Method for detecting activity of mRNA 2 '-O-methyltransferase

A methyltransferase and activity technology, applied in the field of bioengineering, can solve the problems of high experimental environment requirements, cumbersome steps, and high cost

Pending Publication Date: 2022-05-13
NOVOPROTEIN SCI INC
View PDF0 Cites 3 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method has low safety, radioactive contamination, cumbersome steps, high requirements for the experimental environment, and high cost

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for detecting activity of mRNA 2 '-O-methyltransferase
  • Method for detecting activity of mRNA 2 '-O-methyltransferase
  • Method for detecting activity of mRNA 2 '-O-methyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0111] Embodiment 1. The establishment of RNA reaction system

[0112] 1. Sample and material preparation

[0113] a. Materials, reagents and instruments: 2x Fast Pfu Master Mix (2x Fast Pfu Polymerase Mixture), Novoprotein (Inshore Protein Technology Co., Ltd.), Cat. No.: E035; T7 High Yield RNA Transcription Kit (T7 High Yield RNA Transcription Kit) , Novoprotein (Nearshore Protein Technology Co., Ltd.), Cat. No.: E131; Cap1Capping System (Cap1 Capping System), Novoprotein (Nearshore Protein Technology Co., Ltd.), Cat. No.: M082; DEPC-H 2 O, water sterilized after treatment with diethyl coke; freshly prepared 80% ethanol, purified magnetic beads; 0.2ml EP tube, magnetic stand, PCR instrument, Qubit nucleic acid detector, etc. Cap1 capping system includes SAM, GTP, 10× capping reaction buffer, RNase inhibitor, vaccinia virus capping enzyme, mRNA 2'-O-methyltransferase.

[0114] b. Sample: a plasmid containing the gene of interest.

[0115] 2. Design of primers T7-F and T7-...

Embodiment 2

[0139] Embodiment 2: A method for detecting mRNA 2'-O-methyltransferase activity

[0140] 1. Chromatographic conditions and system suitability test:

[0141] Octadecylsilane bonded silica gel is used as filler, chromatographic column: Sepax Bio-C18 analytical column, 4.6×250mm, mobile phase: 5% methanol, 1‰ glacial acetic acid. Flow rate: 1.0ml / min, detection wavelength: 254nm, column temperature: 25°C, injection volume: 10μl.

[0142] 2. Solution preparation:

[0143] Preparation of the test solution: Take 500 μl each of post-transcriptional RNA, Cap0 reaction system, and Cap1 reaction system, filter through a 0.22 μM filter, 12000 rpm, 5 min. Take 10 μl of the supernatant to obtain the test solution.

[0144] Preparation of reference solution: Take 10 mg of SAH powder and dissolve it in 3.25 ml of DEPC-H2O to obtain 8 mM SAH standard solution. Dilute 8mM SAH to 4mM, 2mM, 1mM, 0.5mM, and 0.25mM respectively according to a 2-fold gradient to obtain the reference solution. ...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides a method for detecting the activity of mRNA (messenger ribonucleic acid) 2 '-O-methyltransferase. The method specifically comprises the following steps: (S1) providing a single-stranded RNA molecule with 5 '-triphosphate at the 5'-terminal; (S2) the RNA molecules in the step (S1) are subjected to the following test reactions, the test reactions comprise reactions in a negative test system, a first test system and a second test system, the negative test system does not contain enzyme, and the first test system does not contain enzyme; the first test system comprises Cap0 capping enzyme; the second test system comprises Cap0 capping enzyme and mRNA (messenger ribonucleic acid) 2 '-O-methyltransferase; and (S3) detecting the formation conditions of the SAH in the negative test system, the first test system and the second test system in the step (S2), and comparing with a standard curve to obtain a quantitative result of the SAH. The method is high in safety, free of radioactive contamination, simple in step, low in experimental environment requirement and low in cost.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a method for detecting mRNA 2'-O-methyltransferase activity. Background technique [0002] At present, the main method for detecting the activity of 2'-O-methyltransferase is radioactive labeling of the substrate, using [methyl- 3 H]-SAM characterizes all of the family of methyltransferases utilizing SAM (S-adenosylmethionine). Reaction in [methyl- 3 [ 3 H]CH3 was quantified and then grouped using TLC product separation and radioactivity counting. By using radioactively labeled RNA / nucleotides, changes in the structure of the nucleotide substrate can be observed. This method has low safety, radioactive contamination, cumbersome steps, high requirements for the experimental environment, and high cost. [0003] Therefore, there is a need in this field to develop a method for detecting mRNA 2'-O-methyltransferase activity with high safety, no radioactive pollution, sim...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & AuthorityApplications(China)
IPC IPC(8): C12Q1/48G01N30/02G01N30/72G01N30/86
CPCC12Q1/48G01N30/02G01N30/72G01N30/8634G01N2333/91011G01N2333/91017Y02A50/30
Inventor郑紫君吴亚会郑实朱化星赵曼曼张清仪
OwnerNOVOPROTEIN SCI INC