Show simple item record

dc.contributor.authorBogdanov, Alexei A. Jr.
dc.contributor.authorMetelev, Valeriy G.
dc.contributor.authorZhang, Surong
dc.contributor.authorKumar, Anand T. N.
dc.date2022-08-11T08:10:50.000
dc.date.accessioned2022-08-23T17:21:47Z
dc.date.available2022-08-23T17:21:47Z
dc.date.issued2012-08-01
dc.date.submitted2015-01-05
dc.identifier.citationMol Biosyst. 2012 Aug;8(8):2166-73. doi: 10.1039/c2mb25057h. Epub 2012 Jun 19. <a href="http://dx.doi.org/10.1039/c2mb25057h">Link to article on publisher's site</a>
dc.identifier.issn1742-2051 (Linking)
dc.identifier.doi10.1039/c2mb25057h
dc.identifier.pmid22710322
dc.identifier.urihttp://hdl.handle.net/20.500.14038/48601
dc.description.abstractWe designed and synthesized sensors for imaging transcription factor-DNA interactions using a complementary pair of 21-base pair long oligonucleotides (ODNs) carrying two internucleoside phosphate-linked cyanine fluorophores that can either engage in Forster's resonance energy transfer (FRET) with fluorescence emission or assemble into a ground state quenched dimer with short fluorescence lifetimes (FL). Cyanine fluorophores were linked to ODNs within the NF-kappaB binding site. These sensors were tested in the presence of recombinant p50 and p65 NF-kappaB proteins or constitutively NF-kappaB activating HeLa cell lysates. By using a coherent light excitation source we followed changes in fluorescence lifetime of the donor (Cy5.5) at the donor's excitation and emission light wavelengths, as well as the acceptor (800CW or Cy7 cyanine fluorophores) in FRET mode. We observed increases in the donor lifetime in both emitting (0.08-0.15 ns) and non-emitting quenched (0.21 ns) sensors in response to protein binding. The measurements of lifetimes in FRET mode in quenched pair-carrying ODN duplex sensors showed significant differences in FL of the acceptor cyanine fluorophore between NF-kappaB-containing and NF-kappaB-free samples but not in control sensors with ODN sequences that have decreased binding affinity to NF-kappaB. We anticipate that the observed effects will be instrumental for developing sensors enabling non-invasive imaging in cells that undergo activation of NF-kappaB.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=22710322&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://www.ncbi.nlm.nih.gov/pmc/articles/PMC3544408/pdf/nihms388296.pdf
dc.subjectBinding Sites
dc.subjectCarbocyanines
dc.subjectFluorescence
dc.subjectFluorescence Resonance Energy Transfer
dc.subjectNF-kappa B
dc.subjectOligonucleotide Probes
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectChemical Actions and Uses
dc.subjectChemistry
dc.subjectDiagnosis
dc.subjectInvestigative Techniques
dc.subjectRadiology
dc.titleSensing of transcription factor binding via cyanine dye pair fluorescence lifetime changes
dc.typeJournal Article
dc.source.journaltitleMolecular bioSystems
dc.source.volume8
dc.source.issue8
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/radiology_pubs/88
dc.identifier.contextkey6497742
html.description.abstract<p>We designed and synthesized sensors for imaging transcription factor-DNA interactions using a complementary pair of 21-base pair long oligonucleotides (ODNs) carrying two internucleoside phosphate-linked cyanine fluorophores that can either engage in Forster's resonance energy transfer (FRET) with fluorescence emission or assemble into a ground state quenched dimer with short fluorescence lifetimes (FL). Cyanine fluorophores were linked to ODNs within the NF-kappaB binding site. These sensors were tested in the presence of recombinant p50 and p65 NF-kappaB proteins or constitutively NF-kappaB activating HeLa cell lysates. By using a coherent light excitation source we followed changes in fluorescence lifetime of the donor (Cy5.5) at the donor's excitation and emission light wavelengths, as well as the acceptor (800CW or Cy7 cyanine fluorophores) in FRET mode. We observed increases in the donor lifetime in both emitting (0.08-0.15 ns) and non-emitting quenched (0.21 ns) sensors in response to protein binding. The measurements of lifetimes in FRET mode in quenched pair-carrying ODN duplex sensors showed significant differences in FL of the acceptor cyanine fluorophore between NF-kappaB-containing and NF-kappaB-free samples but not in control sensors with ODN sequences that have decreased binding affinity to NF-kappaB. We anticipate that the observed effects will be instrumental for developing sensors enabling non-invasive imaging in cells that undergo activation of NF-kappaB.</p>
dc.identifier.submissionpathradiology_pubs/88
dc.contributor.departmentDepartment of Radiology
dc.source.pages2166-73


This item appears in the following Collection(s)

Show simple item record