Viral DNA trafficking in cells has large impacts on physiology and disease development. Current methods lack the resolution and accuracy to visualize and quantify viral DNA trafficking at single-molecule resolution. We developed a noninvasive protocol for accurate quantification of viral DNA-genome (vDNA) trafficking in single cells. Ethynyl-modified nucleosides were used to metabolically label newly synthesized adenovirus, herpes virus, and vaccinia virus vDNA, without affecting infectivity. Superresolution microscopy and copper(I)-catalyzed azide-alkyne cycloaddition (click) reactions allowed visualization of infection at single vDNA resolution within mammalian cells. Analysis of adenovirus infection revealed that a large pool of capsid-free vDNA accumulated in the cytosol upon virus uncoating, indicating that nuclear import of incoming vDNA is a bottleneck. The method described here is applicable for the entire replication cycle of DNA viruses and offers opportunities to localize cellular and viral effector machineries on newly replicated viral DNA, or innate immune sensors on cytoplasmic viral DNA.

Mounting evidence supports the presence of biologically relevant G-quadruplexes in single-cell organisms, but the existence of endogenous G-quadruplex structures in mammalian cells remains highly controversial. This is due, in part, to the common misconception that DNA and RNA molecules are passive information carriers with relatively little structural or functional complexity. For those working in the field, however, the lack of available tools for characterizing DNA structures in vivo remains a major limitation to addressing fundamental questions about structure-function relationships of nucleic acids. In this review, we present progress towards the direct detection of G-quadruplex structures by using small molecules and modified oligonucleotides as fluorescent probes. While most development has focused on cell-permeable probes that selectively bind to G-quadruplex structures with high affinity, these same probes can induce G-quadruplex folding, thereby making the native conformation of the DNA or RNA molecule (i.e., in the absence of probe) uncertain. For this reason, modified oligonucleotides and fluorescent base analogues that serve as "internal" fluorescent probes are presented as an orthogonal means for detecting conformational changes, without necessarily perturbing the equilibria between G-quadruplex, single-stranded, and duplex DNA. The major challenges and motivation for the development of fluorescent probes for G-quadruplex structures are presented, along with a summary of the key photophysical, biophysical, and biological properties of reported examples.

Commonly used metabolic labels for DNA, including 5-ethynyl-2′-deoxyuridine (EdU) and BrdU, are toxic antimetabolites that cause DNA instability, necrosis, and cell-cycle arrest. In addition to perturbing biological function, these properties can prevent metabolic labeling studies where subsequent tissue survival is needed. To bypass the metabolic pathways responsible for toxicity, while maintaining the ability to be metabolically incorporated into DNA, we synthesized and evaluated a small family of arabinofuranosylethynyluracil derivatives. Among these, (2′S)-2′-deoxy-2′-fluoro-5-ethynyluridine (F-ara-EdU) exhibited selective DNA labeling, yet had a minimal impact on genome function in diverse tissue types. Metabolic incorporation of F-ara-EdU into DNA was readily detectable using copper(I)-catalyzed azide-alkyne "click" reactions with fluorescent azides. F-ara-EdU is less toxic than both BrdU and EdU, and it can be detected with greater sensitivity in experiments where long-term cell survival and/or deep-tissue imaging are desired. In contrast to previously reported 2′-arabino modified nucleosides and EdU, F-ara-EdU causes little or no cellular arrest or DNA synthesis inhibition. F-ara-EdU is therefore ideally suited for pulse-chase experiments aimed at "birth dating" DNA in vivo. As a demonstration, Zebrafish embryos were microinjected with F-ara-EdU at the one-cell stage and chased by BrdU at 10 h after fertilization. Following 3 d of development, complex patterns of quiescent/senescent cells containing only F-ara-EdU were observed in larvae along the dorsal side of the notochord and epithelia. Arabinosyl nucleoside derivatives therefore provide unique and effective means to introduce bioorthogonal functional groups into DNA for diverse applications in basic research, biotechnology, and drug discovery. T he utilization of chemical techniques to address biological systems is becoming increasingly important in basic research and modern drug discovery (1). One underexplored area at the biology-chemistry interface is the study of nucleic acids in their native environments. Traditional DNA and RNA imaging methodologies have utilized fluorescent fusion proteins, nonspecific stains for nucleic acids, immunostaining of BrdU-labeled DNA, or FISH (2). All of these approaches are limited in terms of their low throughput, large perturbations to native systems, and/or inability to be applied in unmodified cells and organisms.Metabolic labeling of DNA has traditionally been performed using [ 3 H]thymidine or BrdU. These labels are limited in terms of their subsequent visualization, requiring either autoradiography, or DNA denaturation and antibody staining (3). BrdU immunostaining is currently the most commonly used method, but it requires harsh chemical denaturation of cellular DNA and is limited by the poor tissue penetration of the BrdU antibody. In addition, BrdU itself is both toxic and mutagenic when applied at high concentrations, and it can have a negative impact on DNA stability and the cell cycle (4).The recent ...

Recombinant polypeptides and protein domains containing two cysteine pairs located distal in primary sequence but proximal in the native folded or assembled state are labeled selectively in vitro and in mammalian cells using the profluorescent biarsenical reagents FlAsH-EDT 2 and ReAsH-EDT 2 . This strategy, termed bipartite tetracysteine display, enables the detection of protein-protein interactions and alternative protein conformations in live cells. As proof of principle, we show that the equilibrium stability and fluorescence intensity of polypeptide-biarsenical complexes correlates with the thermodynamic stability of the protein fold or assembly. Destabilized protein variants form less stable and less bright biarsenical complexes, which allows discrimination of live cells expressing folded polypeptide and protein domains from those containing disruptive point mutations. Bipartite tetracysteine display may provide a means to detect early protein misfolding events associated with Alzheimer's disease, Parkinson's disease and cystic fibrosis; it may also enable high-throughput screening of compounds that stabilize discrete protein folds.The physical phenomenon known as fluorescence has revolutionized cell biology, and its positive impact on molecular medicine will continue to develop with time. Driving this revolution is the green fluorescent protein (GFP) 1-3 and a rainbow of natural and engineered fluorescent protein (FP) variants that can be fused at the genetic level to proteins expressed in cell cultures and even whole animals 3 . Methodologies based on Förster resonance energy transfer (FRET) that use two or more FPs with overlapping absorption and emission spectra are used frequently as sensors to probe dynamic and complex processes including protein association, metal ion binding, conformational changes and post-translational modifications 4 . However, virtually all sensors based on FRET between FP color variants show dynamic changes in fluorescence intensity (typically 20-50%) that can be smaller than normal variations in cell-to-cell intensity due to differential FP expression 5 and that may be influenced significantly by Mg 2+ -ATP fluctuation 6 . Moreover, the steric bulk and slow folding of FPs limits their spatial and kinetic resolution 3,7 . We selected four structurally characterized polypeptides and protein domains to evaluate whether the Pro-Gly sequence within the linear tetracysteine motif could be replaced with one or more folded proteins while maintaining biarsenical affinity and fluorescence intensity. The feasibility of intramolecular bipartite tetracysteine display was evaluated using avian pancreatic polypeptide (aPP) 20 and Zip4 (ref. 21)-two well-folded polypeptides that were modified to contain one half of the linear tetracysteine motif at each terminus. Intermolecular bipartite tetracysteine display was evaluated using the protein-protein dimerization domains from the basic region leucine zipper (bZIP) proteins GCN4 (ref. 22) and Jun (ref. 23), which were modified to conta...

Aminoglycosides (including neomycin B and tobramycin) exhibit poor uptake by eukaryotic cell lines. When the amines of these natural products are converted into guanidine groups, their cellular uptake is dramatically enhanced. We have synthesized BODIPY-containing aminoglycosides and guanidinoglycosides to evaluate their cellular uptake properties. Fluorescence activated cell sorting (FACS) and fluorescence microscopy are used to compare the membrane translocation and the cellular localization of these compounds. Upon guanidinylation, the cellular uptake efficiencies of tobramycin and neomycin B are enhanced by 10-fold and 20-fold, respectively. Guanidino-neomycin B exhibits a highly efficient uptake, superior to a fluorescent poly-arginine peptide. Interestingly, the cellular uptake of this common transduction peptide is inhibited by guanidine-neomycin B, suggesting a similar uptake mechanism for both the arginine-rich peptides and the guanidinoglycosides.

5-Vinyl-2'-deoxyuridine (VdU) is the first reported metabolic probe for cellular DNA synthesis that can be visualized by using an inverse electron demand Diels-Alder reaction with a fluorescent tetrazine. VdU is incorporated by endogenous enzymes into the genomes of replicating cells, where it exhibits reduced genotoxicity compared to 5-ethynyl-2'-deoxyuridine (EdU). The VdU-tetrazine ligation reaction is rapid (k≈0.02 M(-1) s(-1)) and chemically orthogonal to the alkyne-azide "click" reaction of EdU-modified DNA. Alkene-tetrazine ligation reactions provide the first alternative to azide-alkyne click reactions for the bioorthogonal chemical labeling of nucleic acids in cells and facilitate time-resolved, multicolor labeling of DNA synthesis.

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Metabolic incorporation of azido nucleoside analogues into living cells can enable sensitive detection of DNA replication through copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) and strain-promoted azide-alkyne cycloaddition (SPAAC) "click" reactions. One major limitation to this approach is the poor chemical stability of nucleoside derivatives containing an aryl azide group. For example, 5-azido-2'-deoxyuridine (AdU) exhibits a 4 h half-life in water, and it gives little or no detectable labeling of cellular DNA. In contrast, the benzylic azide 5-(azidomethyl)-2'-deoxyuridine (AmdU) is stable in solution at 37 °C, and it gives robust labeling of cellular DNA upon addition of fluorescent alkyne derivatives. In addition to providing the first examples of metabolic incorporation into and imaging of azide groups in cellular DNA, these results highlight the general importance of assessing azide group stability in bioorthogonal chemical reporter strategies.