A novel molecular rotor facilitates detection of p53-DNA interactions using the Fluorescent Intercalator Displacement Assay.

We have investigated the use of fluorescent molecular rotors as probes for detection of p53 binding to DNA. These are a class of fluorophores that undergo twisted intramolecular charge transfer (TICT). They are non-fluorescent in a freely rotating conformation and experience a fluorescence increase when restricted in the planar conformation.

A conformation-induced fluorescence method for microRNA detection.

MicroRNAs play important roles in a large variety of biological systems and processes through their regulation of target mRNA expression, and show promise as clinical biomarkers. However, their small size presents challenges for tagging or direct detection. Innovation in techniques to sense and quantify microRNAs may aid research into novel aspects of microRNA biology and contribute to the development of diagnostics.

Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions.

We demonstrate the use of fluorescent molecular rotors as probes for detecting biomolecular interactions, specifically peptide-protein interactions. Molecular rotors undergo twisted intramolecular charge transfer upon irradiation, relax via the nonradiative torsional relaxation pathway, and have been typically used as viscosity probes. Their utility as a tool for detecting specific biomolecular interactions has not been explored.

Multispectral labeling of antibodies with polyfluorophores on a DNA backbone and application in cellular imaging.

Most current approaches to multiantigen fluorescent imaging require overlaying of multiple images taken with separate filter sets as a result of differing dye excitation requirements. This requirement for false-color composite imaging prevents the user from visualizing multiple species in real time and disallows imaging of rapidly moving specimens. To address this limitation, here we investigate the use of oligodeoxyfluoroside (ODF) fluorophores as labels for antibodies.

Selective sensor for silver ions built from polyfluorophores on a DNA backbone.

To explore a new modular metal ion sensor design strategy, fluorophores and ligands were incorporated into short DNA-like oligomers. Compound 1 was found to function as a selective sensor for Ag(+) in aqueous buffer, where low micromolar concentrations of Ag(+) induce a red-shifted, turn-on fluorescence signal. Experiments with HeLa cells show that 1 can penetrate cells and yield a signal for intracellular Ag(+).

DNA-polyfluorophore excimers as sensitive reporters for esterases and lipases.

DNA-scaffolded oligodeoxyriboside fluorophores (ODFs) were used as the reporters in turn-on sensing of enzymatic bond-cleaving activity. A tetramer ODF of pyrene deoxynucleosides displayed high quenching efficiency when conjugated via ester linkages with a dabcyl quencher, and yielded large signal increases with several enzymes in vitro and in intact human cells.

Polyfluorophore excimers and exciplexes as FRET donors in DNA.

We describe studies aimed at testing whether oligomeric exciplex and excimer fluorophores conjugated to DNA have the potential to act as donors for energy transfer by the Forster mechanism. Oligodeoxyfluorosides (ODFs) are composed of stacked, electronically interacting fluorophores replacing the bases on a DNA scaffold. The monomer chromophores in the twenty tetramer-length ODFs studied here include pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and a nonfluorescent spacer (S); these are conjugated in varied combinations at the 3' end of a 14mer DNA probe sequence.

Polyfluorophore labels on DNA: dramatic sequence dependence of quenching.

We describe studies carried out in the DNA context to test how a common fluorescence quencher, dabcyl, interacts with oligodeoxynucleoside fluorophores (ODFs)--a system of stacked, electronically interacting fluorophores built on a DNA scaffold. We tested twenty different tetrameric ODF sequences containing varied combinations and orderings of pyrene (Y), benzopyrene (B), perylene (E), dimethylaminostilbene (D), and spacer (S) monomers conjugated to the 3' end of a DNA oligomer. Hybridization of this probe sequence to a dabcyl-labeled complementary strand resulted in strong quenching of fluorescence in 85% of the twenty ODF sequences.

Polyfluorophores on a DNA backbone: a multicolor set of labels excited at one wavelength.

We recently described the assembly of fluorescent deoxyriboside monomers ("fluorosides") into DNA-like phosphodiester oligomers (oligodeoxyfluorosides or ODFs) in which hydrocarbon and heterocyclic aromatic fluorophores interact both physically and electronically. Here we report the identification of a multicolor set of water-soluble ODF dyes that display emission colors across the visible spectrum, and all of which can be simultaneously excited by long-wavelength UV light at 340-380 nm. Multispectral dye candidates were chosen from a library of 4096 tetramer ODFs constructed on PEG-polystyrene beads using a simple long-pass filter to observe all visible colors at the same time.

Studies of oligodeoxyfluorosides (ODFs) as FRET probes for DNA hybridization.

Oligodeoxyfluorosides (ODFs) are a novel system of stacked, electronically interacting fluorophores built on the DNA scaffold. Here we describe early studies of these ODFs as potential universal FRET donors and as reporters of DNA hybridization.

Efficient quenching of oligomeric fluorophores on a DNA backbone.

The quenching properties of a series of oligodeoxyribosides bearing fluorophore ‘bases’ is described. Sequences of adjacent, π-stacked pyrenes exhibit stronger electronic interactions visible in both absorbance and emission spectra than pyrenes that are insulated by intervening adenines. Quenching by N, N′-dimethyl-4,4′-bipyridinium dichloride is efficient for excimer-and exciplex-forming oligomers, with Stern-Volmer constants comparable to conjugated polymer “superquenching” schemes.