Reactivity of parallel-stranded DNA to chemical modification reagents

Abstract

Four 25-nt long oligonucleotides containing dA and dT (D1, D2, D3, and D4) which are capable of forming parallel-stranded (ps) or antiparallel-stranded (aps) duplexes have been synthesized [Rippe, K., Ramsing, N.B. and Jovin, T.M. (1989) Biochemistry 28, 9536-9541]. In the present study, the OsO4-pyridine complex (Os,py), diethyl pyrocarbonate (DEPC), KMnO4, and the 1,10-phenanthroline-cuprous complex [(OP)2Cu+] were used to investigate the conformation-dependent reactivity of ps, aps, and single-stranded (ss) oligonucleotides. The products were analyzed by polyacrylamide gel electrophoresis with single-nucleotide resolution. The results confirm the duplex nature of the ps combinations of oligonucleotides and reveal structural differences in comparison with the aps molecules. Under conditions in which ss-DNA is substantially sensitive to Os,py, both the ps and aps duplexes are very unreactive. A similar result was observed with KMnO4 and DEPC, although with the latter reagent the modification pattern of the labeled strands D1* and D4* was slightly different for the parallel than for the antiparallel duplex. The (OP)2Cu+ complex efficiently cleaves the aps but not the ps duplex and shows a preference for TAT steps. We also tested the effect of monovalent and divalent cation concentration on the chemical reactivity of the ps, aps and ss species. Elevated NaCl concentration leads to a dramatic increase in the Os,py and KMnO4 modification of ss molecules and the ps, but not the aps, duplex. We attribute the apparent reaction with ps-DNA to a destablization of this conformation under the conditions of reaction. In contrast, all reactions with DEPC are somewhat depressed at high salt concentrations. The effects of MgCl2 and temperature on the chemical reactivity with Os,py were also determined. The helix-coil transition of both the ps and the aps duplexes can be monitored by chemical modification with the OsO4-pyridine reagent.