Direct visualization of DNA affinity landscapes using a highthroughput sequencing instrument
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Burge_Direct measurement.pdf
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1.42 MB
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Checksum (MD5)
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Author(s)
Nutiu, Razvan
Friedman, Robin Carl
Luo, Shujun
Khrebtukova, Irina
Silva, David
Li, Robin
Zhang, Lu
Schroth, Gary P.
Burge, Christopher B.
Alternative Title
Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument
Date Issued
June 2011
Journal
Nature Biotechnology
Publisher
Nature Publishing Group
Citation
Nutiu, Razvan et al. “Direct Measurement of DNA Affinity Landscapes on a High-throughput Sequencing Instrument.” Nature Biotechnology 29.7 (2011): 659–664. Web.
Version
Author's final manuscript
Abstract
Several methods for characterizing DNA-protein interactions are available, but none have demonstrated both high throughput and quantitative measurement of affinity. Here we describe 'high-throughput sequencing'-'fluorescent ligand interaction profiling' (HiTS-FLIP), a technique for measuring quantitative protein-DNA binding affinity at unprecedented depth. In this approach, the optics built into a high-throughput sequencer are used to visualize in vitro binding of a protein to sequenced DNA in a flow cell. Application of HiTS-FLIP to the protein Gcn4 (Gcn4p), the master regulator of the yeast amino acid starvation response, yielded [approx]440 million binding measurements, enabling determination of dissociation constants for all 12-mer sequences having submicromolar affinity. These data revealed a complex interdependency between motif positions, allowed improved discrimination of in vivo Gcn4p binding sites and regulatory targets relative to previous methods and showed that sets of genes with different promoter affinities to Gcn4p have distinct functions and expression kinetics. Broad application of this approach should increase understanding of the interactions that drive transcription.
MIT Department
Massachusetts Institute of Technology. Computational and Systems Biology Program
Massachusetts Institute of Technology. Department of Biological Engineering
Massachusetts Institute of Technology. Department of Biology
Terms of Use
Creative Commons Attribution-Noncommercial-Share Alike 3.0
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DOI of Published Version
https://doi.org/10.1038/nbt.1882
