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Related Experiment Videos

Screening for oligonucleotide binding affinity by a convenient fluorescence competition assay.

J G Harrison1, X Liu, S Balasubramanian

  • 1University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 1EW, UK.

Nucleic Acids Research
|August 14, 1999
PubMed
Summary

A new fluorescence assay enables high-throughput screening of molecules that bind single-stranded DNA. This method accurately measures binding affinity, useful for discovering new DNA-binding compounds.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Assay Development

Background:

  • Developing efficient methods for screening DNA-binding molecules is crucial.
  • Existing assays may lack the throughput or accuracy needed for large-scale screening.

Purpose of the Study:

  • To describe a novel homogeneous quenched fluorescence assay system.
  • To enable high-throughput screening (HTS) of DNA conjugates that bind to single-stranded DNA (ssDNA).

Main Methods:

  • Utilized a competitive homogeneous quenched fluorescence system.
  • Generated fluorescence signal via competitive binding to a target ssDNA strand.
  • Employed a quencher probe on the target strand and a fluorescent probe on a complementary strand.

Main Results:

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  • The assay's fluorescence signal is directly related to the binding affinity of the sample molecule.
  • Competitive analysis of peptide-oligonucleotide conjugates showed good correlation with UV melting data.
  • Demonstrated the assay's utility for screening ssDNA-binding molecules.

Conclusions:

  • The developed assay is effective for HTS of ssDNA-binding molecules.
  • This method provides a reliable way to assess binding affinities.
  • The assay has broad applicability in drug discovery and molecular biology research.