Influence of loop residues on the relative stabilities of DNA hairpin structures

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Abstract

We have determined the relative stabilities and melting behaviors of DNA hairpin structures as a function of the nonbonded residues in the loop. The specific family of hairpin structures we investigated in this work is formed by the 16-mer sequence d[CGAACG(X)4CGTTCG], where X is deoxyadenosine, deoxycytidine, deoxyguanosine, or deoxythymidine. As shown below, this 16-mer can fold back on itself to form a family of DNA hairpin structures that possess a common hexameric stem duplex and a nonbonded loop of 4 nucleotides. For the hairpin structures investigated in this work, we varied the loop composition from all purine residues to all pyrimidine residues X CGAACG X GCTTGC X X. We thermodynamically characterized the relative stabilities and melting profiles of these hairpin structures by a combination of spectroscopic and calorimetric techniques. To establish a thermodynamic 'baseline', we also conducted parallel studies on the isolated hexameric duplex d[(CGAACG)·(CGTTCG)], which corresponds to the common stem duplex present in each hairpin structure. Our spectroscopic and calorimetric data reveal the following: (i) The hairpin structure with four dT residues in the loop exhibits the highest melting temperature, while the corresponding hairpin structure with four dA residues in the loop exhibits the lowest melting temperature. (ii) The free energy data at 25°C reveal the following order of DNA hairpin stability for the four structures studied here: T loop > C loop > G loop > A loop. In other words, the pyrimidine-looped hairpins of four residues are more stable than the purine-looped hairpin. (iii) The loop-dependent order of hairpin stability is paralleled by a similar trend in the calorimetrically determined transition enthalpies for hairpin disruption. Thus, the enhanced stability of the pyrimidine-looped hairpin structures relative to purine-looped hairpin structures is enthalpic in origin. To develop insight into the molecular basis for the thermodynamic differences, proton NMR spectroscopy was used to probe for structural disparities between the most stable hairpin structure (T loop) and the least stable hairpin structure (A loop). Two-dimensional nuclear Overhauser enhancement spectroscopy revealed connectivities between the residues in the stem duplexes of both hairpin structures that are consistent with B-form DNA. In addition, the nonbonded residues in both the T and A loops exhibited the same connectivity patterns. However, on the 5' side of the stem-loop junction, the T-loop residue exhibited a connectivity with the adjacent base pair of the stem duplex that is not observed for the corresponding A-loop residue. This difference in connectivities at the stem-loop junction may provide a structural basis for our observation that the T-looped hairpin structure is more stable than the corresponding A-looped hairpin structure.

Original languageEnglish (US)
Pages (from-to)6242-6246
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume85
Issue number17
DOIs
StatePublished - 1988

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