Spectral properties and DNA targeting features of a thiazole orange-peptide bioconjugate.

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

Biomacromolecules

journal_title

Biomacromolecules

authors

Thompson M

doi

10.1021/bm070334h

subject

Has Abstract

pub_date

2007-11-01 00:00:00

pages

3628-33

issue

11

eissn

1525-7797

issn

1526-4602

journal_volume

8

pub_type

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