Highly efficient labeling of DNA polymerases by a binary system of photoaffinity Reagents

摘要

A binary system of photoaffinity reagets was proposed earlier for highly efficient labeling of DNA polymerases by 5'-[~(32)P]DNA primers.In the present study we demonstrate the feasibility of this approach to increase the efficinecy of DNA polymerase labeling.A photoactive 2,3,5,6-tetrafluoro-4-azidobenzoyl (FAB) group was incorporated at the 3'-end of 5'-[[~(32P]DNA primers synthesized by DNA polymerase beta or Tte in the presence of one of the dTTP analogs-FAB-4- dUTP,FAB-9-dUTP,or FAB-4-ddUTP.The reaction mixture was irradiated by light with wave length of 334-365 nm (direct labeling) or 365-450 nmin the presence of photosensitizer,one of dTTP analogs containing a pyrene moiety,Pyr-6-dUTP of Pyr-8-dUTP.In the case of the binarysystem of photoaffinity reagents,a FAB group is activated by energy transfer from sensitizer localized in the dNTP-binding site of DNA polymerase in the triple complex,comprised by reagent,DNA polymerase,and Pyr-6(8)-dUTP.Direct activation of the FAB group under these conditions is negligible.The most efficient photolableing of DNA polymerases wasobserved with a primer containing a FAB-4-dUMP group at the 3'-end,and Pyr-6-dUTP as a photosensitizer.Using 10-fold molar excess of photoreagent to DNA polymerase beta,the labeling efficiency was shown to achieve 60%,whichis 2-fold higher than the efficiency of the direct DNA polymerase labeling under harsher conditions (334-365nm).