Screening methods for compounds that inhibit or stimulate helicase enzyme activity
AssigneeBoard of Trustees of the University of Arkansas
InventorsKevin Raney, Patrick Morris, Regina Dennis
Life of this patent
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Abstract
The gp4l and Dda helicases were found to significantly enhance the dissociation rate of streptavidin from biotin-labeled oligonucleotides in an ATP dependent reaction, demonstrating that these enzymes are capable of imparting a significant force on a molecule blocking their path. The present invention describes an assay for studying enzymatic activity of a helicase using the rate of dissociation of streptavidin from biotinylated oligonucleotides.
Claims12
A method of screening for compounds that inhibit or stimulate helicase enzyme activity, comprising the steps of:
- (a) combining under appropriate conditions:
- (i) a helicase enzyme capable of unidirectional translocation on an oligonucleotide, and
- (ii) a biotinylated single-stranded oligonucleotide bound to streptavidin, thereby producing helicase-associated streptavidin-biotinylated-oligonucleotide;
- (b) contacting a sample of said helicase-associated streptavidin-biotinylated-oligoiucleotide with a compound, thereby producing a compound-treated helicase-associated streptavidin-biotinylated-oligonucleotide sample;
- and
- (c) measuring the amount of dissociation of said biotinylated oligonucleotide from streptavidin in said compound-treated helicase-associated streptavidin-biotinylated-oligonucleotide sample and an untreated helicase-associated streptavidin-biotinylated-oligonucleotide sample, wherein less dissociation of said biotinylated oligonucleotide from said streptavidin in said compound-treated helicase-associated streptavidin-biotinylated-oligonucleotide sample than in said untreated helicase-associated streptavidin-biotinylated-oligonucleotide sample indicates the compound inhibits helicase enzyme activity, wherein greater dissociation of said biotinylated oligonucleotide from said streptavidin in said compound-treated helicase-associated streptavidin-biotinylated-oligonucleotide sample than in said untreated helicase-associated streptavidin-biotinylated-oligonucleotide sample is indicative of a compound that stimulates said helicase enzyme activity.
A method of releasing a streptavidin-captured, biotinylated oligonucleotide, comprising the steps of:
- (a) contacting a biotinylated oligonucleotide with a streptavidin that has normal or decreased affinity for biotin, thereby forming a streptavidin-captured biotinylated oligonucleotide;
- (b) contacting said streptavidin-captured biotinylated oligonucleotide with a helicase, wherein said helicase releases said streptavidin from said biotinylated oligonucleotide;
- and
- (c) collecting said biotinylated oligonucleotide released from said streptavidin.
Dependencies are inferred from the claim text.