Poster abstracts

Poster number 38 submitted by Shiqin Miao

A novel structural probe: fluorescent-labeled bPNA(+)

Shiqin Miao (Department of Chemistry and Biochemistry), Jie Mao (Department of Chemistry and Biochemistry), Yufeng Liang (Department of Chemistry and Biochemistry), Christopher DeSantis (Department of Chemistry and Biochemistry)

Abstract:
We offer a unique labeling method, using fluorescent-labeled bPNA(+) as a probe for monitoring long-range RNA interactions. We employ a novel compact DNA recognition element, t2M, which comprises two melamines on a linear, 5-atom, sp3-hybridized motif displayed on various scaffolds and forms a base triple with two thymine or uracil bases. This work explores the recognition of nucleic acids by the t2M motif for versatile molecular targeting applications. First, we confirmed the binding affinity and stoichiometry of t2M and t4M with unstructured, T-rich DNAs to form hairpin-like structures. Second, we exploited the ability of t2M and t4M to act as functional switches in deactivated hammerhead ribozymes. We found that hammerhead ribozymes mutated by stem or loop replacement with a U-rich sequence in stems II and III could have bond scission function restored by t2M/t4M. Third, we tested the utility of the t2M motif in multivalent recognition by displaying this motif on a native peptide backbone (bPNA(+)). We accomplished scalable synthesis of bPNA(+) and observed sub-nanomolar Kd. Finally, we applied fluorescent-labeled bPNA(+) as a probe for monitoring long-range RNA interactions. We use a hexapeptide and decapeptide bPNA(+), labeled with a FRET pair, respectively, to label RNA secondary structures to provide FRET reporters of RNA-RNA interactions. Initially, we confirmed the site-selectivity of bPNA(+) as driven by length-matching using U-sites of different sizes and bPNA(+) of the corresponding lengths in the ColE1 kissing loop system by FRET. Next, we designed an RNA host containing the well-studied GAAA tetraloop-receptor interaction as a proof-of-concept system. This novel probe has been successfully applied to RNAse P and HIV-1 systems in RNA-center collaborations. Our work will show that the t2M motif can recognize T/U rich sequences as well as act as a structural probe.

References:
Mao J, DeSantis C, Bong D. Small Molecule Recognition Triggers Secondary and Tertiary Interactions in DNA Folding and Hammerhead Ribozyme Catalysis. J Am Chem Soc 2017;139:9815–8.

Keywords: RNA recognition motif , Structural probe, Functional switch