Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

Summary

This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.

Abstract

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (•OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.¹ When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA¹ and RNA² with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein¹ and RNA-protein complexes. Equilibrium³ and kinetic⁴ transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., 'single-hit kinetics') results in the uniform sampling of each nucleotide of the polymer.⁵

In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H₂O₂ (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O₂ (we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.

Protocol

1. Preparation of Footprinting Reagents

1. Prepare a 10x reaction buffer containing 100 mM sodium cacodylate, 1 mM EDTA, and 1 M KCl. Adjust the pH to 7.4. Filter the buffer using a 0.2 μM acetate filter device (Nalgene). Remark: do not pipet RNA directly into 10x buffer.
2. Prepare the titration reaction mix for each reaction as indicated in Table 1. The volume of the titration mix (1x buffer and Mg(II) at the desired concentration) should be 90 μl, before adding 10μl of RNA in 1x buffer.
3. Prepare a RNAse T1 digestion buffer containing 6.63M Urea, 20mM sodium citrate, 1mM EDTA, 0.25 μg/μl tRNA, 0.025% xylene cyanol, and 0.025% bromphenol blue....

Discussion

Hydroxyl radical footprinting is a valuable tool to assess the solvent accessible surface area of nucleic acids. Qualitative and quantitative formation of tertiary structure¹⁴ can be followed as a function of parameters such as ion type and concentration, pH, temperature, binding proteins or folding co-factors. The compelling combination of a straight forward and inexpensive protocol and the resulting solvent accessibility and folding information on a single-nucleotide level makes this method very attractive. .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Name	Company	Cat#
Sodium Cacodylate(Caution! Toxic)	Sigma-Aldrich	C4945-25g
EDTA (0.5 M)	Ambion	AM9260G
DEPC treated water	Ambion	AM9915G
Sodium Acetate (3 M)	Ambion	AM9740
MgCl2 (1 M)	Ambion	AM9530G
Urea	Ambion	AM9902
Sodium Citrate	Sigma-Aldrich	W302600
tRNA	Sigma-Aldrich	R-7876
Sodium-L-ascorbate	Sigma-Aldrich	A7631-25g
Fe(NH4)2(SO4)2 . 6 H2O	Sigma-Aldrich	F1543-500g
RNase T1	Fermentas	EN0541
Hydrogen Peroxide (30%)	Sigma-Aldrich	349887