Nucleic Acid Structure Characterization by Small Angle X‐Ray Scattering (SAXS)

Jordan E. Burke1, Samuel E. Butcher1

1 Department of Biochemistry, University of Wisconsin, Madison, Wisconsin
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 7.18
DOI:  10.1002/0471142700.nc0718s51
Online Posting Date:  December, 2012
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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 przovided. Curr. Protoc. Nucleic Acid Chem. 51:7.18.1‐7.18.18. © 2012 by John Wiley & Sons, Inc.

Keywords: nucleic acid structure; SAXS; NMR; RNA folding; molecular modeling

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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Sample Preparation
  • Basic Protocol 2: SAXS Data Acquisition
  • Basic Protocol 3: SAXS Data Processing and Analysis
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Sample Preparation

  • Nucleic acid sample
  • SAXS buffer
  • Tris base, 99% pure
  • 0.3 M sodium acetate, pH 5.2
  • Ethanol
  • Potassium hydroxide (KOH), 89% pure
  • Liquid nitrogen
  • 15‐mL Centrifuge tubes (Falcon)
  • 90°C heating block
  • 1‐L, 0.22‐µm filter flask (Millipore or GE Healthcare)
  • 0.5‐ and 15‐mL centrifugal filters, regenerated cellulose (Amicon, Ultra)
  • Centrifuge
  • HiLoad 16/60 Superdex 75 or 200 prep‐grade column (GE Healthcare)
  • FPLC system (such as ÄKTAFPLC) equipped with online UV monitor at 254 nm, 100‐µL loading loop, and fraction collector
  • 100‐µL syringe with 23‐mm, 22‐G, blunt‐end needle (Hamilton)
  • Nanodrop ND‐1000 spectrophotometer or other UV spectrophotometer
  • 0.5‐ to 3‐mL dialysis cassettes (Slide‐A‐Lyzer, Thermo Scientific)
  • 0.20‐µm syringe filters (Sartorius Stedim)
  • 0.5‐ or 1.5‐mL microcentrifuge tubes (Eppendorf)
  • 0.22‐µm SpinX cellulose acetate filters (CoStar)
  • Microcentrifuge
  • Additional reagents and equipment for non‐denaturing polyacrylamide gel electrophoresis (PAGE; unit 10.4)

Basic Protocol 2: SAXS Data Acquisition

  • Silver(I) behenate (AgBe)
  • Reserved filtered dialysate (see protocol 1)
  • Purified nucleic acid (see protocol 1)
  • Standard sample: lysozyme (Fluka), glucose isomerase (Hampton research), or standard RNA sample (Table 7.18.1)
  • SAXS instrument (∼67 to 100 cm sample‐to‐detector distance with 1‐ to 1.5‐mm quartz capillary and glassy carbon)
  • Additional detector for WAXS (30 cm sample‐to‐detector distance), optional
    Table 7.8.1   MaterialsSAXS Standard Samples

    Molecule (conditions) Mol. wt. (kDa) Rg (Å) (± 1) Reference
    Lysozyme (8 mg/mL, 40 mM acetic acid, pH 4.0, 50 mM NaCl) 14.7 13 Voets et al. ( )
    Glucose isomerase (5 mg/mL, 100 mM Tris⋅Cl, pH 8.0, 1 mM MgCl 2) 173 33 Kozak ( )
    Tetraloop‐receptor RNA (PDB ID: 2JYJ) (1 mg/mL, 50 mM Tris⋅Cl, pH 7.0, 150 mM NaCl, 2 mM MgCl 2) 28.2 25 Zuo et al. ( )

Basic Protocol 3: SAXS Data Processing and Analysis

  • ATSAS software package (http://www.embl‐ for the appropriate operating system(s)
  • Sample data files: 2JYJ_APS_SAXS_WAXS_1mgml.dat and 2JYJ_Nanostar_SAXS_1mgml_4h.dat (see Supplemental Files at
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Literature Cited

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Supplementary Material

Sample data files for the tetraloop-receptor RNA complex: