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    Hairpin-like fluorescent probe for imaging of NF-kappaB transcription factor activity

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    Authors
    Metelev, Valeriy G.
    Zhang, Surong
    Tabatadze, David
    Bogdanov, Alexei A. Jr.
    UMass Chan Affiliations
    Department of Radiology
    Document Type
    Journal Article
    Publication Date
    2011-04-01
    Keywords
    Binding Sites
    DNA
    Fluorescence Resonance Energy Transfer
    Fluorescent Dyes
    Humans
    Models, Molecular
    *Molecular Imaging
    Molecular Structure
    NF-kappa B p50 Subunit
    Oligodeoxyribonucleotides
    Recombinant Proteins
    Transcription Factor RelA
    Chemicals and Drugs
    Investigative Techniques
    Medicinal-Pharmaceutical Chemistry
    Radiology
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    Link to Full Text
    http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3086100/
    Abstract
    Three oligodeoxyribonucleotides (ODN) covalently labeled with near-infrared (NIR) fluorochromes were synthesized and characterized with a goal of comparing in vitro a hairpin-based and a duplex-based FRET probe designed for the detection of human recombinant NF-kappaB p50/p65 heterodimer binding to DNA. Using deoxyguanosine phosphoramidite with a phosphorus-linked aminoethylene (diethylene glycol) hydrophilic linker, we synthesized ODNs with internucleoside reactive sites. The hairpin loop amino linker was modified with IRDye 800CW (FRET acceptor), and the 3'-end was modified with Cy5.5 (FRET donor) using a dithio-linker. To obtain a duplex probe, we conjugated Cy5.5 and 800CW to complementary strands at the distance of ten base pairs in the resultant duplex. No quenching of dyes was observed in either probe. The FRET efficiency was higher in the duplex (71%) than in the hairpin (56%) due to a more favorable distance between the donor and the acceptor. However, the hairpin design allowed more precise ratiometric measurement of fluorescence intensity changes as a result of NF-kappaB p50/p65 binding to the probe. We determined that as a result of binding there was a statistically significant increase of fluorescence intensity of Cy5.5 (donor) due to a decrease of FRET if normalized by 800CW intensity measured independently of FRET. We conclude that the hairpin based probe design allows for the synthesis of a dual fluorescence imaging probe that renders signal changes that are simple to interpret and stoichiometrically correct for detecting transcription factor-DNA interactions.
    Source
    Bioconjug Chem. 2011 Apr 20;22(4):759-65. doi: 10.1021/bc100553e. Epub 2011 Mar 21. Link to article on publisher's site
    DOI
    10.1021/bc100553e
    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/48596
    PubMed ID
    21417216
    Related Resources
    Link to Article in PubMed
    ae974a485f413a2113503eed53cd6c53
    10.1021/bc100553e
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