Poster abstracts

Poster number 16 submitted by Shiqin Miao

bPNA probes of RNA tertiary interactions

Shiqin Miao (Department of Chemistry and Biochemistry, Center for RNA Biology), Ila Marathe (Department of Chemistry and Biochemistry), Venkat Gopalan (Department of Chemistry and Biochemistry), Dennis Bong (Department of Chemistry and Biochemistry, Center for RNA Biology)

Abstract:
We demonstrate herein the use of bifacial peptide nucleic acids (bPNAs) as fluorescent probes of RNA loop-loop and protein interactions. Fluorophore-labeled bifacial peptide nucleic acid can bind to U-rich domains in folded RNA to label loop regions with fluorophores that can serve as probes for monitoring long-range RNA interactions. This is demonstrated with previously reported bPNA scaffolds, as well as a new, branched sidechain bPNA we call bPNA(+). This new scaffold displays two melamines on the lysine sidechain, with each melamine targeting two thymine or uracil bases for base triple formation. First, we accomplished scalable synthesis of bPNA(+) and observed sub-nanomolar Kd. Second, we applied fluorophore-labeled bPNA and bPNA(+) as probes 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. Notably, dye-labeled bPNA(+) exhibits unexpected strong fluorescence turn on to signal both RNA and protein binding: a 2-fold turn on is observed when forming the ColE1 kissing loop complex and an 8-fold turn on is seen when Rop protein interacts with the kissing complex. We anticipate that this fluorescence turn on could be used in measurements of RNA-RNA and RNA-protein interactions and binding affinity measurements.

References:
1. Mao J., DeSantis C., Bong D. (2017). Small molecule recognition triggers secondary and tertiary interactions in DNA folding and hammerhead ribozyme catalysis.J. Am. Chem. Soc., 139, 9815-9818.
2. Zeng Y., Pratumyot Y., Piao X, and Bong D. (2012). Discrete assembly of synthetic peptide-DNA triplex structures from polyvalent melamine-thymine bifacial recognition. J. Am. Chem. Soc. 134, 832-835

Keywords: structural probe, RNA recognition, fluorescence turn on