Book of Abstracts: Albany 2007
June 19-23 2007
FRET and Beyond: High-Resolution Approaches Based on Target-Assembled Exciplexes for Nucleic Acid Detection
Many detection methods for DNA and RNA depend on some form of Fluorescence Resonance Energy Transfer (FRET). Efficient FRET depends on the spectral overlap of fluorescence donor and acceptor, the angle their transition dipoles bear to each other and, crucially, on the distance between them, this being optimal between 10 and 100 Å. As the rise in B-DNA per base pair is around 3.4 Å, this means that FRET operates at a resolution of around 3-30 bp. Clearly, this is not well matched to detecting Single Nucleotide Polymorphisms (SNPs) and other single base pair differences. In addition, the background signal with current fluorophores is large as the usual Stokes shifts achieved with conventional fluorophores is usually around 23 nm at best. This presentation will describe approaches initially based on novel FRET methods between Ru-based donors and Os-based acceptors (1), and then on excimers (2-4) and more recently on exciplexes (5-7) to improve on both the spatial resolution and the background problem. Using novel exciplex constructs, we are now able to detect a single Cytochrome P450 SNP relative to wild type embedded in a 3000 bp sequence of DNA.
References and Footnotes
Kenneth T. Douglas*
Wolfson Centre for Rational Structure-Based Design of Molecular Diagnostics