Characterization of labeled oligonucleotides using enzymatic digestion and tandem mass spectrometry

A simple and powerful method for the determination of labeling sites on oligodeoxynucleotides (ODN) has been developed. The method is based on the finding that nuclease P1 (NP1) digestions of label-containing ODNs produce site-specific products: 5′-labeled ODNs produce label-nucleotide (L-N); 3′-labeled ODN produces phosphorylated label (pL); and a label in between the ODN termini produces pL-N. Mass spectrometry spectra of these products from the digestion mixture can be easily utilized 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 products generate site-specific fragmentation patterns upon collision-induced dissociation. These patterns can be easily recognized and used for the identification of labeling sites of ODNs with unknown label structures. 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 single or multiple labels, and for ODNs with the same or different labels within an ODN.