Poster abstracts
Poster number 43 submitted by Ila Marathe
Fluorescence turn-on upon annealing of cyanine dye-labeled bPNAs to uridine-tract-containing RNAs
Ila Marathe (Department of Chemistry and Biochemistry, The Ohio State University), Shiqin Miao (Department of Chemistry and Biochemistry, The Ohio State University), Yehong Qiu (Department of Chemistry and Biochemistry, The Ohio State University), Khan Cox, Michael Poirier (Department of Physics, The Ohio State University), Dennis Bong (Department of Chemistry and Biochemistry, The Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Single-molecule fluorescence resonance energy transfer (smFRET) spectroscopy is a powerful technique for studying the assembly of ribonucleoprotein (RNP) complexes. However, site-specific fluor-labeling of long RNAs remains a major technical challenge, and often requires multiple purification steps to remove unincorporated labels. Thus, facile labeling approaches are needed to broaden the utility of smFRET for uncovering RNP assembly and function. To study assembly of archaeal RNase P (comprised of one RNA and five proteins) using ensemble and single-molecule FRET, we used cyanine dye-labeled bifacial peptide nucleic acids (Cy-bPNAs), which bind with high affinity to uridine-tracts (U-tracts)1, 2. The RNase P RNA (RPR) was modified to contain an internal U-tract, and fluor-labeling of the RPR with Cy-bPNA was achieved through a simple heating and cooling step. During the course of these ensemble FRET experiments, we unexpectedly observed that the fluorescence of Cy-bPNA alone is diminished to near baseline but the addition of a U-tract-containing RPR increases the Cy-bPNA’s fluorescence up to 200-fold, an unprecedented enhancement for Cy dyes. This ‘fluorescence turn-on’ has been observed with multiple RNAs (<100 - 380 nt) bearing U-tracts of different lengths at terminal or internal positions, and under varying salt, pH, and temperature conditions. Moreover, preliminary sm-fluorescence experiments revealed strong fluorescence resulting from the binding of Cy-bPNA to an immobilized U-tract-containing RPR. The bPNA-based method for labeling RNAs has broad utility for fluorescence-based techniques including smFRET, and the turn-on phenomenon has potential for further exploration in bio-sensing applications.
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: Fluorescence, bPNA, RNA fluor-labeling