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Results for “DNA-binding fluorescent probes”

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PubMed2026

HoT auto-blinking probes enable real-time, super-resolution chromatin imaging in live cells and tissues.

Single-molecule localization microscopy (SMLM) enables visualization of chromatin architecture at nanoscale resolution. However, high-performance DNA probes suitable for SMLM in both live cells and tissues remain limited. We developed Hoechst-6-Carboxytetramethylrhodamine (6-TAMRA) derivative (HoT) probes-rhodamine-based derivatives conjugated to a Hoechst moiety-through structural fine-tuning of rhodamine spirocyclization. HoTs are self-assembling, auto-blinking probes with excellent photostability and high temporal resolution. They permeate live cells, enabling long-term, real-time nanoscopic chromatin imaging in live and fixed cells and in tissue sections. In live cells, we identified nanoscale features in the 3D organization of chromatin and quantified DNA fiber kinetics at high resolution. We quantified DNA compaction in single cells within retinal and colon cancer sections. OligoSTORM (stochastic optical reconstruction microscopy)-labeled gene loci can be visualized and measured within their HoT-labeled chromatin footprints. Our work provides powerful tools for investigating chromatin structure and functions in living cells and tissues, with applications ranging from cancer diagnosis to retinal regeneration.

Chromatin↗

PubMed1978

Fluorescence and chemical studies on the interaction of Escherichia coli DNA-binding protein with single-stranded DNA.

Nanosecond and steady-state fluorescence spectoscopy were used to probe the environment of the tryptophan residues of Escherichia coli DNA-binding protein. A spectral shift and a change in quantum yield of the protein upon binding to DNA or oligonucleotides indicate that the tryptophan residues are near or at the DNA binding site. The observation of two excited-state lifetimes of the protein indicates that there is heterogeneity in the microenvironments of these tryptophan residues. The "short-lifetime" tryptophan residues are more sensitive to the interaction with DNA than the "long-lifetime" residues. The results of solute-perturbation studies with iodide or acrylamide indicate that there are tryptophan residues near the surface of the protein which are heterogeneous in their accessibility to these quenchers and that they become less accessible after DNA binding. Also, lysine residues of the protein have been shown to be essential to DNA binding by chemical-modification studies. Tyrosine, arginine, and cysteine residues appear not to be involved in this binding process. From studies of the decay of fluorescence anisotropy of the binding protein in the presence and absence of DNA, it has been concluded that (a) the tetrameric binding protein does not dissociate into subuniits upon binding to the oligonucleotide d(pT)16 and (b) the binding protein-fd DNA complex possesses "local flexibility" and, therefore, cannot be described as a continuous, rigid rod.

Coliphages↗

PubMed1978

DNA Bifunctional intercalators. 2. Fluorescence properties and DNA binding interaction of an ethidium homodimer and an acridine ethidium heterodimer.

An ethidium homodimer and acridine ethidium heterodimer have been synthesized (Gaugain, B., Barbet, J., Oberlin, R., Roques, B. P., & Le Pecq, J. B. (1978) Biochemistry 17 (preceding paper in this issue)). The binding of these molecules to DNA has been studied. We show that these dimers intercalate only one of their chromophores in DNA. At high salt concentration (Na+ greater than 1 M) only a single type of DNA-binding site exists. Binding affinity constants can then be measured directly using the Mc Ghee & Von Hippel treatment (Mc Ghee, J. D., & Von Hippel, P. H. (1974) J. Mol. Biol. 86, 469). In these conditions the dimers cover four base pairs when bound to DNA. Binding affinities have been deduced from competition experiments in 0.2 M Na+ and are in agreement with the extrapolated values determined from direct DNA-binding measurements at high ionic strength. As expected, the intrinsic binding constant of these dimers is considerably larger than the affinity of the monomer (ethidium dimer K = 2 X 10(8) M-1; ethidium bromide K = 1.5 X 10(5) M-1 in 0.2 M Na+). The fluorescence properties of these molecules have also been studied. The efficiency of the energy transfer from the acridine to the phenanthridinium chromophore, in the acridine ethidium heterodimer when bound to DNA, depends on the square of the AT base pair content. The large increase of fluorescence on binding to DNA combined with a high affinity constant for nucleic acid fluorescent probes. In particular, such molecules can be used in competition experiments to determine the DNA binding constant of ligands of high binding affinity such as bifunctional intercalators.

Acridines↗