A method for the detection of discontinuous RNA G-quadruplexes

A non-continuous, quadruplex technology, applied in the biological field, can solve the problem that the detection method needs to be improved

Active Publication Date: 2020-05-22
INST OF CHEM CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, current methods for the detection of discontinuous RNA G-quadruplexes still need to be improved

Method used

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  • A method for the detection of discontinuous RNA G-quadruplexes
  • A method for the detection of discontinuous RNA G-quadruplexes
  • A method for the detection of discontinuous RNA G-quadruplexes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 prepares sample

[0038] The samples used in the following examples are as follows:

[0039] Sample 1: non-continuous RNA G-quadruplex sample, the non-continuous RNAG-quadruplex used in the following examples are Spinach (SEQ ID NO:1), Bulges (SEQ ID NO:2), tRNA- Ala (SEQ ID NO:3) and tRNA-Cys (SEQ ID NO:4).

[0040] Nucleotide sequence shown:

[0041] GCAGCCGGCUUGUUGAGUAGAGUGUGAGCUCCGUAACUGGUCGCGUCGACGCGACCGAAUGAAAUGGUGAAGGACGGGUCCAGCCGGCUGC (SEQ ID NO: 1).

[0042] UUGUGGUGGGUGGGUGGGU (SEQ ID NO:2).

[0043] GGG GGUGUAGCUCAGUGGUAGAGCGCGUGC (SEQ ID NO: 3).

[0044] GGGGUAUAGCUCAGUGGUAGAGCAUUUGA (SEQ ID NO: 4).

[0045] All nucleotide sequences were synthesized by Guangzhou Ruibo Biological Co., Ltd. Dissolve the sequence in Tris-HCl (with K + ) solution, the final concentration of the RNA solution (SEQ ID NO:1-4) is 20 micromole / liter, and after being heated to 90°C, and then naturally lowered to room temperature, samples 1, 2, 3 and 4 were obtained...

Embodiment 2

[0052] Example 2 Detection of non-continuous RNA G-quadruplex

[0053] 1. Preparation of reaction solution (solution A, solution B, solution C)

[0054] 1) Solution A

[0055] Add 4 μl of 200 μmol / L high-purity aqueous solution of Thioflavin T to a 2 mL reaction tube, then add 40 μL of 20 μmol / L sample 4 (pH 7.2), and then use Tris-HCl (containing K + ) to 400 microliters, and mixed to obtain solution A, wherein the molar ratio of discontinuous RNA G-quadruplex to Thioflavin T is 2:1.

[0056] 2) Solution B

[0057] Add 4 μl of 200 μmol / L high-purity aqueous solution of Thioflavin T to a 2 mL reaction tube, then add 40 μL of 20 μmol / L sample 5 (pH 7.2), and then use Tris-HCl (containing K + ) to 400 microliters, and mix well to obtain solution B, wherein the molar ratio of single-stranded RNA to Thioflavin T is 2:1.

[0058] 3) Solution C

[0059] Add 4 microliters of 200 micromol / liter high-purity aqueous solution of Thioflavin T to a 2mL reaction tube, and then use Tris...

Embodiment 3

[0068] Example 3 Detection of non-continuous RNA G-quadruplex

[0069] 1. Preparation of reaction solution (solution A, solution B, solution C)

[0070] 1) Solution A

[0071] Add 4 μl of 200 μmol / L high-purity aqueous solution of Thioflavin T to a 2 mL reaction tube, then add 80 μL of 20 μmol / L sample 3 (pH 7.2), and then use Tris-HCl (containing K + ) to 400 microliters, and mixed to obtain solution A, wherein the molar ratio of discontinuous RNA G-quadruplex to Thioflavin T was 2:1.

[0072] 2) Solution B

[0073] Add 4 μl of 200 μmol / L high-purity aqueous solution of Thioflavin T to a 2 mL reaction tube, then add 80 μL of 20 μmol / L sample 5 (pH 7.2), and then use Tris-HCl (containing K + ) to 400 microliters, and mix well to obtain solution B, wherein the molar ratio of single-stranded RNA to Thioflavin T is 2:1.

[0074] 3) Solution C

[0075] Add 4 microliters of 200 micromol / liter high-purity aqueous solution of Thioflavin T to a 2mL reaction tube, and then use Tri...

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Abstract

The invention discloses an application of thioflavin-T to preparation of a kit, a method for determining whether a discontinuous RNA G-quadruplex exists in a to-be-detected sample and a kit for detecting the discontinuous RNA G-quadruplex. The method for determining whether the discontinuous RNA G-quadruplex exists in the to-be-detected sample comprises the following steps: (1) the to-be-detected sample makes contact with thioflavin-T, a mixed solution is obtained, and the fact that the to-be-detected sample contains RNA is determined in advance; (2) fluorescence spectrum analysis is performed on the mixed solution; (3) whether the discontinuous RNA G-quadruplex exists in the to-be-detected sample is determined on the basis of a fluorescence spectrum analysis result. The method has the characteristics of being simple and convenient to operate and high in sensitivity and accuracy.

Description

technical field [0001] The present invention relates to the field of biotechnology. In particular, the present invention relates to methods for the detection of non-continuous RNA G-quadruplexes. More specifically, the present invention relates to the use of Thioflavin T in the preparation of a kit, a method for determining whether there is a non-continuous RNA G-quadruplex in a test sample, and a kit for detecting a non-continuous RNA G-quadruplex. Background technique [0002] The RNA G-quadruplex is a secondary structure formed by guanine-rich RNA sequences. The basic unit of the structure is a G-tetrad planar structure formed by three or four guanine bases through Hoogsteen hydrogen bonds. In the presence of cations (such as potassium ions or sodium ions), a unique parallel-structured G-quadruplex is formed through the stacking of multiple G planes. In the study of human transcriptional genes, it was found that many genes closely related to the occurrence and developme...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486G01N2021/6417
Inventor 唐亚林许淑娟李骞
Owner INST OF CHEM CHINESE ACAD OF SCI
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