Dataset Information

High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.

ABSTRACT: 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.

SUBMITTER: Mitra S

PROVIDER: S-EPMC2441812 | biostudies-other | 2008 Jun

REPOSITORIES: biostudies-other

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High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.

Mitra Somdeb S Shcherbakova Inna V IV Altman Russ B RB Brenowitz Michael M Laederach Alain A

Nucleic acids research 20080513 11

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 implemen ...[more]

PMID: 18477638

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Project description:The major information-carrying macromolecules in the cell, DNA, RNA, and protein, carry an additional layer of information on top of their sequence in the form of modifications of residues. The modifications provide additional functional groups and impact the structure and function of the molecules. Cellular RNA molecules contain more than 100 different modifications and are found in all domains of life and in all major classes of RNA in eukaryotic organisms. Together these modifications constitute the epitranscriptome of which two-thirds are methylations with 2’-O methylation of the ribose moiety of the nucleotide as the most abundant. Many aspects of ribose methylation are underexplored because the existing methods for their detection are laborious and can only address a few modification sites at a time. Here, we introduce RiboMeth-Seq, a high-throughput sequencing based method and applies it to yeast ribosomal RNA. We detect all of the known as well as one new methylation site and provide evidence for hypomethylation at specific residues. Furthermore we demonstrate that many methylation events are interdependent and outline the timing of modifications during ribosome biogenesis. Our results demonstrate a novel and efficient approach to understanding of the role of modifications in ribosomal RNA folding and ribosome function. Recent evidence point to changes in ribose methylation patterns in cancer ribosomes and we anticipate that RiboMeth-Seq can be applied here as well as to other diseases in which ribosomes are affected, including the heritable ribosomopathies. Yeast RNA was degraded at denaturing conditions into small fragments, long enough to be mapped by sequence alignment to the reference genome (20-40 nucleotides). The fragments were ligated to RNA oligos using a tRNA ligase that was mutated to remove its kinase activity, reverse transcribed and the cDNA used as input for Ion semiconductor sequencing. The first and last nucleotides of the inserts were recorded using the full sequence for mapping and the read-ends plotted against the sequence. 2'-O-Methylated RNA positions are protected from cleavage, and read ends from such positions are therefore underrepresented compared to the surrounding positions.

Dataset Information

High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.

Publications

High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis.

Similar Datasets

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