Direct measurement of DNA affinity landscapes on a high-throughput sequencing instrument
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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 ~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.
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Normalized fluorescence intensity is proportional to poly(A)-tract length after accounting for cluster density.
“To account for the density of each cluster, this raw intensity is normalized to that of the fluorescent bases added during sequencing by synthesis12, thereby yielding a normalized fluorescence-intensity for the poly(A) tail of each transcript, paired with a sequencing read that identifies its poly(A) site and thus the gene of origin.”
From Poly(A)-tail profiling reveals an embryonic switch in translational control (2014) · cites this paper as [12]
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- Authors
- Nutiu, R., Friedman, R. C., Luo, S., Khrebtukova, I., Silva, D., Li, R., Zhang, L., Schroth, G. P., & Burge, C. B.
- Affiliations
- Massachusetts Institute of Technology (US)
- Illumina (United States) (US)
- Corresponding author
- Burge, Christopher B
- Venue
- Nature Biotechnology
- Published
- 2011-06-26
- Volume
- 29
- Issue
- 7
- Pages
- 659-664
- ISSN
- 0733-222X (ISSN-L)
- 1087-0156
- 1546-1696
- PMC
- PMC3134637
- OpenAlex
- W2069985409
- Cited by
- 194
- References
- 32
- Citations by year
- 2026: 7 · 2025: 5 · 2024: 8 · 2023: 6 · 2022: 8 · 2021: 7 · 2020: 9 · 2019: 17 · 2018: 14 · 2017: 21 · 2016: 14 · 2015: 15 · 2014: 20 · 2013: 18 · 2012: 22
- FWCI
- 8.30
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-sa)
- Funders
- National Human Genome Research Institute (R01HG002439)
- National Institute of General Medical Sciences (R01GM085319)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Diffusion and Search Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- protein-DNA binding affinity, GCN4, DNA-binding motifs, dissociation constant, promoter binding sites, gene expression prediction, transcriptional regulation, yeast
- MeSH terms
- Chromatography, Affinity, DNA, DNA-Binding Proteins, Equipment Design, Spectrometry, Fluorescence, Sequence Analysis, DNA, Equipment Failure Analysis, Protein Interaction Mapping
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