Base specificity of oligonucleotide digestion by calf spleen phosphodiesterase with matrix-assisted laser desorption ionization analysis.
Calf spleen phosphodiesterase cleaves oligonucleotide strands in a stepwise manner from the 5' end and can be used in combination with matrix-assisted laser desorption ionization (MALDI) mass spectrometry to perform ladder sequencing. The relative intensities of ladder peaks in the mass spectra of a series of 5-mers and 7-mers show that the rate of digestion is influenced by strand sequence. Sequences terminating in A or G at the 5' end are found to react two to three times faster than sequences terminating in C or T. The reactivity of the terminal base is also influenced by the sequence beyond the 5' end. When the third base from the 5' end is A or G, removal of the first and second bases is faster than when the third base is C or T. A method is described which permits reaction rates to be quantitatively determined from the time dependences of ladder peaks in the MALDI spectra. A similar approach could be used for mechanistic studies.
Bentzley
C M
CM
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716, USA.
Johnston
M V
MV
Larsen
B S
BS
D000818:Animals; D001483:Base Sequence; D002417:Cattle; D066298:In Vitro Techniques; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013154:Spleen / Q000201:enzymology; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D010727:Phosphoric Diester Hydrolases