Influence of dangling thymidine residues on the stability and structure of two DNA duplexes

Abstract
We have employed temperature-dependent UV spectroscopy, circular dichroism (CD), 400-MHz proton nuclear magnetic resonance (NMR), and computer modeling to characterize both structurally and thermodynamically the influence of unpaired, dangling thymidine residues (T) on the thermal stability and melting behavior of two DNA core duplexes. The specific DNA double helices that we have investigated in this work are core duplexes {d(GC)3}2 (I) and {d(CG)3}2 (IV), 3'' dangling T derivatives {d[(GC)3TT]}2 (II) and {d[(CG)3TT]}2 (V), and 5'' dangling T derivatives {d[TT(CG)3]}2 (III) and {d[TT(CG)3]}2 (VI). Our experimental data allow us to reach the folowing conclusions: (1) For both core duplexes (I and IV), the addition of dangling T residues on either the 5'' or 3'' end causes an increase in the optical melting temperature tm (2) For both core duplexes, 5'' dangling T residues induce a greater increase in the optical tm''s than 3'' dangling T residues. (3) For both cores duplexes, the increase in tm induced by the addition of dangling T residues is enthalpic in origin, with 5'' dangling T residues inducing a greater increase in the van''t Hoff transition enthalpy than 3'' dangling T''s. (4) Dangling T residues cause downfield shifts in all of the nonexchangeable aromatic protons of the {d(GC)3}2 core duplex (I), with the 5'' T residues inducing the largest shifts. For the most part, this trend doses not hold with the {d(GC)3}2 core duplex (IV). (5) For both core duplexes, the addition of dangling T residues causes an increase in the NMRtm''s of almost all the nonexchangeable aromatic protons of the core duplex. This increase generally is greater when the dangling T residues are attached to the 5'' end of the core duplex. (6) For both core duplexes, the unpaired T residues exhibit NMR melting temperatures that are close to the global optical tm''s of the duplex structures. (7) At low temperatures, the dangling T residues exhibit chemical shifts that are consistent with their stacking on the core duplex. (8) CD spectra both core duplexes and their corresponding dangling T derivatives are consistent with all six structures existing in the B conformation. These experimental results, in conjunction with computer modeling, support a picture in which the dangling T residues increase the thermal stability of their core duplexes by causing an increase in enthalpically favorable interactions. We propose that these enthalpically favorable interactions result from two effects: (i) stacking of the dangling T residues on the base-paired termini of the core duplex and (ii) an increase in stacking within the core duplex induced by the presence of the dangling ends. Significantly, the increase in thermal stability and transition enthalphy induced by the dangling thymidine residues is greatest when the unpaired residues are positioned on the 5'' end of the core duplexes. The Turner group has shown that dangling ends on RNA core duplexes exhibit the opposite positional dependence [Petersheim, M., and Turner, D.H. (1983) Biochemistry 22, 256-263; Freier, S.M., Alkema, D., Sinclair, A., Neilson, T., and Turner, D. H. (1985) Biochemistry 24, 4533-4539], with 3'' rather than 5'' dangling residues exerting the greatest influence.