Shenliang Wang

Direct Fluorescence Monitoring of DNA Base Excision Repair

Uracil is an undesired component of DNA, as it arises from spontaneous deamination of cytosine.[1] This hydrolysis reaction promotes mutations, since the resulting U-G pair can be misread during DNA replication. As a result, multiple cellular enzymes have evolved to detect uracil in DNA and remove it prior to replication.[2] In E. coli uracil DNA glycosylase (UDG) enzyme functions to guard the bacterial genome. In humans, similar enzyme activities exist, including the proteins UNG1/2, SMUG, and TDG.[3] These enzymes flip uracil out of the DNA helix and cleave it from its deoxyribose sugar, leaving an abasic site in its place.[4]

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.

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.

Monitoring eukaryotic and bacterial UDG repair activity with DNA-multifluorophore sensors

We report the development of simple fluorogenic probes that report on the activity of both bacterial and mammalian uracil–DNA glycosylase (UDG) enzymes. The probes are built from short, modified single-stranded oligonucleotides containing natural and unnatural bases. The combination of multiple fluorescent pyrene and/or quinacridone nucleobases yields fluorescence at 480 and 540 nm (excitation 340 nm), with large Stokes shifts of 140–200 nm, considerably greater than previous probes. They are strongly quenched by uracil bases incorporated into the sequence, and they yield light-up signals of up to 40-fold, or ratiometric signals with ratio changes of 82-fold, on enzymatic removal of these quenching uracils. We find that the probes are efficient reporters of bacterial UDG, human UNG2, and human SMUG1 enzymes in vitro, yielding complete signals in minutes. Further experiments establish that a probe can be used to image UDG activity by laser confocal microscopy in bacterial cells and in a human cell line, and that signals from a probe signalling UDG activity in human cells can be quantified by flow cytometry. Such probes may prove generally useful both in basic studies of these enzymes and in biomedical applications as well.

DNA-Polyfluorophores for Real-Time Multicolor Tracking of Dynamic Biological Systems

Dye-ing to live: Spectral limitations of common organic dyes make it difficult or impossible to visualize and follow multiple biological components in rapidly moving systems. The development of a multispectral set of improved DNA-scaffolded fluorophores is described. Their use in multicolor cellular imaging (see scheme) and in tracking of biological motions on the subsecond timescale is demonstrated.

Templated chemistry for monitoring damage and repair directly in duplex DNA

We report the fluorogenic detection of the product of base excision repair (an abasic site) in a specific sequence of duplex DNA. This is achieved by DNA-templated chemistry, employing triple helix-forming probes that contain unnatural nucleobases designed to selectively recognize the site of a missing base. Light-up signals of up to 36-fold were documented, and probes could be used to monitor enzymatic removal of a damaged base.

DNA-polyfluorophore Chemosensors for Environmental Remediation: Vapor-phase Identification of Petroleum Products in Contaminated Soil

Contamination of soil and groundwater by petroleum-based products is an extremely widespread and important environmental problem. Here we have tested a simple optical approach for detecting and identifying such industrial contaminants in soil samples, using a set of fluorescent DNA-based chemosensors in pattern-based sensing. We used a set of diverse industrial volatile chemicals to screen and identify a set of five short oligomeric DNA fluorophores on PEG-polystyrene microbeads that could differentiate the entire set after exposure to their vapors in air. We then tested this set of five fluorescent chemosensor compounds for their ability to respond with fluorescence changes when exposed to headgas over soil samples contaminated with one of ten different samples of crude oil, petroleum distillates, fuels, lubricants and additives. Statistical analysis of the quantitative fluorescence change data (as Δ(R,G,B) emission intensities) revealed that these five chemosensors on beads could differentiate all ten product mixtures at 1000 ppm in soil within 30 minutes. Tests of sensitivity with three of the contaminant mixtures showed that they could be detected and differentiated in amounts at least as low as one part per million in soil. The results establish that DNA-polyfluorophores may have practical utility in monitoring the extent and identity of environmental spills and leaks, while they occur and during their remediation.

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.

Sampling variance of a T-year flood estimated by curve-fitting

The sampling variance of a T-year flood when estimated using a curve-fitting method results from the errors in hydrologic observations, plotting positions, and model-fitting. This paper develops a method to quantify the contribution of plotting positions to the sampling variance of the T-year flood magnitude. Application of the method to 150 flood-flow data sets of 41 rivers in the Peoples Republic of China show that the errors due to plotting positions contribute more to the sampling variance than others.

Correction to “Genetically Encoded Multispectral Labeling of Proteins with Polyfluorophores on a DNA Backbone”

A internal review of our NMR data in the published Supporting Information (SI) file revealed that one of the authors (V.S.) had digitally removed peaks of impurities and solvents from some of the spectra. To correct this, we now provide a new version of the SI file with the unaltered spectra. Two new authors (K.M.C., S.A.C.) have recharacterized the original haloalkyl reagent B8 and have confirmed its identity by NMR and mass spectrometry. In addition, we have confirmed the identity of one of the original dyes by MALDI-TOF mass spectrometry, and used it successfully to label bacteria expressing a HaloTag fusion; these data are added to the corrected SI file. We stand by the conclusions of the article, and we regret the publication of the altered characterization data. We also correct the author list to include the scientists (K.M.C. and S.A.C.) who worked to independently check the data and conclusions. The new author list should read as follows: Vijay Singh, Shenliang Wang, Ke Min Chan, Spencer A. Clark, and Eric T. Kool*