The Impact of Base Stacking on the Conformations and Electrostatics of Single-Stranded DNA

Single-stranded DNA (ssDNA) is notable for its interactions with ssDNA binding proteins (SSBs) during fundamentally important biological processes including DNA repair and replication. Previous work has begun to characterize the conformational and electrostatic properties of ssDNA in association with SSBs. However, the conformational distributions of free ssDNA have been difficult to determine. To capture the vast array of ssDNA conformations in solution, we pair small angle X-ray scattering with novel ensemble fitting methods, obtaining key parameters such as the size, shape and stacking character of strands with different sequences. Complementary ion counting measurements using inductively coupled plasma atomic emission spectroscopy are employed to determine the composition of the ion atmosphere at physiological ionic strength. Applying this combined approach to poly dA and poly dT, we find that the global properties of these sequences are very similar, despite having vastly different propensities for single-stranded helical stacking. These results suggest that a relatively simple mechanism for the binding of ssDNA to non-specific SSBs may be at play, which explains the disparity in binding affinities observed for these systems

Both helix topology and counterion distribution contribute to the more effective charge screening in dsRNA compared with dsDNA

The recent discovery of the RNA interference mechanism emphasizes the biological importance of short, isolated, double-stranded (ds) RNA helices and calls for a complete understanding of the biophysical properties of dsRNA. However, most previous studies of the electrostatics of nucleic acid duplexes have focused on DNA. Here, we present a comparative investigation of electrostatic effects in RNA and DNA. Using resonant (anomalous) and non-resonant small-angle X-ray scattering, we characterized the charge screening efficiency and counterion distribution around short (25 bp) dsDNA and RNA molecules of comparable sequence. Consistent with theoretical predictions, we find counterion mediated screening to be more efficient for dsRNA than dsDNA. Furthermore, the topology of the RNA A-form helix alters the spatial distribution of counterions relative to B-form DNA. The experimental results reported here agree well with ion-size-corrected non-linear Poisson–Boltzmann calculations. We propose that differences in electrostatic properties aid in selective recognition of different types of short nucleic acid helices by target binding partners