Abstract
RNA-protein interactions play critical roles in gene regulation, but methods to quantitatively analyze these interactions at a large scale are lacking. We have developed a high-throughput sequencing–RNA affinity profiling (HiTS-RAP) assay by adapting a high-throughput DNA sequencer to quantify the binding of fluorescently labeled protein to millions of RNAs anchored to sequenced cDNA templates. Using HiTS-RAP, we measured the affinity of mutagenized libraries of GFP-binding and NELF-E–binding aptamers to their respective targets and identified critical regions of interaction. Mutations additively affected the affinity of the NELF-E–binding aptamer, whose interaction depended mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depended primarily on secondary structure.
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Acknowledgements
We thank R. Friedman (Institut Pasteur) for providing scripts to extract intensity data, B. Mohanty (Medical University of South Carolina) for providing vectors containing the tus gene, K. Szeto and D. Shalloway for advice on data analysis, W. Zipfel and A. Singh for help in understanding the optics of the GAIIx, C.T. Waters and H. Kwak for bioinformatics advice, the Cornell sequencing core facility for help in learning the GAIIx, and H. Craighead (Cornell University) and the members of the Lis lab for helpful discussions on experimental design and the manuscript. This work was supported by US National Institutes of Health grants GM090320 and DA030329 to J.T.L.
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Contributions
Initial idea: A.O. and J.T.L. Experimental design: J.M.T., A.O., D.G., G.P.S. and J.T.L. Experimental implementation: J.M.T., A.O. and J.M.P. HiTS-RAP experiments and bioinformatics analysis: J.M.T. Project coordination: J.T.L. Paper writing: J.M.T., A.O., J.M.P. and J.T.L.
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D.G. and G.P.S. are employees of Illumina, Inc.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–9 and Supplementary Notes 1–4 (PDF 13434 kb)
Supplementary Table 1
All GFPapt mutants measured by HiTS-RAP (XLSX 109 kb)
Supplementary Table 2
HiTS-RAP measured GFPapt mutants verified by HiTS-RAP (XLSX 9 kb)
Supplementary Table 3
All GFPapt single base substitution mutants measured by HiTS-RAP (XLSX 40 kb)
Supplementary Table 4
All GFPapt double base substitution mutants measured by HiTS-RAP (XLSX 52 kb)
Supplementary Table 5
All NELFapt mutants measured by HiTS-RAP (XLSX 536 kb)
Supplementary Table 6
All NELFapt single base substitution mutants measured by HiTS-RAP (XLSX 36 kb)
Supplementary Table 7
All NELFapt double base substitution mutants measured by HiTS-RAP (XLSX 674 kb)
Supplementary Table 8
Primers used in this work (XLSX 10 kb)
Supplementary Software
XML recipe used for the GFPapt run, and the Python program used for processing data, through fitting Kd values. (ZIP 8 kb)
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Tome, J., Ozer, A., Pagano, J. et al. Comprehensive analysis of RNA-protein interactions by high-throughput sequencing–RNA affinity profiling. Nat Methods 11, 683–688 (2014). https://doi.org/10.1038/nmeth.2970
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DOI: https://doi.org/10.1038/nmeth.2970
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