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. We hypothesized that intercalation of a molecular rotor between DNA base pairs would result in a fluorescence turn-on signal. Upon displacement by a DNA binding protein, measurable loss of signal would facilitate use of the molecular rotor in the fluorescent intercalator displacement (FID) assay. A panel of probes was interrogated using the well-established p53 model system across various DNA response elements. A novel, readily synthesizable molecular rotor incorporating an acridine orange DNA intercalating group (AO-R) outperformed other conventional dyes in the FID assay. It enabled relative measurement of p53 sequence-specific DNA interactions and study of the dominant-negative effects of cancer-associated p53 mutants. In a further application, AO-R also proved useful for staining apoptotic cells in live zebrafish embryos.

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. By introducing an additional stem loop into the fluorescent RNA Spinach and altering its 3' and 5' ends, we have generated a new RNA, Pandan, that functions as the basis for a microRNA sensor. Pandan contains two sequence-variable stem loops that encode complementary sequence for a target microRNA of interest. In its sensor form, it requires the binding of a target microRNA in order to reconstitute the RNA scaffold for fluorophore binding and fluorescence. Binding of the target microRNA resulted in large changes in fluorescence intensity. The median fold change in fluorescence observed for the sensors tested was ∼50-fold. Pandan RNA sensors exhibit good signal-to-noise ratios, and can detect their target microRNAs within complex RNA mixtures.

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. Using the well characterized p53-Mdm2 interaction as a model system, we designed a 9-(2-carboxy-2-cyanovinyl) julolidine-based p53 peptide reporter, JP1-R, which fluoresces conditionally only upon Mdm2 binding. The reporter was used in a rapid, homogeneous assay to screen a fragment library for antagonists of the p53-Mdm2 interaction, and several inhibitors were identified. Subsequent validation of these hits using established secondary assays suggests increased sensitivity afforded by JP1-R. The fluorescence of molecular rotors contingent upon target binding makes them a versatile tool for detecting specific biomolecular interactions.

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. ODFs are short DNA-like oligomers with fluorophores replacing the DNA bases and can be assembled in many colors with excitation at a single wavelength. A DNA synthesizer was used to construct several short ODFs carrying a terminal alkyne group and having emission maxima of 410-670 nm. We developed a new approach to antibody conjugation, using Huisgen-Sharpless cycloaddition, which was used to react the alkynes on ODFs with azide groups added to secondary antibodies. Multiple ODF-tagged secondary antibodies were then used to mark primary antibodies. The set of antibodies was tested for spectral characteristics in labeling tubulin in HeLa cells and revealed a wide spectrum of colors, ranging from violet-blue to red with excitation through a single filter (340-380 nm). Selected sets of the differently labeled secondary antibodies were then used to simultaneously mark four antigens in fixed cells, using a single image and filter set. We also imaged different surface tumor markers on two live cell lines. Experiments showed that all colors could be visualized simultaneously by eye under the microscope, yielding multicolor images of multiple cellular antigens in real time.

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(+). This suggests a broadly applicable approach to developing sensors for a wide variety of cations.

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. In the absence of an acceptor chromophore, many of the ODF-DNAs show broad, unstructured long-wavelength emission peaks characteristic of excimer and exciplex excited states, similar to what has been observed for unconjugated ODFs. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Forster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Interestingly, only specific sequences of ODFs were able to act as donors, while others did not, even though their emission wavelengths were similar. The new FRET donors possess large Stokes shifts, which can be beneficial for multiple applications. In addition, all ODFs can be excited at a single wavelength; thus, ODFs may be candidates as "universal FRET donors", thus allowing multicolor FRET of multiple species to be carried out with one excitation.

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. The high efficiency of quenching was also established by their large Stern-Volmer constants (K(SV)) of between 2.1 x 10(4) and 4.3 x 10(5) M(-1), measured with a free dabcyl quencher. Interestingly, quenching of ODFs displayed strong sequence dependence. This was particularly evident in anagrams of ODF sequences; for example, the sequence BYDS had a K(SV) that was approximately two orders of magnitude greater than that of BSDY, which has the same dye composition. Other anagrams, for example EDSY and ESYD, also displayed different responses upon quenching by dabcyl. Analysis of spectra showed that apparent excimer and exciplex emission bands were quenched with much greater efficiency compared to monomer emission bands by at least an order of magnitude. This suggests an important role played by delocalized excited states of the pi stack of fluorophores in the amplified quenching of fluorescence.

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. We resynthesized and characterized a set of 23 ODFs containing one to four individual chromophores and included 2-3 spacer monomers to increase aqueous solubility and minimize aggregation. Emission maxima of this set range from 376 to 633 nm, yielding apparent colors from violet to red, all of which can be visualized directly. The spectra of virtually all ODFs in this set varied considerably from the simple combination of monomer components, revealing extensive electronic interactions between the presumably stacked monomers. In addition, comparisons of anagrams in the set (isomers having the same components in a different sequence) reveal the importance of nearest-neighbor interactions in the emissive behavior. Preliminary experiments with human tumor (HeLa) cells, observing two ODFs by laser confocal microscopy, showed that they can penetrate the outer cellular membrane, yielding cytoplasmic localization. In addition, a set of four distinctly colored ODFs was incubated with live zebrafish embryos, showing tissue penetration, apparent biostability, and no apparent toxicity. The results suggest that ODF dyes may be broadly useful as labels in biological systems, allowing the simultaneous tracking of multiple species by color, and allowing visualization in moving systems where classical fluorophores fail.

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.