Proc. SPIE. 5699, Imaging, Manipulation, and Analysis of Biomolecules and Cells: Fundamentals and Applications III
KEYWORDS: Confocal microscopy, Microscopes, Sensors, Luminescence, Molecules, Energy transfer, Quantum efficiency, Distance measurement, Fluorescence resonance energy transfer, Molecular energy transfer
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.