Characterization of Page Bands from 3′-Labeled Short DNA Fragments Resulting from Oxidative Cleavage by “Mn-TMPyP/KHSO₅”. Drastic Modifications of Band Migrations by 5′-End Sugar Residues

Summary

Recent experiments with the chemical nuclease Mn-TMPyP/KHSO₅ showed highly specific cleavage on both 3′-sides of AT base pair triplets of double stranded oligonucleotides.¹ Hydroxylation at the 5′-carbon of a deoxyribose represents the initial damage and leaves a 3′-phosphate and a 5′-aldehyde at the ends. Careful examination of PAGE bands resulting from oxidative cleavage of two selected double-stranded oligodeoxyribonucleotides which have been labeled either at 5′ or 3′ ends indicated that 5′-end-labeled fragments with a 3′-phosphate terminus were unsensitive to different chemical or thermal treatments and migrated according to the corresponding fragments obtained by Maxam-Gilbert sequencing. Whereas 3′-end-labeled fragments with a 5′-aldehyde terminus (-CHO) migrated differently after reduction (-CH₂OH), oxidation (-COOH), heating (5′-P terminus and release of free base and furfural), heating and alkaline phosphatase treatment (5′-OH terminus and loss of free base, furfural and inorganic phosphate). The precise knowledge of the chemical lesion at the oxidized site of DNA allowed us to localize the exact position of the break and attribute the structures of the different fragments observed on PAGE analysis after the manganese porphyrin-mediated cleavage of two ODNs containing A.T rich sequences.