Linked Life Data

21417216

Source:http://linkedlifedata.com/resource/pubmed/id/21417216

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2011-4-20
pubmed:abstractText
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-?B 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-?B 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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
1520-4812
pubmed:author
pubmed:issnType
Electronic
pubmed:day
20
pubmed:volume
22
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
759-65
pubmed:dateRevised
2011-9-26
pubmed:meshHeading
pubmed:year
2011
pubmed:articleTitle
Hairpin-like fluorescent probe for imaging of NF-?B transcription factor activity.
pubmed:affiliation
Laboratory of Molecular Imaging Probes, Department of Radiology, University of Massachusetts Medical School, 55 Lake Ave North,Worcester MA 01655, USA.
pubmed:publicationType
Journal Article, Research Support, N.I.H., Extramural