Abstract:
:The molecular recognition features of a DNA-sensitive fluorescent bioconjugate capable of targeting a specific DNA sequence with high efficiency are described. The bioconjugate combines a polypeptide from the Tc3 transposase DNA-binding domain with the dsDNA-sensitive fluorophore thiazole orange. Fluorescence spectroscopy and circular dichroism reveal that the polypeptide moiety determines the DNA sequence specificity as the intercalating dye makes nonspecific contributions to binding affinity. The conjugated thiazole orange is able to intercalate and fluoresce when the peptide binds at concentrations where little fluorescence is observed from either the bioconjugate alone or the bioconjugate mixed with DNA lacking the target sequence. Fluorescence studies indicate this molecular probe is sequence specific, binds the native Tc3 DNA target sequence with nanomolar affinity (KD approximately 15 nM), and is able to discriminate multiple point mutations in the cognate DNA site. The attachment of a sequence-specific binding peptide onto a functional probe provides a viable strategy for construction of synthetic enzymes and repressors, and facilitates dynamic studies of protein-DNA interactions.
journal_name
Biomacromoleculesjournal_title
Biomacromoleculesauthors
Thompson Mdoi
10.1021/bm070334hsubject
Has Abstractpub_date
2007-11-01 00:00:00pages
3628-33issue
11eissn
1525-7797issn
1526-4602journal_volume
8pub_type
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journal_title:Biomacromolecules
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