Nucleic acid structure characterization by small angle X-ray scattering (SAXS)

Abstract

Small angle X-ray scattering (SAXS) is a powerful method for investigating macromolecular structure in solution. SAXS data provide information about the size and shape of a molecule with a resolution of ∼2 to 3 nm. SAXS is particularly useful for the investigation of nucleic acids, which scatter X-rays strongly due to the electron-rich phosphate backbone. Therefore, SAXS has become an increasingly popular method for modeling nucleic acid structures, an endeavor made tractable by the highly regular helical nature of nucleic acid secondary structures. Recently, SAXS was used in combination with NMR to filter and refine all-atom models of a U2/U6 small nuclear RNA complex. In this unit, general protocols for sample preparation, data acquisition, and data analysis and processing are given. Additionally, examples of correctly and incorrectly processed SAXS data and expected results are provided.

Figure 1

General scheme for characterization of RNA using small angle X-ray scattering. Ovals indicate procedures discussed in other publications (see Strategic Planning and Commentary).

Figure 2

A. X-ray scattering of a 28.2 kDa RNA at 1 mg/ml concentration from either a synchrotron source (Advanced Photon Source or APS) or bench top source (Bruker Nanostar) for 4 hours (see Basic Protocol 2). Synchrotron data were averaged over a total of 3 min. from 4 individual 45 sec. exposures. The bench top data were collected from a single 4 hour exposure. B. SAXS and WAXS data from the same RNA demonstrating accurate and inaccurate buffer subtraction (see Basic Protocol 3).

Figure 3

Guinier plot of an aggregated protein (black diamonds), an RNA with interparticle repulsion (gray triangles) or an RNA sample of good quality (gray squares) (see Basic Protocol 3).

Figure 4

Kratky plot of three different RNA molecules with unique global folds (see Basic Protocol 3).

Figure 5

Pair distance distribution functions of the same three RNA molecules as in Fig. 4.

Figure 6

Ab initio structure (gray spheres) of a 28.2 kDa RNA with the jointly refined NMR/SAXS structure (Zuo et al., 2008) superimposed using the Supcomb20 algorithm (Kozin and Svergun, 2001).