Fluorescent nucleobases as tools for studying DNA and RNA.

Understanding the diversity of dynamic structures and functions of DNA and RNA in biology requires tools that can selectively and intimately probe these biomolecules. Synthetic fluorescent nucleobases that can be incorporated into nucleic acids alongside their natural counterparts have emerged as a powerful class of molecular reporters of location and environment. They are enabling new basic insights into DNA and RNA, and are facilitating a broad range of new technologies with chemical, biological and biomedical applications. In this Review, we will present a brief history of the development of fluorescent nucleobases and explore their utility as tools for addressing questions in biophysics, biochemistry and biology of nucleic acids. We provide chemical insights into the two main classes of these compounds: canonical and non-canonical nucleobases. A point-by-point discussion of the advantages and disadvantages of both types of fluorescent nucleobases is made, along with a perspective into the future challenges and outlook for this burgeoning field.

Luminescent Carbon Dot Mimics Assembled on DNA.

Nanometer-sized fragments of carbon in the form of multilayer graphene ("carbon dots") have been under highly active study for applications in imaging. While offering advantages of low toxicity and photostability, such nanomaterials are inhomogeneous and have limited wavelengths of emission. Here we address these issues by assembling luminescent aromatic C16-C38 hydrocarbons together on a DNA scaffold in homogeneous, soluble molecular compounds. Monomer deoxyribosides of five different aromatic hydrocarbons were synthesized and assembled into a library of 1296 different tetramer compounds on PEG-polystyrene beads. These were screened for photostability and a range of emission colors using 365 nm excitation, observing visible light (>400 nm) emission. We identified a set of six oligomers (DNA-carbon assemblies, DNA-CAs) with exceptional photostability that emit from 400 to 680 nm in water, with Stokes shifts of up to 110 nm, quantum yields ranging from 0.01 to 0.29, and fluorescence lifetimes from 3 to 42 ns. In addition, several of these DNA-CAs exhibited white emission in aqueous solution. The molecules were used in multispectral cell imaging experiments and were taken up into cells passively. The results expand the range of emission properties that can be achieved in water with all-hydrocarbon chromophores and establish the use of the DNA scaffold to arrange carbon layers in homogeneous, rapidly synthesized assemblies.

Color-Change Photoswitching of an Alkynylpyrene Excimer Dye.

We describe a photoswitchable DNA-based dimeric dye that visibly changes fluorescence from green to blue upon UV irradiation. A novel bis-alkyne-dependent [2+2+2] cycloaddition is proposed as a mechanism for the color change in air. The photoinduced structural switching results in spatial separation of stacked pyrene units, thereby causing selective loss of the excimer emission. We demonstrate and suggest several applications for this novel photoswitch.

DNA as an environmental sensor: detection and identification of pesticide contaminants in water with fluorescent nucleobases.

Environmental contaminants pose a substantial health risk in many areas of the world. One of these risks is contamination of water with toxic organic species, such as herbicides and insecticides. Here we describe the discovery and properties of a set of fluorescent chemosensors that respond to micromolar concentrations of a broad range of common organic pesticides. The chemosensors are short DNA-like oligomers with fluorophores replacing DNA bases that are assembled via a DNA synthesizer. We screened a library of 1296 tetrameric compounds on polystyrene microbeads, and identified a set of chemosensor sequences that respond strongly to a set of structurally varied pesticide analytes. We show that ten chemosensors on beads can be used to detect and identify 14 different common pesticides at 100 µM, using the pattern of fluorescence intensity and wavelength changes. Limits of detection for two analytes were as low as 2 µM. The chemosensors are shown to function successfully in a practical setting, correctly identifying unknown pesticide contaminants in water from Felt Lake, California. The results establish a simple, low cost strategy for sensing environmental spills of toxic organics.

In Vitro Fluorogenic Real-Time Assay of the Repair of Oxidative DNA Damage.

The repair of oxidative damage to DNA is essential to avoid mutations that lead to cancer. Oxidized DNA bases, such as 8-oxoguanine, are a main source of these mutations, and the enzyme 8-oxoguanine glycosylase 1 (OGG1) is the chief human enzyme that excises 8-oxoguanine from DNA. The activity of OGG1 has been linked to human inflammation responses and to cancer, and researchers are beginning to search for inhibitors of the enzyme. However, measuring the activity of the enzyme typically requires laborious gel-based measurements of radiolabeled DNAs. Here we report the design and properties of fluorogenic probes that directly report on the activity of OGG1 (and its bacterial homologue Fpg) in real time as the oxidized base is excised. The probes are short, modified DNA oligomers containing fluorescent DNA bases and are designed to utilize 8-oxoguanine itself as a fluorescence quencher. Screening of combinations of fluorophores and 8-oxoguanine revealed two fluorophores, pyrene and tCo, that are strongly quenched by the damaged base. We tested 42 potential probes containing these fluorophores: the optimum probe, OGR1, yields a 60-fold light-up signal in vitro with OGG1 and Fpg. It can report on oxidative repair activity in mammalian cell lysate and with bacterial cells overexpressing a repair enzyme. Such probes might prove useful in quantifying enzyme activity and performing competitive inhibition assays.

Fast alpha nucleophiles: structures that undergo rapid hydrazone/oxime formation at neutral pH.

Hydrazones and oximes are widely useful structures for conjugate formation in chemistry and biology, but their formation can be slow at neutral pH. Kinetics studies were performed for a range of structurally varied hydrazines, and a surprisingly large variation in reaction rate was observed. Structures that undergo especially rapid reactions were identified, enabling reaction rates that rival orthogonal cycloaddition-based conjugation chemistries.

Genetically encoded multispectral labeling of proteins with polyfluorophores on a DNA backbone.

Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.