Pentaprobe: a comprehensive sequence for the one‐step detection of DNA‐binding activities

• Published in: Nucleic acids research Vol. 31; no. 20; p. e124
• Main Authors: Kwan, Ann H. Y., Czolij, Robert, Mackay, Joel P., Crossley, Merlin
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 15.10.2003; Oxford Publishing Limited (England)
• Subjects: Algorithms; Base Sequence; Binding Sites - genetics; Binding, Competitive; BKLF protein; DNA - genetics; DNA - metabolism; DNA-Binding Proteins - metabolism; Electrophoretic Mobility Shift Assay; Eos protein; Molecular Sequence Data; NAR Methods Online; Oligonucleotide Probes - genetics; Oligonucleotide Probes - metabolism; Pegasus protein; Protein Binding; PU.1 protein
• ISSN: 0305-1048; 1362-4962; 1362-4962
• DOI: 10.1093/nar/gng124

The rapid increase in the number of novel proteins identified in genome projects necessitates simple and rapid methods for assigning function. We describe a strategy for determining whether novel proteins possess typical sequence‐specific DNA‐binding activity. Many proteins bind recognition sequences of 5 bp or less. Given that there are 45 possible 5 bp sites, one might expect the length of sequence required to cover all possibilities would be 45 × 5 or 5120 nt. But by allowing overlaps, utilising both strands and using a computer algorithm to generate the minimum sequence, we find the length required is only 516 base pairs. We generated this sequence as six overlapping double‐stranded oligonucleotides, termed pentaprobe, and used it in gel retardation experiments to assess DNA binding by both known and putative DNA‐binding proteins from several protein families. We have confirmed binding by the zinc finger proteins BKLF, Eos and Pegasus, the Ets domain protein PU.1 and the treble clef N‐ and C‐terminal fingers of GATA‐1. We also showed that the N‐terminal zinc finger domain of FOG‐1 does not behave as a typical DNA‐binding domain. Our results suggest that pentaprobe, and related sequences such as hexaprobe, represent useful tools for probing protein function.