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Single Molecule Fluorescence Resonance Energy Transfer of a RNA Kissing Complex

Published

Author(s)

Peter Yim, Xiaoyi Y. Zhang, E S. DeJong, J M. Carroll, John P. Marino, Lori S. Goldner

Abstract

We used single molecular fluorescence resonance energy transfer (FRET) to understand the conformation of a RNA loop to loop complex, termed a kissing complex. The kissing complex formed by two Escherichia Coli (ColE1) plasmid-encoded transcripts, RNA I and RNA II, is a critical intermediate in multi-step hybridization pathway which controls plasmid replication. Small model RNA hairpins (R1inv and R2inv) derived from RNA I and RNA II were used in our study. To obtain quantitative measurements of intramolecular distances, we employ donor-only single molecule FRET. This technique is described in detail and used to study a small double helical RNA with known structure. The RNA duplexes were labeled with donor and acceptor dyes on their 5 ends and immobilized on a glass surface. Fluorescence from the donor and acceptor dyes was collected and measured by photon counting detectors in two spectrally separated channels in a custom built confocal microscope. The donor-only FRET measurement was used to determine the distance between 5 ends of the RNA.
Proceedings Title
Society of Photo-Optical Instrumentation Engineers | Proceedings | Biomedical Spectroscopy, Microscopy, and Imaging
Conference Dates
January 22-27, 2005
Conference Title
Society of Photo-Optical Instrumentation Engineers

Keywords

confocal microscope, Cy5, FRET, RNA, single molecule

Citation

Yim, P. , Zhang, X. , DeJong, E. , Carroll, J. , Marino, J. and Goldner, L. (2005), Single Molecule Fluorescence Resonance Energy Transfer of a RNA Kissing Complex, Society of Photo-Optical Instrumentation Engineers | Proceedings | Biomedical Spectroscopy, Microscopy, and Imaging (Accessed July 16, 2024)

Issues

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Created January 27, 2005, Updated June 13, 2017