Abstract
A competitive homogeneous quenched fluorescence assay system is described for the high throughput screening of DNA conjugates that bind to single-stranded DNA. Fluorescence signal is generated by competitive binding of the sample molecule to a target strand labelled with a quencher probe, which is otherwise hybridised to a complementary strand containing a fluorescent probe. Thus fluorescence generated is related to the affinity of the sample. Competitive analysis of a number of peptide-oligonucleotide conjugates gave data that correlated well with the corresponding UV melting data. The assay will be useful for screening of large numbers of potential single-stranded binding molecules.
Full Text
The Full Text of this article is available as a PDF (128.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Cardullo R. A., Agrawal S., Flores C., Zamecnik P. C., Wolf D. E. Detection of nucleic acid hybridization by nonradiative fluorescence resonance energy transfer. Proc Natl Acad Sci U S A. 1988 Dec;85(23):8790–8794. doi: 10.1073/pnas.85.23.8790. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Harrison J. G., Balasubramanian S. Synthesis and hybridization analysis of a small library of peptide-oligonucleotide conjugates. Nucleic Acids Res. 1998 Jul 1;26(13):3136–3145. doi: 10.1093/nar/26.13.3136. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lockhart D. J., Dong H., Byrne M. C., Follettie M. T., Gallo M. V., Chee M. S., Mittmann M., Wang C., Kobayashi M., Horton H. Expression monitoring by hybridization to high-density oligonucleotide arrays. Nat Biotechnol. 1996 Dec;14(13):1675–1680. doi: 10.1038/nbt1296-1675. [DOI] [PubMed] [Google Scholar]
- Nazarenko I. A., Bhatnagar S. K., Hohman R. J. A closed tube format for amplification and detection of DNA based on energy transfer. Nucleic Acids Res. 1997 Jun 15;25(12):2516–2521. doi: 10.1093/nar/25.12.2516. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schena M., Shalon D., Heller R., Chai A., Brown P. O., Davis R. W. Parallel human genome analysis: microarray-based expression monitoring of 1000 genes. Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10614–10619. doi: 10.1073/pnas.93.20.10614. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tyagi S., Kramer F. R. Molecular beacons: probes that fluoresce upon hybridization. Nat Biotechnol. 1996 Mar;14(3):303–308. doi: 10.1038/nbt0396-303. [DOI] [PubMed] [Google Scholar]