Description of Invention:
Pteridines are naturally occurring, highly fluorescent compounds (Quantum yields 0.88-0.40) that are structurally similar to purines and that were first isolated from butterfly wings in 1889. The pteridine nucleoside analogs developed by NCI scientist Hawkins and co-workers are structurally similar to guanosine (3-MI and 6-MI) or adenosine (6-MAP). These analogs are stable, can be formulated as phosphoramidites and are incorporated into oligonucleotides as a direct substitute for a purine base using automated DNA synthesis. The fluorescence properties of these probes are directly impacted by the chemistry of neighboring bases and reflect changes in tertiary structure due to interactions with proteins, RNA or DNA. Even subtle changes in base stacking or base pairing can be observed through changes in fluorescence intensity, lifetimes, energy transfer or anisotropy, making these pteridines ideally suited for the study of DNA/DNA and DNA/protein interactions.
Several applications have been further developed using this technology and one such application causes the pteridine probe to "bulge" out of the base stacking environment as it anneals to a target sequence which does not contain a base pairing partner for the pteridine. Prior to binding to the bulge-forming target strand the fluorescence of the probe is very quiet, only "lighting up" when bound to a specific sequence. This highly specific technique results in a dramatic increase in fluorescence intensity of up to 27 fold, is very rapid, does not require separation of oligonucleotides in a mixture and has been used in the development of a PCR product detection system. The specific nature of the "bulge hybridization" technique may be used to overcome some of the issues caused by non-specific probe binding in standard chip technology. (For a review see: Hawkins, M. (2003) Fluorescent Nucleoside Analogues as DNA Probes, in DNA Technology. J. R. Lakowicz. New York, Kluwer Academic/Plenum Publishers Vol 7 151-175.) More recent applications have shown that the stability and brightness of the guanosine analogy 3-MI are suitable for studies requiring probe detection at the single molecule level and studies using 6-MAP and 2-photon counting excitation demonstrate the adenosine analog's usefulness as a UV probe.
The pteridine nucleoside analogs provide a unique opportunity to use native-like, stable and highly fluorescent probes in the development of further refined, quantitative approaches to the study of DNA/DNA and DNA/protein interactions. The pteridine nucleoside patent portfolio is available for licensing and provides composition and methods of use claims for these versatile fluorophores.
Inventors:
Mary E. Hawkins (NCI) et al.
Patent Status:
DHHS Reference No. E-181-1993/0 --
U.S. Patent 5,525,711 issued 11 Jun 1996
U.S. Patent 5,612,468 issued 18 Mar 1997
