Screening for oligonucleotide binding affinity by a convenient fluorescence competition assay.
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.
Harrison
J G
JG
University Chemical Laboratory, Cambridge University, Lensfield Road, Cambridge CB2 1EW, UK.
Liu
X
X
Balasubramanian
S
S
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D005456:Fluorescent Dyes; D013058:Mass Spectrometry; D009691:Nucleic Acid Denaturation; D009693:Nucleic Acid Hybridization; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D004247:DNA