Title

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

UMMS Affiliation

Department of Radiology

Publication Date

4-2011

Document Type

Article

Subjects

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

Disciplines

Chemicals and Drugs | Investigative Techniques | Medicinal-Pharmaceutical Chemistry | Radiology

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.

Rights and Permissions

Citation: Bioconjug Chem. 2011 Apr 20;22(4):759-65. doi: 10.1021/bc100553e. Epub 2011 Mar 21. Link to article on publisher's site

Journal/Book/Conference Title

Bioconjugate chemistry

Related Resources

Link to Article in PubMed

PubMed ID

21417216