METHOD FOR DETERMINING THE ACTIVITY OF EXCISION REPAIR ENZYMES OF DNA BASES IN HUMAN CELLS Russian patent published in 2023 - IPC G01N33/52 G01N21/64 

FIELD: biochemistry.

SUBSTANCE: the invention relates to biochemistry. A method is described for determining the activity of excision repair enzymes of DNA bases of the group: АРЕ1, UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1 or OGG1 in human cells. It includes mixing of substrate, buffer and cell extract in the ratio of 1:1 and recording changes in the fluorescence intensity of the substrate using a fluorimeter, followed by processing the obtained kinetic curves using a program. The method differs in that the following DNA-probes are used as a substrate: 5-FAM-GCTCA(F)GTACAGAGCTGTTTTTCAGCTCTGTACGTGAGCps-BHQ1-3, 5-FAM-GCTCA(U)GTACAGAGCTGTTTTTCAGCTCTGTACGTGAGCps-BHQ1-3, 5-FAM-GCTCA(εA)GTACAGAGCTGTTTTTCAGCTCTGTACGTGAGCps-BHQ1-3, 5-FAM-GCTCA(Tg)GTACAGAGCTGTTTTTCAGCTCTGTACGTGAGCps-BHQ1-3 or 5-FAM-GCTCA(oxoG)GTACAGAGCTGTTTTTCAGCTCTGTACGTGAGCps-BHQ1-3, which are oligodeoxyribonucleotides forming a hairpin and carrying a FAM/BHQ1 FRET pair at the ends of the chain. At the same time, the 3-terminal internucleotide phosphate group is replaced with a thiophosphate one to prevent nonspecific 3-5-exonuclease degradation of the DNA probe in the cell extract.

EFFECT: increasing the accuracy and reliability of the results of determining the activity of DNA repair enzymes in humans, expanding the functionality of the known method, and applying this method to analyze activity in human cells.

5 cl, 5 dwg, 1 tbl, 5 ex