Sensing of transcription factor binding via cyanine dye pair fluorescence lifetime changes.
Summary of "Sensing of transcription factor binding via cyanine dye pair fluorescence lifetime changes."
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 Förster'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-κB binding site. These sensors were tested in the presence of recombinant p50 and p65 NF-κB proteins or constitutively NF-κB 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-κB-containing and NF-κB-free samples but not in control sensors with ODN sequences that have decreased binding affinity to NF-κB. We anticipate that the observed effects will be instrumental for developing sensors enabling non-invasive imaging in cells that undergo activation of NF-κB.
Affiliation
The Laboratory of Molecular Imaging Probes, S6-434, Department of Radiology, University of Massachusetts Medical School, 55 Lake Ave North, Worcester, MA 01655, USA. Alexei.Bogdanov@umassmed.edu.
Journal Details
This article was published in the following journal.
Name: Molecular bioSystems
ISSN: 1742-2051
Pages: 2166-73
Links
- PubMed Source: http://www.ncbi.nlm.nih.gov/pubmed/22710322
- DOI: http://dx.doi.org/10.1039/c2mb25057h
Medical and Biotech [MESH] Definitions
Transcription Factor Dp1
A transcription factor that possesses DNA-binding and E2F-binding domains but lacks a transcriptional activation domain. It is a binding partner for E2F TRANSCRIPTION FACTORS and enhances the DNA binding and transactivation function of the DP-E2F complex.
Core Binding Factor Beta Subunit
A non-DNA binding transcription factor that is a subunit of core binding factor. It forms heterodimeric complexes with CORE BINDING FACTOR ALPHA SUBUNITS, and regulates GENETIC TRANSCRIPTION of a variety of GENES involved primarily in CELL DIFFERENTIATION and CELL CYCLE progression.
Transcription Factor Tfiia
An RNA POLYMERASE II specific transcription factor. It may play a role in transcriptional activation of gene expression by interacting with the TATA-BOX BINDING PROTEIN component of TRANSCRIPTION FACTOR TFIID.
Core Binding Factors
Heterodimeric transcription factors containing a DNA-binding alpha subunits, (CORE BINDING FACTOR ALPHA SUBUNITS), along with a non-DNA-binding beta subunits, CORE BINDING FACTOR BETA SUBUNIT. Core Binding Factor regulates GENETIC TRANSCRIPTION of a variety of GENES involved primarily in CELL DIFFERENTIATION and CELL CYCLE progression.
Core Binding Factor Alpha 2 Subunit
A transcription factor that dimerizes with the cofactor CORE BINDING FACTOR BETA SUBUNIT to form core binding factor. It contains a highly conserved DNA-binding domain known as the runt domain. Runx1 is frequently mutated in human LEUKEMIAS.
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