Nucleic Acids Research Advance Access published online on May 13, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn267
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Methods Online |
High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis
1Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, 2Departments of Bioengineering and Genetics, 300 Pasteur Drive, Stanford University, Stanford, CA 94305 and 3Developmental Genetics and Bioinformatics, Wadsworth Center, Albany NY 12208, USA
*To whom correspondence should be addressed. Tel: +1 518 486 4103; Fax: +1 518 402 2288; Email: alain{at}wadsworth.org.
Received March 24, 2008. Revised April 17, 2008. Accepted April 21, 2008.
The use of capillary electrophoresis with fluorescently labeled nucleic acids revolutionized DNA sequencing, effectively fueling the genomic revolution. We present an application of this technology for the high-throughput structural analysis of nucleic acids by chemical and enzymatic mapping (‘footprinting’). We achieve the throughput and data quality necessary for genomic-scale structural analysis by combining fluorophore labeling of nucleic acids with novel quantitation algorithms. We implemented these algorithms in the CAFA (capillary automated footprinting analysis) open-source software that is downloadable gratis from https://simtk.org/home/cafa. The accuracy, throughput and reproducibility of CAFA analysis are demonstrated using hydroxyl radical footprinting of RNA. The versatility of CAFA is illustrated by dimethyl sulfate mapping of RNA secondary structure and DNase I mapping of a protein binding to a specific sequence of DNA. Our experimental and computational approach facilitates the acquisition of high-throughput chemical probing data for solution structural analysis of nucleic acids.