CHARACTERIZATION OF LABELED OLIGONUCLEOTIDES USING ENZYMATIC DIGESTION AND TANDEM MASS-SPECTROMETRY

A simple and powerful method for the determination of labeling sites o n oligodeoxynucleotides (ODN) has been developed. The method is based on the finding that nuclease P1 (NP1) digestions of label-containing O DNs produce site-specific products: 5'-labeled ODNs produce label-nucl eotide (L-N); 3'-labeled ODN produces phosphorylated label (pL); and a label in between the ODN termini produces pL-N. Mass spectrometry spe ctra of these products-from the digestion mixture can be easily utiliz ed for structural verification of labeled ODNs such as DNA probes. We also developed a method for the determination of the labeling sites of ODNs with unknown label structures. In this method, NP1 digestion pro ducts generate site-specific fragmentation patterns upon collision-ind uced dissociation. These patterns can be easily recognized and used fo r the identification of labeling sites of ODNs with unknown label stru ctures. When an ODN is internally labeled, phosphodiesterase digestion may be used to determine the exact labeling site (sequence location). It was demonstrated that these methods can be applied for ODNs with s ingle or multiple labels, and for ODNs with the same or different labe ls within an ODN. (C) 1998 American Society for Mass Spectrometry.