Friday, June 9, 2023
6/9/2023
Summary/Abstract. RNA-targeting approaches have received considerable attention, and remain major areas of focus in both basic research and therapeutic development. We have reported TBP6.7, a laboratory evolved U1A-derived RNA Recognition Motif (RRM), that binds HIV-1 trans-activation response (TAR) hairpin (KD ~5 nM), has no endogenous human RNA hairpin targets, and inhibits TAR-dependent transcription. More recently, we reported the 1.8Å crystal structure of the TBP6.7-TAR hairpin complex, which led to structure-guided development of a cyclic peptide inhibitor of TAR-dependent transcription. Researchers have used TBP6.7 as a central component in CRISPR-Cas-Inspired RNA Targeting System (CIRTS), a programmable RNA modifying platform. CIRTS consists of an effector domain, TAR-binding domain (TBP6.7), and a single-stranded RNA shield protein domain. When mixed with a guide strand RNA (gRNA) consisting of a target sequence complementary region flanked by TAR RNA hairpin, sequence-specific mRNA targeting (degradation or gene editing) is achieved. However, a single TBP6.7:TAR binding pair, responsible for forming the necessary CIRTS fusion protein-gRNA complex, dramatically limits number of mRNAs that can be targeted – to one per experiment. Using a structure-guided approach, we will evolve new RRMs that bind TAR-derived RNA hairpins. Following biophysical characterization of these complexes, they will be incorporated into the CIRTS platform and mRNA degradation or gene editing will be measured using standard reporter systems (Dickinson lab). In addition to TAR, we have evolved RRMs that bind the oncogenic precursor microRNA pre-miR-21. Preliminary flow cytometry data indicates that a number of these proteins potently and selectively bind pre-miR-21. We will use ELISA, SPR, and ITC to characterize these new protein-RNA interactions, engineer and test mutant forms of these new RRM-RNA pairs to better understand the requirements for binding, and measure RRM-dependent inhibition of DICER pre-miR-21 splicing. Collectively, this research will lead to the development of new RNA-targeted therapeutic leads, expand our understanding of sequence-selective RNA recognition, and develop new protein-RNA binding partners for incorporation into the mRNA gene editing platform CIRTS – enabling multiplexed editing of specific mRNAs in a single cell. Finally, funding for this research will expand research opportunities for undergraduate and graduate researchers at Delaware State University – an Historically Black College and University (HBCU). Given the makeup of our undergraduate and graduate student population, this research will enrich the training of young researchers and scholars who are historically underrepresented in Science, Technology, Engineering, and Mathematics (STEM).
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