Fluorescence resonance energy transfer in near-infrared fluorescent oligonucleotide probes for detecting protein-DNA interactions
UMass Chan Affiliations
Department of Cell BiologyDepartment of Radiology
Laboratory of Molecular Imaging Probes
Document Type
Journal ArticlePublication Date
2008-03-14Keywords
DNAFluorescence Resonance Energy Transfer
Hydrolysis
Molecular Structure
Oligonucleotides
Protein Binding
Proteins
Life Sciences
Medicine and Health Sciences
Metadata
Show full item recordAbstract
Optical imaging in the near-infrared (NIR) range enables detecting ligand-receptor interactions and enzymatic activity in vivo due to lower scattering and absorption of NIR photons in the tissue. We designed and tested prototype NIR fluorescent oligodeoxyribonucleotide (ODN) reporters that can sense transcription factor NF-kappaB p50 protein binding. The reporter duplexes included donor NIR Cy5.5 indodicarbocyanine fluorochrome linked to the 3' end of the first ODN and NIR acceptor fluorochromes (indodicarbocyanine Cy7 or, alternatively, a heptamethine cyanine IRDye 800CW) that were linked at the positions +8 and +12 to the complementary ODN that encoded p50 binding sites. Both Cy7 and 800CW fluorochromes were linked by using hydrophilic internucleoside phosphate linkers that enable interaction between the donor and the acceptor with no base-pairing interference. We observed efficient fluorescence resonance energy transfer (FRET) both in the case of Cy5.5-Cy7 and Cy5.5-800CW pairs of fluorochromes, which was sensitive to the relative position of the dyes. Higher FRET efficiency observed in the case of Cy5.5-Cy7 pair was due to a larger overlap between the ODN-linked Cy5.5 emission and Cy7 excitation spectra. Fluorescent mobility shift assay showed that the addition of human recombinant p50 to ODN duplexes resulted in p50 binding and measurable increase of Cy5.5 emission. In addition, p50 binding provided a concomitant protection of FRET effect from exonuclease-mediated hydrolysis. We conclude that NIR FRET effect can be potentially used for detecting protein-DNA interactions and that the feasibility of detection depends on FRET efficacy and relative fluorochrome positions within ODN binding sites.Source
Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4156-61. Epub 2008 Mar 12. Link to article on publisher's site
DOI
10.1073/pnas.0800162105Permanent Link to this Item
http://hdl.handle.net/20.500.14038/39267PubMed ID
18337505Related Resources
ae974a485f413a2113503eed53cd6c53
10.1073/pnas.0800162105