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Method of inhibiting expression of target mRNA using sirna consisting of nucleotide sequence complementary to said target mRNA

一种核苷酸、序列的技术，应用在使用由互补于靶mRNA的核苷酸序列组成的siRNA抑制靶mRNA表达领域，能够解决不同、不能反映等问题

Inactive Publication Date: 2010-12-15
BIONEER
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the structure of mRNA obtained by in vitro transcription may be different from the structure of mRNA in cells, and many proteins can bind to mRNA in cells, so that the experimental results obtained by using in vitro transcribed mRNA cannot reflect the real results

Method used

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  • Method of inhibiting expression of target mRNA using sirna consisting of nucleotide sequence complementary to said target mRNA
  • Method of inhibiting expression of target mRNA using sirna consisting of nucleotide sequence complementary to said target mRNA
  • Method of inhibiting expression of target mRNA using sirna consisting of nucleotide sequence complementary to said target mRNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0159] Example 1 Comparison with traditional siRNA design methods

[0160] In order to test the performance of the method of optimizing siRNA design using the relative binding energy mode of the present invention, the siRNA design optimization method was compared with the siRNA design scoring method disclosed in Patent No. WO2004 / 045543 (Functional and Hyperfunctional siRNA, published on June 3, 2004). The siRNA efficiency scoring method disclosed in the many algorithms of patent number WO2004 / 045543 is performed according to the following equation 6:

[0161] [equation 6]

[0162] Relative functionality of siRNA = -(GC / 3)+(AU 15-19 )-(T m20℃ )*3-(G 13 )*3-(C 19 )+(A 19 )*2+(A 3 )+(U 10 )+(A 13 )-(U 5 )-(A 11 )

[0163]Three datasets from the Khvorova and Amarzguioui paper, using one dataset from the Amarzguioui paper instead of the two datasets from the Khvorova paper used to evaluate relative binding energy patterns as experimental data to compare the two scores...

Embodiment 2

[0168] Embodiment 2 Inhibition experiment of survivin gene expression

[0169] Through the method of optimizing siRNA design based on the relative binding energy mode of the present invention, 36 siRNAs that inhibit the expression of survivin gene were designed, and then the inhibition experiment of survivin gene expression was carried out. Based on the expression inhibition rate of 75%, the resulting data set was divided into effective group / ineffective group. Here, the three datasets in the Khvorova and Amarzguioui paper are used as training sets, and the survivin dataset is used as a test set. The same method as in Example 1 was used to score siRNA, and the statistical program R was used to calculate the predicted success rate of siRNA efficiency by LDA (Linear Discriminant Analysis) and QDA (Quadratic Discriminant Analysis). Results The prediction success rate of both LDA and QDA was 0.64, showing almost the same results as Example 1 (see Table 9).

[0170] Table 9

[0...

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Abstract

A inhibition method of target mRNA expression includes: (a) obtaining binding energy of a double combination section on a dsRNA sequence of all combination comprising complementary nucleotides to a random target mRNA; (b) dividing the binding energy into four sections on the dsRNA sequence of each combination to obtain a difference of the mean binding energy between each section and convert into a score of a relative combination energy pattern; (c) selecting siRNA whose inhibition efficiency to target mRNA is expected to be high by applying the converted score to the dsRNA sequence with otherfactors that affect the efficiency of siRNA; and (d) inhibiting target mRNA expression using the selected siRNA. As a result, a researcher or an experimenter can analyze patterns of a relative binding energy on base sequences of unknown siRNA without actual experiments to determine whether the siRNA is effective or ineffective rapidly, thereby design and production efficiency of siRNA can be maximized and target mRNA can be effectively inhibited with efficient siRNA to the target mRNA.

Description

technical field [0001] The present invention generally relates to a method for inhibiting expression of target mRNA using small interfering RNA (hereinafter referred to as "siRNA"), and more specifically, the present invention relates to a method for inhibiting expression of target mRNA using siRNA, the method comprising analyzing candidate siRNA The relative binding energy pattern between adjacent and non-adjacent portions of the nucleotide sequence, the step of screening for complementary siRNAs predicted to show the greatest efficiency of on-target inhibition, and the step of using the screened siRNAs to inhibit expression of the target mRNA. Background technique [0002] RNA interference (hereinafter referred to as "RNAi") refers to a phenomenon in which a target mRNA in the cytoplasm is decomposed by double-stranded RNA (hereinafter referred to as "dsRNA") having a nucleotide sequence complementary to the target mRNA. In 1998, after Fire and Mello first discovered the p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68A61K31/7088A61K48/00C12N15/09C12N15/113G16B20/20G16B20/50
CPCC12N15/113C12N15/111C12N2310/14G06F19/18C12N2320/11G16B20/00G16B20/50G16B20/20
Inventor 崔泳哲朴翰浯郑素林金永柱金尚洙朴城敏金相喆尹圭晚崔庆玉姜孝晋
Owner BIONEER