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

A fast method to detect and sequence photomodifted oligodeoxynucleotides (O DNs) by exonuclease digestion and matrix-assisted laser desorption/ionizati on (MALDI) mass spectrometry (MS) is reported. Upon treatment of modified O DNs with both phosphodiesterase I and phosphodiesterase H, the digestion st ops at the sites of photomodification. Post-source decay (PSD) of MALDI-pro duced 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 symmetr ical or identical compositions at the 3' and 5' ends, respectively. Studies have also been done to follow the kinetics for enzyme degradation of photo modified 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. (C) 2001 American So ciety for Mass Spectrometry.