Matrix-assisted laser desorption/ionization mass spectrometry for locating abasic sites and determining the rates of enzymatic hydrolysis of model oligodeoxynucleotides.
A method using a combination of exonuclease enzymatic digestion and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was developed to locate model abasic sites in a series of model 21-base oligodeoxynucleotides in which a nucleobase was replaced by a hydrogen atom. The exonuclease digestion rate, with either snake venom phosphodiesterase (SVP) or bovine spleen phosphodiesterase (BSP), clearly slows as the digestion approaches the abasic sites and stops as it reaches it. An oligodeoxynucleotide containing an abasic site in which OH replaces the nucleobase shows similar results. MALDI mass spectra taken at appropriate times during the course of hydrolysis are the basis for rate measurements, and the kinetics also reveal the location of the abasic site. A mathematical treatment of the time-dependent MALDI data was implemented to obtain rate curves and rate constants for the enzymatic digestion of both modified and unmodified oligodeoxynucleotides.
Zhang
L K
LK
Department of Chemistry, Washington University, St. Louis, Missouri 63130, USA.
Rempel
D
D
Gross
M L
ML
D001080:Apurinic Acid / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000378:metabolism*; D011119:Polynucleotides / Q000737:chemistry / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D009841:Oligonucleotides; D011119:Polynucleotides; C001790:apyrimidinic acid; D001080:Apurinic Acid; D004247:DNA; D005092:Exonucleases; C022650:spleen exonuclease; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom