA interference-inducing nucleic acid is invented by base pairing at the 6th nucleotide position from the 5' end of the "pivot".

[0070] First, in order to compare the on-target and off-target effects mediated by the deformed siRNA molecules according to the present invention and conventio...

Embodiment 2

[0079] Comparison of target gene inhibition and off-target effects caused by siRNA molecule ribonucleotide spacer (rSpacer) replacement

[0080] To see whether the results in Example 1 above can be replicated by a similar spacer containing a ribonucleotide backbone, the nucleotides at positions 2-7 of siRL are replaced by a ribonucleic acid spacer (rSpacer) (pi-r ), this position was previously shown to eliminate off-target effects, and here the targeting activity of siRL was detected by calculating IC50 according to the same method as in Example 1.

[0081] results, such as image 3 As shown in a and 3c, each rSpacer substitution at positions 2-7 from the 5' end showed a maximum inhibition rate (Imax) greater than or equal to 54% relative to untransformed (WT). Of these, each variant except 2pi-r (applied to site 2) had less on-target inhibitory activity than 6pi. Especially 2pi-r showed superior on-target inhibition than 6pi, but still as image 3 The inhibition of off-ta...

Embodiment 3

[0084] Comparison of target gene inhibition and off-target effects caused by implantation of siRNA molecule deoxynucleotide spacer (dSpacer)

[0085] The sequence configuration in the seed region contains a deoxynucleotide spacer (dSpacer). When interacting with the target gene RNA, inconsistent matching should occur in a non-base part of the siRNA, which is passed in Example 1 and Example 2. The substitution in is made. Therefore, when a dSpacer is implanted in the seed region of siRNA, the non-base can form a bulge, such as Figure 4 as shown in a. Therefore, changes in on-target and off-target effects were investigated for the case of such dSpacer implants. Initially, the targeting effect of a siRL comprising a dSpacer implanted between positions 2-7 from the 5' end, where positions 2 to 7 are in the above example, can be detected by measuring the IC50 as in Example 1 Reported to eliminate off-targets.

[0086] Therefore, if Figure 4 In b, each implantation of dSpacer...

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Abstract

The present invention relates to a nucleic acid inducing RNA interference modified for preventing off-target, and a use thereof, and more specifically, to a nucleic acid inducing RNA interference, modified by replacing, by a spacer capable of no base pairing, 5' terminal and 3' terminal regions in at least one single strand of the double strand of the nucleic acid inducing RNA interference. The nucleic acid inducing RNA interference of the present invention can provide: a nucleic acid having a novel modified form for preventing an off-target effect generated when inhibiting the expression of a target gene by an RNA interference phenomenon; and a method for selectively inhibiting the expression of a target gene by using the same. In addition, the nucleic acid inducing RNA interference according to the present invention solves the inaccuracy and side effects due to off-target, which are the problems of a known nucleic acid inducing RNA interference, by providing a nucleic acid inducing RNA interference as a novel modified form having target selectivity and specificity by preventing an off-target effect while inhibiting the expression of a target gene, and thus can be used, without worries, in the research of a technique for inhibiting the expression of a gene and in gene therapy.

Description

technical field [0001] The present invention relates to RNA interference-inducing nucleic acids and uses thereof, and more particularly, to RNA interference-inducing nucleic acids contained in at least one single strand of the double strands of RNA interference-inducing nucleic acids in the 5' end and 3' end regions separated by Substituted deformation. Background technique [0002] siRNA (small interfering RNA) has been widely used as a technology to inhibit the expression of the expected target gene by interference, but, as its inevitable disadvantage, other genes will also be inhibited non-specifically, and the generation of off-target effects will bring False research findings and treatment side effects are very worrying. This off-target effect is caused by the artificially introduced siRNA of the AGO (Argonaute) protein, which plays a central role in RNA interference, being treated the same as the microRNA existing in the cell. Therefore it is called microRNA-like off...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113A61K48/00
CPCC12N15/111C12N15/113C12N2310/14C12N2310/141C12N2310/31C12N2310/332C12N2310/533C12N2320/53C12N2310/531C12N15/1137C12N2310/344C12N2310/321C12N2310/3521A61K48/00C12N15/1131
Inventor 池晟旭张恩淑
Owner ENCODEGEN CO LTD
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