High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.
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.
Mitra
Somdeb
S
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Shcherbakova
Inna V
IV
Altman
Russ B
RB
Brenowitz
Michael
M
Laederach
Alain
A
D000465:Algorithms; D000818:Animals; D018983:DNA Footprinting / Q000379:methods*; D003850:Deoxyribonuclease I; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D017665:Hydroxyl Radical; D007438:Introns; D008958:Models, Molecular; D015335:Molecular Probes; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D012984:Software; D013768:Tetrahymena / Q000235:genetics
D005456:Fluorescent Dyes; D015335:Molecular Probes; D009711:Nucleotides; D017665:Hydroxyl Radical; D012313:RNA; D003850:Deoxyribonuclease I