Counting Ions around DNA with Anomalous Small-Angle X-ray Scattering

Abstract

The majority of charge compensating ions around nucleic acids forms a diffuse counterion "cloud" that is not amenable to investigation by traditional methods that rely on rigid structural interactions. Although various techniques have been employed to characterize the ion atmosphere around nucleic acids, only Anomalous Small-Angle X-ray Scattering (ASAXS) provides information about the spatial distribution of ions. Here, we present an experimentally straightforward extension of ASAXS to count the number of ions around nucleic acids.The number and spatial distribution of small, positively-charged ions around highly negatively charged DNA or RNA provide entropic contributions to the free energy of binding in the cell1. Thus, salt concentrations and condensed counterions greatly affect the conformation, stability and binding affinity of nucleic acids2 -4. Although much attention has focused on the study of specifically bound charged molecules (e.g. bound ions, polyamines, charged protein surfaces), the majority of charge compensating ions form a diffuse counterion 'cloud'5 that is not amenable to investigation by traditional methods like x-ray crystallography or ligand-binding chemistry. Although various techniques have been employed to characterize the ion atmosphere around nucleic acids, only Anomalous SmallAngle X-ray Scattering (ASAXS) provides information about the spatial distribution of ions6. In fact, we recently used ASAXS to highlight differing ion distributions around comparably sequenced double-stranded DNA and RNA helices7.Here, we present an experimentally straightforward extension of ASAXS to count the number of ions around nucleic acids while measuring the ion-nucleic acid spatial distribution. The technique is simple and, in principle, can easily be implemented in any energy-tunable SAXS beamline with energy resolution appropriate for ASAXS or multiplewavelength anomalous diffraction (MAD) experiments. Application of this technique requires absolute calibration of two experimental parameters: scattering intensities and changes in the near-edge scattering factors resulting from resonant effects. These quantities can be readily obtained from a variety of calibration standards8 , 9. Most critically, ion counting via ASAXS does not rely on computing small differences between ion numbers in two solutions (e.g. one with and the other without the nucleic acid), as is often needed in ion-counting experiments involving ion-sensitive dyes10 and buffer-exchange11. All the necessary information is derived from measurements on the same sample, thereby extending ion-counting into regimes where equilibrium dialysis may not be applicable, for example when characterizing nucleic acids in difficult-to-dialyze osmolyte solutions. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptTo demonstrate the feasibility of this approach, we measured SAXS signals at several x-ray energies, close to but below the absorption edge of the ions of interest. Previous studies show that the number and dis...

Extracting water and ion distributions from solution x-ray scattering experiments

“…The proposed analysis complements recent experimental techniques (such as ion counting 12 and anomalous SAXS 16 ) by providing not only the number excess of particles but also their distribution in real space. It is, however, worth restating the fact that our decomposition requires an independent knowledge of the structure of solute, which is assumed to be rigid; it cannot be used (in its current form) for systems with significant conformational heterogeneity or disorder, primarily because uncertainties in computing the scattering from the solute would probably lead to large uncertainties in extracting solvent information.…”

“…Changing the energy of the incident beam in an anomalous SAXS (ASAXS) experiment is one approach, varying the atomic scattering factor of a given ion. [14][15][16]18,34,35 Another approach uses heavy ion replacement, assuming the ion and water distributions are similar for the same type of ions (alkalies, for example). 14,20 By doing this, onlyF ion is allowed to vary while the co-ion and hydration terms are fixed.…”

“…11,12 The q = 0 limit of small-angle X-ray scattering (SAXS) can provide similar excess counts for both water and ions. 13 Anomalous small angle X-ray scattering (ASAXS) data in principle yield additional information about the extent of perturbations of the ion/water environment, [14][15][16][17] but the ASAXS signal is known to be intertwined with all components in the system, complicating the analysis. 13,18 Efforts to extract the contribution from ions to ASAXS profiles date back to 2003 with the work of Ballauff and coworkers.…”

“…Both the excess number (as in ASAXS and ioncounting experiments 12,16 ) and (low-resolution) information about the actual distribution can be obtained for ions and water molecules. The correlation between solute-ion and solute-water can be displayed in both reciprocal space (as partial intensities) and real space (as interatomic distribution functions).…”

Accurate small and wide angle x-ray scattering profiles from atomic models of proteins and nucleic acids

“…The ASAXS measurements were done at the CHESS Cline station as described in our previous work 46,47 with the following experimental upgrades: a Pilatus 100 K photoncounting area-detector was used for collecting scattered images, the liquid samples was placed in a 3 mm diameter quartz capillary (Hampton Research, Aliso Viejo, CA) and oscillated during x-ray exposures to prevent radiation damage, a semitransparent beamstop consisting of a stack of Molybdenum foils (Goodfellow, Coraopolis, PA) was mounted inside the flight-tube and a quartz flow cell with a 0.1-mm path length (Starna Cells, Atascadero, CA) was placed in-line with the x-ray capillary to facilitate accurate DNA concentration measurements using a fiber-coupled UV spectrometer (Avantes, Broomfield, CO). The sample to detector distance was 0.96 m (0.024 < q < 0.5 Å −1 ).…”