Single molecule fluorescence resonance energy transfer measurements

Fluorescence resonance energy transfer (FRET) is a spectroscopic means of obtaining distance information over a range up to 80 A in solution. It is based on the dipolar coupling between the electronic transition moments of a donor and acceptor fluorophore attached at known positions on the RNA species of interest. It can be applied in ensembles of molecules, either by steady-state fluorescence or by lifetime measurements, but it is also very appropriate for single-molecule studies. In addition to the provision of distance information, recent studies have emphasized the orientation dependence of energy transfer. 

A third application for fluorescent PNA probes is to introduce fluorescent labels into the RNA site specifically (20). For example, PNA probes have been hybridized at exonic sites that flank consecutive splice sites in a pre-mRNA from yeast. Fdrster resonance energy transfer (FRET) donor and acceptor dyes were attached covalently to the PNAs, and low FRET efficiencies were observed when the PNAs were hybridized to the pre-mRNA. However, when hybridized to the mRNA produced by splicing, large increases in ERET have been observed both in bulk solution and on a glass slide where single-molecule measurements could be made. As in the EISH applications, the ability to use short PNA probes to deliver the fluorescent dye to a desired location decreased the likelihood that the PNA wiU disrupt the structure and the function of the RNA that is under investigation. 

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