Interpreting wide-angle X-ray scattering data using DNA duplexes as model systems

Source abstract

Small-angle X-ray scattering (SAXS) provides important information about global biomolecular solution structures yet is resolution limited. Extension to higher scattering angles through wide-angle X-ray scattering (WAXS) extends the resolution of solution measurements to single ångströms; however, interpretation of WAXS profiles is challenging. Here we establish a practical framework for interpreting WAXS measurements in terms of repeated real-space distances and specific structural motifs in nucleic acids. We compare experimentally measured scattering profiles from two 25-base-pair DNA duplexes, an AT-rich construct and a mixed-sequence duplex. Their SAXS profiles are similar but they exhibit strikingly different WAXS signatures. By systematically varying the reciprocal-space cutoff prior to indirect Fourier transforms, we show how increasing information content sharpens real-space resolution and reveals periodic features associated with base stacking and duplex geometry. Real-space distance distributions identify the dominant atomic contributors to these features and directly connect them to backbone separations, groove widths, and long-range stacking periodicities that vary in a sequence-dependent manner. Together, these results establish WAXS as a powerful tool for probing nucleic acid architecture and, in conjunction with time-resolved measurements, dynamics, and demonstrate that WAXS measurements provide a robust and generalizable route to extracting high-resolution structural information from solution measurements.