Determination of photomodified oligodeoxynucleotides by exonuclease digestion, matrix-assisted laser desorption/ionization and post-source decay mass spectrometry

Abstract

A fast method to detect and sequence photomodified oligodeoxynucleotides (ODNs) by exonuclease digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is reported. Upon treatment of modified ODNs with both phosphodiesterase I and phosphodiesterase II, the digestion stops at the sites of photomodification. Post-source decay (PSD) of MALDI-produced ions from two enzymatic digestion end products distinguishes isomers such as 5-d(T[cis-syn]TAAGC) and 5-d(CGAAT[cis-syn]T), which have symmetrical or identical compositions at the 3 and 5 ends, respectively. Studies have also been done to follow the kinetics for enzyme degradation of photomodified ODNs. The calculated rate constants from a mathematical treatment of the time-dependent MALDI data clearly show that the enzymatic digestion rate slows as the enzyme approaches the modified site.