Determination of platinated purines in oligoribonucleotides by limited digestion with ribonucleases T1 and U2

Abstract

Platinum complexes which are known to react preferentially with guanine (G) and adenine (A) bases of oligonucleotides can be used as tools to analyze their tertiary structures and eventually to cross-link them. However, this requires efficient methods to allow the identification and quantification of the corresponding adducts which have so far been developed only for oligodeoxyribonucleotides. Maxam–Gilbert type digestions cannot be used for RNAs and HPLC techniques would require too large amounts of expensive material for separation and further characterization. We report a method to determine platination sites on oligoribonucleotides based on the cleavage activity of ribonucleases T1 and U2. To test the method, these enzymes were first used under conditions of limited digestion on 5-mer oligoribonucleotides platinated at a single defined purine. The phosphodiester bond on the 3 ′ side of platinated G or A appeared fully resistant to cleavage by ribonuclease T1 or U2, respectively. An inhibitory effect was also observed due to neighboring platinated purines, which decreases with their distance (−2, −1, +1, +2) from the cleavage site and with the enzyme concentration. The method allowed the identification and quantification of the platination sites of a 17-mer oligoribonucleotide, based on the analysis of the mixture of monoplatinated adducts.