We describe the engineering of reversible fluorescence photoswitching in DNA with high-density substitution, and its applications in advanced fluorescence microscopy methods. High-density labeling of DNA with cyanine dyes can be achieved by polymerase chain reaction using a modified DNA polymerase that has been evolved to efficiently incorporate Cy3- and Cy5-labeled cytosine base analogues into double-stranded DNA. The resulting biopolymer, "CyDNA", displays hundreds of fluorophores per DNA strand and is strongly colored and highly fluorescent, although previous observations suggest that fluorescence quenching at such high density might be a concern, especially for Cy5. Herein, we first investigate the mechanisms of fluorescence quenching in CyDNA and we suggest that two different mechanisms, aggregate formation and resonance energy transfer, are responsible for fluorescence quenching at high labeling densities. Moreover, we have been able to re-engineer CyDNA into a reversible fluorescence photoswitchable biopolymer by using the properties of the Cy3-Cy5 pair. This novel biopolymer constitutes a new class of photoactive DNA-based nanomaterial and is of great interest for advanced microscopy applications. We show that reversible fluorescence photoswitching in CyDNA can be exploited in optical lock-in detection imaging. It also lays the foundations for improved and sequence-specific super-resolution fluorescence microscopy of DNA.
Guinea‐pigs were sensitized with 3 injections of ovalbumin (OA) (1 or 10 μg per animal) using Al(OH) 3 and pertussis vaccine as adjuvants at two week intervals. Sensitized guinea‐pigs were challenged with an aerosol of OA (0.1%) over a one hour period and both airway reactivity and cellular content of bronchoalveolar lavage (BAL) fluid were assessed at intervals for up to 7 days. Guinea‐pigs sensitized with 1 μg of ovalbumin responded to an aerosol of OA with increased pulmonary airway eosinophilia, which was evident 1 day after challenge and was present for up to 7 days. Airway hyperreactivity was not detectable in these animals. Guinea‐pigs sensitized with 10 μg of ovalbumin responded to an aerosol of OA with increased pulmonary airway neutrophilia and eosinophilia and with increased airway reactivity which was maximal between 8 and 24 h after exposure to OA. Depletion of circulating platelets or neutrophils, by use of selective antisera, did not alter either the magnitude of eosinophilia or the intensity of airway reactivity in sensitized guinea‐pigs (10 μg) exposed to an aerosol of OA. Pretreatment of sensitized guinea‐pigs (10 μg) for 6 days with AH 21–132, aminophylline, dexamethasone or ketotifen inhibited pulmonary airway eosinophilia, but did not diminish airway hyperreactivity. Neither eosinophil accumulation nor development of airway hyperreactivity was influenced by treatment with mepyramine or salbutamol over a 6 day period before OA inhalation. Although eosinophilia may occur in association with increased airway reactivity in this animal model, there is no evidence of a causal relationship.
We report an approach for visualizing DNA sequence and using these 'DNA barcodes' to search complex mixtures of genomic material for DNA molecules of interest. We demonstrate three applications of this methodology; identifying specific molecules of interest from a dataset containing gigabasepairs of genome; identification of a bacterium from such a dataset and, finally, by locating infecting virus molecules in a background of human genomic material. As a result of the dense fluorescent labelling of the DNA, individual barcodes of the order 40 kb pairs in length can be reliably identified. This means DNA can be prepared for imaging using standard handling and purification techniques. The recorded dataset provides stable physical and electronic records of the total genomic content of a sample that can be readily searched for a molecule or region of interest.
Growth inhibition studies were done on an encapsulated and non-encapsulated strain of Cryptococcus neoformans at the minimal inhibitory concentration and one-half the minimal inhibitory concentration of ketoconazole and amphotericin B alone and in combination. Growth of both strains was significantly inhibited by ketoconazole, amphotericin B, and the combined drugs at the minimal inhibitory concentration of each drug over a 5-day period. Calculation of the expected inhibition of growth for both strains with both drugs showed antagonism at 24 h followed by an additive effect and synergy for the remaining 4 days of the assay. Although similar results were obtained for both strains with one-half the minimal inhibitory concentration, an additive effect was observed with the drug combination at 24 h for the encapsulated strain, and an antagonistic effect was observed with the non-encapsulated strain.
A quantitative understanding of how conformational transitions contribute to enzyme catalysis and specificity remains a fundamental challenge. A suite of biophysical approaches was used to reveal several transient states of the enzyme–substrate complexes of the model DNA cytosine methyltransferase M.HhaI. Multidimensional, transverse relaxation-optimized nuclear magnetic resonance (NMR) experiments show that M.HhaI has the same conformation with noncognate and cognate DNA sequences. The high-affinity cognatelike mode requires the formation of a subset of protein–DNA interactions that drive the flipping of the target base from the helix to the active site. Noncognate substrates lacking these interactions undergo slow base flipping, and fluorescence tracking of the catalytic loop corroborates the NMR evidence of a loose, nonspecific binding mode prior to base flipping and subsequent closure of the catalytic loop. This slow flipping transition defines the rate-limiting step for the methylation of noncognate sequences. Additionally, we present spectroscopic evidence of an intermediate along the base flipping pathway that has been predicted but never previously observed. These findings provide important details of how conformational rearrangements are used to balance specificity with catalytic efficiency.
Optical mapping relies on the preparation of fluorescent DNA. DNA must be imaged with good signal to noise and therefore the background of unwanted DNA fragments, fluorescent dyes and other reagents need to be removed. We use deterministic lateral displacement to separate 48.5 kbp DNA from 50 kbp molecules from a background of shorter digested fragments. In both cases improving signal to noise during imaging.
We review various methods for analysing time-resolved fluorescence data acquired using the time-correlated single photon counting method in an attempt to evaluate their benefits and limitations. We have applied these methods to both experimental and simulated data. The relative merits of using deterministic approaches, such as the commonly used iterative reconvolution method, and probabilistic approaches, such as the smoothed exponential series method, the maximum entropy method and recently proposed basis pursuit denoising (compressed sensing) method, are outlined. In particular, we show the value of using multiple methods to arrive at the most appropriate choice of model. We show that the use of probabilistic analysis methods can indicate whether a discrete component or distribution analysis provides the better representation of the data.
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