Hairpin-like fluorescent probe for imaging of NF-kappaB transcription factor activity
UMass Chan Affiliations
Department of RadiologyDocument Type
Journal ArticlePublication Date
2011-04-01Keywords
Binding SitesDNA
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
Metadata
Show full item recordAbstract
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 siteDOI
10.1021/bc100553ePermanent Link to this Item
http://hdl.handle.net/20.500.14038/48596PubMed ID
21417216Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1021/bc100553e