A DNA Assay Based on Fluorescence Resonance Energy Transfer and DNA Triplex Formation

Abstract

Fluoresence resonance energy transfer (FRET) results from nonradiative coupling of two fluorophores and reports on distances in the range 10–100 Å. It is therefore a suitable probe to determine distances in RNA molecules and define their global structure, to follow kinetics of RNA conformational changes during folding in real time, to monitor ion binding, or to analyze conformational equilibria and assess the thermodynamic stability of tertiary structure conformers. Along with the basic principles of steady-state and time-resolved fluorescence resonance energy transfer measurements, approaches to investigate RNA conformational transitions and folding are described and illustrated with selected examples. The versatility of FRET-based techniques has recently been demonstrated by implementations of FRET in high-throughput screening of potential drugs as well as studies of energy transfer that monitor RNA conformational changes on the single-molecule level.