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An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription David W Crawford, Brett D. Blakeley, Po-Han Chen, Chringma Sherpa, Stuart F.J. Le Grice, Ite A. Laird-Offringa, and Brian R McNaughton ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00145 • Publication Date (Web): 02 Jun 2016 Downloaded from http://pubs.acs.org on June 3, 2016
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ACS Chemical Biology Crawford, Blakeley, & McNaughton, et al. -1
An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription David W. Crawford,1 Brett D. Blakeley,1 Po-Han Chen,3 Chringma Sherpa,4 Stuart F.J. Le Grice,4 Ite A. Laird-Offringa,3 and Brian R. McNaughton*,1,2 1
Department of Chemistry, Colorado State University, Fort Collins, Colorado, USA. Department of Biochemistry & Molecular Biology, Colorado State University, Fort Collins, Colorado, USA. 3 Department of Surgery and Department of Biochemistry & Molecular Biology, USC/Norris Comprehensive Cancer Center, Keck School of Medicine, Los Angeles, California, USA. 4 Basic Research Laboratory, National Cancer Institute, Frederick, Maryland, USA 2
*corresponding author:
[email protected] ACS Paragon Plus Environment
ACS Chemical Biology
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Abstract
Potent and selective recognition and modulation of disease-relevant RNAs remains a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition, and showed that as few as one or two mutations can result in moderate affinity (low µM dissociation constant) for the human immunodeficiency virus (HIV) Trans-Activation Response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here we use yeast display and saturation mutagenesis of established RNA binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant ~0.5 nM), but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5´-untranslated region, inhibits the interaction between TAR RNA and an HIV Trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents – small molecule, nucleic acid, or protein - reported to date, and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition—and modulation—of a disease-relevant RNA.
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ACS Chemical Biology Crawford, Blakeley, & McNaughton, et al. -3
Ribonucleic acids (RNAs) play a critical role in functionally diverse cellular processes, including many that are disease-relevant.(1-3) Ligands that bind these RNAs have value as basic research tools, possible therapeutic application, and have the potential to illuminate the molecular requirements for their selective recognition. Traditionally, research has focused on developing new small organic molecules (MW
