Dynamics, thermodynamics, and structural investigations of nucleic acids using site-specific spin-labeling and electron paramagnetic resonance

Using site-specific Electron Paramagnetic Resonance (EPR)-active nitroxide spin-probes, the continuous wave (CW)-EPR spectra of DNA and RNA in aqueous buffers were recorded at several temperatures. In some experiments, the submicrosecond bending dynamics of DNA and RNA were measured. Based on a weakly bending rod (WBR) theory, a sub-microsecond dynamic bending contribution to the total equilibrium persistence length of duplex DNA (500 A) was measured to be 1500 to 1700 A. A modified WBR theory allowed for differentiation between dinucleotide pairs and a 0.85- to 1.15-fold range in flexibilities is reported for six force constant ratios. Methylphosphonated DNA were found to be 1.2 to 1.4 more flexible than the unmodified analogs, depending on placement of the methylphosphonate (MP). Duplex DNA containing a single cis-platin lesion were found to be 1.9-fold more flexible than the same sequences containing no lesion. A 14-mer duplex DNA with a one base bulge showed a 1.99-fold greater local flexibility at the bulge site. 12-mer RNAs containing a three-base bulge and labeled at four different locations around the bulge showed distinctly different dynamics.;The change in dynamics between a free and a protein bound DNA were measured. The DNA in the protein-DNA complex, ADR1-DNA, showed an increase in DNA mobility at the spin-probe, indirect evidence that this two zinc-finger protein may be similar to others in its class in that it may be unwinding the DNA upon binding.;To obtain structural information, the theory of interacting spins was used to design and analyze the interaction between two spin-labels close enough in space to mix their individual spin states. Distance and orientation were measured between two EPR spin-labels placed in a duplex DNA.;Lastly, the CW-EPR spectra were analyzed as a superposition of two conformational states to determine the thermodynamics of base interconversion at a bulge site and the thermodynamics of monomer, dimer, and trimer (n-mer) duplex stacking of a 14-mer duplex DNA in solution.