Determination of photomodified oligodeoxynucleotides by exonuclease digestion, matrix-assisted laser desorption/ionization and post-source decay mass spectrometry.
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.
Zhang
L K
LK
Department of Chemistry, Washington University, St Louis, Missouri 63130, USA.
Ren
Y
Y
Rempel
D
D
Taylor
J S
JS
Gross
M L
ML
D005092:Exonucleases / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry*; D010777:Photochemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D009841:Oligonucleotides; D005092:Exonucleases