Oligodeoxynucleotides containing 4-thiothymidine and 6-thiodeoxyguanosine as affinity labels for the Eco RV restriction endonuclease and modification methylase

摘要

4-Thlothymidine and 6-thiodeoxyguanosine were incorporated into synthetic dodecamers containing the recognition site d(GATATC) of the enzymes Eco RV endonuclease and Eco RV methyltransferase. Upon irradiation with long wavelength UV light (340?360 nm), these oligodeoxynucleotides were photochemic-ally crosslinked to both enzymes. The yields were up to 35% with the methyltransferase, but lower (up to 6%) with the endonuclease. Oligodeoxynucleotides containing 4-thiothymidine generally gave higher yields of crosslinking than those containing 6-thiodeoxyguanosine. Although both specific (i.e. those containing the d(GATATC) sequence) and non-specific (lacking this sequence) photoreactive oligodeoxynucleotides gave rise to crosslinked products, the use of a non-reactive, competitive substrate oligodeoxynucleotlde suppressed the crosslinking, indicating that the reaction takes place at the enzymes' active sites. Oligodeoxynucleotides containing 4-thiocyanatothymidine or 6-thio-cyanatodeoxyguanosine were also prepared by treatment of the title oligomers with CNBr and KCN. The dodecamers containing 4-thiocyanatothymidine were found to covalently modify both enzymes under study, with levels of crosslinking reaching up to 42% with the endonuclease and up to 12% with the methyltransferase. No crosslinking was observed with oligodeoxynucleotides containing 6-thiocyanatodeoxy-guanosine.