The three-dimensional context of a double helix determines the fluorescence of the internucleoside-tethered pair of fluorophores

Abstract

We report a general phenomenon of the formation of either a fluorescent, or of an entirely quenched oligodeoxynucleotide (ODN) duplex system by hybridizing pairs of complementary ODNs with identical chemical composition. The ODNs carried internucleoside tether-linked cyanines, where the cyanines were chosen to form a Förster's resonance energy transfer (FRET) doner/acceptor pair. The fluorescent and quenched ODN duplex systems differed only in that the cyanines linked to the respective ODNs s were linked either closer to the 5′-, or closer to the 3′-ends of the molecule. In either case however, the dyes were separated by an identical number (7 or 8) of base pairs. Characterization by molecular modeling and energy minimization using a conformational search algorithm in a molecular operating environment (MOE) revealed that linking of the dyes closer to the 5′-ends resulted in their reciprocal orientation across the major groove which allowed a closely interacting dye pair to be formed. This overlap between the donor and acceptor dye molecules resulted in changes of absorbance spectra consistent with the formation of H-aggregates. Conversly, dyes linked closer to 3′-ends exhibited emissive FRET and formed a pair of dyes that interacted with the DNA helix only weakly. Induced CD spectra analysis suggested that interaction with the double helix was weaker than in the case of the closely interacting cyanine dye pair. Linking the dyes such that the base pair separation was 10 or 0 favored energy transfer with subsequent acceptor emission. Our results suggest that when interpreting FRET measurements from nucleic acids, the use of a “spectroscopic ruler” principle which takes into account the 3D helical context of the double helix will allow more accurate interpretation of fluorescence emission.

Fluorocarbons Enhance Intracellular Delivery of Short STAT3-sensors and Enable Specific Imaging

“…The gels were imaged on an EpiChem III system (UVP BioImaging System, Upland, CA 91786) equipped with a Hamamatsu CCD camera and the obtained TIF images of gels were analyzed using ImageJ (NIH software) 42 . Fluorescence lifetime was measured as described in 45 , 46 using 520-540 nm excitation and 580 nm long-pass filter; time resolved fluorescence was registered using a CCD camera coupled to a gated-image-intensifier (LaVision Gmbh, Germany) that provides a 300 ps time resolution.…”

Near-infrared oligonucleotide duplex sensors for imaging rapidly activated transcription factors in vitro and in situ

“…The chemical structure differences between various pairs of F and Q molecules in their ability to interact with each other allow fine tuning of quenching effects essential for molecular target identification. If fluorescence of F spectrally overlaps with Q [7]. Upon binding with NF-B there is a dissociation of the pair with resultant favorable orientation of transition dipole vectors (shown with arrows).…”

“…The increased cellular tropism of oligonucleotide duplexes [45] and hairpins suggests that structure design and optimization of decoys can potentially greatly increase their biological activity. The sensor carries two interacting fluorophores [7] (in the major groove, rendered interacting surfaces are shown), and two short perfluorinated (PF) ODN end-modification tails [8]. B -the measurements of FL of a near-infrared fluorophore in the near-infrared range determined from mono-exponential decay curves can be easily separated from the short FL values of excitation pulse diffusely propagating through the tissue; C-pseudo-color FL maps of donor/acceptor pair linked to ODN-MS obtained in FRET mode (ex 650 nm/em 800 nm) showing the change of FL of the acceptor fluorophore after ODN-MS binding to TF.…”

“…Activation in the cytoplasm provides a high concentration of binding centers for ODN-MS. Upon binding, the fluorophores linked to ODN-MS disengage from the non-fluorescent (Haggregates) they form within the major groove of the ODN duplex [7], then associate with interacting amino acid residues (e.g. arginine) within the TF binding site in such a way as to generate fluorescence.…”

Substrate-Based Near-Infrared Imaging Sensors Enable Fluorescence Lifetime Contrast via Built-in Dynamic Fluorescence Quenching Elements

“…These sensors are known to be strongly affected by static quenching including radiative and non-radiative resonance energy transfer, and non-radiative energy transfer either to structurally different acceptors or to molecules of the same kind, which enables exceptional flexibility in designing FLT-based sensors for imaging 7, 12 . The simplest case of static quenching is a consequence of interaction of the individual cyanine molecules with the formation of intermolecular H -aggregates in polar solvents 12–15 . The separation of the fluorophores usually occurs as a consequence of enzyme-mediated sensor breakdown, resulting in the emission of a light photon, as opposed to non-emissive transfer within the originally quenched substrate-based sensors 16, 17 .…”