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.