Monitoring equilibrium changes in RNA structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting.

Ravichandra Bachu, Frances Camille S Padlan, Sara Rouhanifard, Michael Brenowitz, Jörg C. Schlatterer

Research output: Contribution to journalArticle

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. When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA and RNA 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-protein and RNA-protein complexes. Equilibrium and kinetic 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. 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 H(2)O(2) (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O(2)(we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number56
StatePublished - 2011
Externally publishedYes

Fingerprint

RNA
Hydroxyl Radical
Monitoring
Nucleotides
DNA
Nucleic Acid Probes
Proteins
Tetrahymena
Catalytic RNA
Molecules
Kinetics
Nucleic acids
Nucleic Acids
Binders
Isotherms
Data reduction
Polymers
Sampling
Catalysts

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Monitoring equilibrium changes in RNA structure by 'peroxidative' and 'oxidative' hydroxyl radical footprinting. / Bachu, Ravichandra; Padlan, Frances Camille S; Rouhanifard, Sara; Brenowitz, Michael; Schlatterer, Jörg C.

In: Journal of visualized experiments : JoVE, No. 56, 2011.

Research output: Contribution to journalArticle

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