deoxyribonuclease assay
deoxyribonuclease assay
With the development of in vitro molecular experimental techniques, many DNA molecules with different catalytic functions have been discovered in recent years. These catalytic DNA molecules called deoxyribonucleases can catalyze many chemical reactions such as cleavage, ligation, and porphyrin ring metallization, and they also have DNA kinase activity. This experiment is from "RNA Laboratory Guidebook", edited by Xiaofei Zheng.
Operation method
deoxyribonuclease assay
Principle
With the development of in vitro molecular experimental techniques, many DNA molecules with different catalytic functions have been discovered in recent years. These catalytic DNA molecules, known as deoxyribonucleases, can catalyze many chemical reactions such as cleavage, ligation, and porphyrin ring metallization, and also have DNA kinase activity.
Materials and Instruments
Deoxyribonuclease Move I. Materials and equipment For more product details, please visit Aladdin Scientific website.
Deoxyribozyme cutting buffer Termination buffer Liposomes
Polyacrylamide gel electrophoresis apparatus Radiographic autoradiography apparatus Water baths
1. Polyacrylamide gel electrophoresis device.
2. Radiographic autoradiography apparatus.
3. water bath.
4. deoxyribozyme (modified or unmodified) obtained by chemical synthesis.
5. substrate RNA for in vitro cleavage by deoxyribonuclease is obtained by in vitro transcription.
6. 2X deoxyribozyme cleavage buffer: 50 mmol/L Tris (pH 7.5), 10 mmol/L MgCl2, 150 mmol/L NaCl, 0.01% SDS.
7. Termination buffer: 95% deionized formamide, 0.05% bromophenol blue, 0.05% xylene cyan, 10 mmol/L EDTA.
8. Liposomes.
II. Methods of operation
1. Design of "10-23" deoxyribonuclease.
(1) Use RNA secondary structure analysis software to simulate the secondary structure of the target RNA, and select the main loop and single-stranded protruding region as the preferred cutting site. Although the accuracy of the current secondary structure prediction software needs to be improved, the prediction results can still be used as a reference for selecting the target site and designing the deoxyribozyme.
(2) Selective cleavage reaction occurs between purines (G or A) and pyrimidines (U or C) in the RNA molecule, and the start codon AUG is generally taken as one of the preferred candidate cleavage sites.
(3) For long-stranded RNA substrates, both the length of the deoxyribozyme arm and the stability of the deoxyribozyme substrate binding should be considered. Usually the arm length of deoxyribonuclease is 7~9 nucleotides, when the arm is too short, the binding of deoxyribonuclease and substrate is unstable, which is not conducive to the binding of deoxyribonuclease and substrate; when the arm is too long, the binding is too strong, which is not conducive to the separation of deoxyribonuclease and cleavage product, which also affects the efficiency of the enzyme. Of course, in considering the length of the nuclear arm, but also consider the base composition of the nuclease.
(4) The deoxyribonuclease applied inside the cell can be modified by sulfur modification or methoxyl modification of several nucleotides at both ends of its arms to improve the stability of deoxyribonuclease inside the cell.
(5) Generally, it is necessary to design 3-8 candidate deoxyribonucleases for experimental screening.
(6) Some methods have been developed for screening accessible sites, and it is also possible to design deoxyribonucleases for the accessible sites of the RNA obtained to improve the accuracy of the design (see antisense nucleic acid technology).
2. Validation of in vitro cleavage activity of "10-23" type deoxyribonucleases
(1) Reaction system for deoxyribozyme cleavage of substrate RNA. Take 0.5 nmol/L~0.5 μmol/L deoxyribonuclease and 0.2 μmol/L substrate and add them into 2X deoxyribonucleic cleavage buffer, incubate at 37℃ for 10 min, then mix them together and incubate at 37℃ for 1 h. The reaction system is as follows: (1) "10-23" type deoxyribonuclease cleavage substrate RNA reaction system.
(2) Add the termination buffer to end the reaction, and analyze the cleavage activity of deoxyribonuclease by polyacrylamide gel electrophoresis.
(3) Silver staining or autoradiography (if the RNA substrate is labeled with a radioisotope).
3. Validation of the in vivo inhibitory activity of "10-23" type deoxyribonuclease on target RNA gene expression
(1) Transfection of cells. Add sterile deoxyribonuclease directly into cell culture flasks at a concentration of 2-20 μmol/L. If liposomes are used to transfect cells, the deoxyribonuclease concentration can be reduced to 0.1-1 μmol/L. Set up a negative control.
(2) Collect the cells after 48~72 h of incubation.
(3) Extract RNA, and then use RT-PCR or Northern blot to detect the inhibition of target RNA.
(4) Cells were lysed, proteins were extracted, and Western blot was used to detect changes in the protein levels of target mRMA expression.