Pyrite footprinting of RNA

► RNA structure is mapped by pyrite mediated OH footprinting. ► Repetitive experiments can be done in a powdered pyrite filled cartridge. ► High OH reactivity of nucleotides imply dynamic role in Diels–Alderase catalysis. In RNA, function follows form. Mapping the surface of RNA molecules with chemi...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2012-08, Vol.425 (2), p.374-378
Main Authors: Schlatterer, Jörg C., Wieder, Matthew S., Jones, Christopher D., Pollack, Lois, Brenowitz, Michael
Format: Article
Language:English
Subjects:
60 APPLIED LIFE SCIENCES
ARCHITECTURE
CRYSTAL STRUCTURE
CUTTING
DNA
ENZYMES
Footprinting
Hydroxyl radical
HYDROXYL RADICALS
Iron - chemistry
IRON SULFIDES
MOLECULAR DYNAMICS METHOD
NUCLEOTIDES
PYRITE
RNA
RNA Folding
RNA, Catalytic - chemistry
Sequence Analysis, RNA - methods
Sulfides - chemistry
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Summary:► RNA structure is mapped by pyrite mediated OH footprinting. ► Repetitive experiments can be done in a powdered pyrite filled cartridge. ► High OH reactivity of nucleotides imply dynamic role in Diels–Alderase catalysis. In RNA, function follows form. Mapping the surface of RNA molecules with chemical and enzymatic probes has revealed invaluable information about structure and folding. Hydroxyl radicals (OH) map the surface of nucleic acids by cutting the backbone where it is accessible to solvent. Recent studies showed that a microfluidic chip containing pyrite (FeS2) can produce sufficient OH to footprint DNA. The 49-nt Diels–Alder RNA enzyme catalyzes the C–C bond formation between a diene and a dienophile. A crystal structure, molecular dynamics simulation and atomic mutagenesis studies suggest that nucleotides of an asymmetric bulge participate in the dynamic architecture of the ribozyme’s active center. Of note is that residue U42 directly interacts with the product in the crystallized RNA/product complex. Here, we use powdered pyrite held in a commercially available cartridge to footprint the Diels–Alderase ribozyme with single nucleotide resolution. Residues C39 to U42 are more reactive to OH than predicted by the solvent accessibility calculated from the crystal structure suggesting that this loop is dynamic in solution. The loop’s flexibility may contribute to substrate recruitment and product release. Our implementation of pyrite-mediated OH footprinting is a readily accessible approach to gleaning information about the architecture of small RNA molecules.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2012.07.100