Conformational Equilibria of Bulged Sites in Duplex DNA Studied by EPR Spectroscopy

Abstract

Conformational flexibility in nucleic acids provides a basis for complex structures, binding, and signaling. One-base bulges directly neighboring single-base mismatches in nucleic acids can be present in a minimum of two distinct conformations, complicating the examination of the thermodynamics by calorimetry or UV-monitored melting techniques. To provide additional information about such structures, we demonstrate how electron paramagnetic resonance (EPR) active spin-labeled base analogues, base-specifically incorporated into the DNA, are monitors of the superposition of different bulge-mismatch conformations. EPR spectra provide information about the dynamic environments of the probe. This information is cast in terms of "dynamic signatures" that have an underlying basis in structural variations. By examining the changes in the equilibrium of the different states across a range of temperatures, the enthalpy and entropy of the interconversion among possible conformations can be determined. The DNA constructs with a single bulge neighboring a single-base mismatch ("bulge-mismatches") may be approximately modeled as an equilibrium between two possible conformations. This structural information provides insight into the local composition of the bulge-mismatch sequences. Experiments on the bulge-mismatches show that basepairing across the helix can be understood in terms of purine and pyrimidine interactions, rather than specific bases. Measurements of the enthalpy and entropy of formation for the bulgemismatches by differential scanning calorimetry and UV-monitored melting confirm that the formation of bulge-mismatches is in fact more complicated than a simple two-state process, consistent with the base-specific spectral data that bulge-mismatches exist in multiple conformations in the pre-melting temperature region. We find that the calculations with the nearest-neighbor (NN) model for the two likely conformations do not correlate well with the populations of structures and thermodynamic parameters inferred from the base-specific EPR dynamics probe. We report that the base-specific spin probes are able to identify a bi-stable, temperature dependent, switching between conformations for a particular complex bulged construct.* To whom correspondence should be addressed: Phone: (206) 543-1773, FAX: (206) 616-6250, email: robinson@chem.washington.edu SUPPORTING INFORMATION AVAILABLE:Figures illustrating: two-component fit of EPR spectra; calculation of α from UV melting curves; rigid 14-mer simulated, FAST, and SLOW spectra at 0, 20, and 40 °C; results of fitting spectra to FAST and "closest match Ç/X" spectra; NN calculation comparisons; spectra and analysis of doubly spin-labeled samples for distance measurements. This material is available free of charge via the Internet at

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