Single RNA kissing complexes studied by fluorescence resonance energy transfer

Peter B. Yim; Xiaoyi Zhang; Eric S. DeJong; Jennifer M. Carroll; John P. Marino; Lori S. Goldner

doi:10.1117/12.590896

29 March 2005 Single RNA kissing complexes studied by fluorescence resonance energy transfer

Peter B. Yim, Xiaoyi Zhang, Eric S. DeJong, Jennifer M. Carroll, John P. Marino, Lori S. Goldner

Proceedings Volume 5699, Imaging, Manipulation, and Analysis of Biomolecules and Cells: Fundamentals and Applications III; (2005) https://doi.org/10.1117/12.590896
Event: SPIE BiOS, 2005, San Jose, CA, United States

Abstract

We have used single molecular-pair fluorescence resonance energy transfer (FRET) to probe the conformation of a RNA loop-loop “kissing complex” formed by two small RNA hairpins (R1inv and R2inv) derived from Escherichia coli (ColE1) plasmid-encoded transcripts, RNA I and RNA II. This RNA kissing complex is a critical intermediate in a multi-step hybridization pathway which controls plasmid replication. Biotinylated RNA molecules were labeled with donor and acceptor dyes on their 5' ends and immobilized on a biotinylated surface using streptavidin. Fluorescence from the donor and acceptor dyes was collected and measured by photon counting detectors in two spectrally separated channels in a customized confocal microscope. Quantitative measurement of intramolecular distances between 5' ends of the RNA was obtained using donor-only single molecule FRET. This donor-only single molecule FRET technique is described in detail and validated through determination of the distance between 5' ends of 8mer A-form RNA helices of known structure.

Citation Download Citation

Peter B. Yim, Xiaoyi Zhang, Eric S. DeJong, Jennifer M. Carroll, John P. Marino, and Lori S. Goldner "Single RNA kissing complexes studied by fluorescence resonance energy transfer", Proc. SPIE 5699, Imaging, Manipulation, and Analysis of Biomolecules and Cells: Fundamentals and Applications III, (29 March 2005); https://doi.org/10.1117/12.590896

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