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BioSpotlight

2009; Future Science Ltd; Volume: 47; Issue: 4 Linguagem: Inglês

10.2144/000113231

1940-9818

Patrick C.H. Lo, Kristie Nybo,

Microbial Metabolic Engineering and Bioproduction

BioTechniquesVol. 47, No. 4 BioSpotlightOpen AccessBioSpotlightPatrick C.H. Lo & Kristie NyboPatrick C.H. Lo & Kristie NyboPublished Online:25 Apr 2018https://doi.org/10.2144/000113231AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail Fluorescence Polarization Assay: Heterogeneous Is IngeniousWhile there are numerous techniques for measuring protein-ligand interactions, most of these are not well-suited for high-throughput screening applications, such as the identification of potential inhibitors of specific protein-protein interactions. One suitable technique is the fluorescence polarization (FP) assay, which detects the binding in solution of a fluorescently-labeled analyte to a binding partner through an increase in fluorescent polarization caused by a reduction in the rotational mobility of the analyte in the binding complex. A limitation of this homogeneous FP assay, which takes place in solution, is that the binding partner needs to be much larger than the labeled analyte in order to detect the fluorescence polarization change upon binding. In this issue, A. Reichers, J. Schmidt, B. König, and A. Bosserhoff at the University of Regensburg (Regensburg, Germany) have developed a high-throughput version of the FP assay that permits the detection and measurement of binding of either low or high molecular weight partners to a target protein. In their assay, which they call heterogeneous transition metal–based fluorescence polarization (HTFP), instead of fluorescently labeling the molecules that are to be screened for binding to a protein, the protein itself is fluorescently-labeled with the transition metal complex tris(2,2′-bipyridine)ruthenium (II), which has a sufficiently long fluorescence lifetime to detect the loss of fluorescence polarization in unbound proteins up to 500 kDa. The fluorescently-labeled protein is bound to a known ligand which is attached to the surface of wells in a multiwell plate, thereby restricting its ability to rotate and resulting in an FP signal. However, if an added test molecule competes for binding to the same site on the protein as the attached ligand, the protein is then released into solution, which increases its rotational mobility and causes a loss of FP signal. The ability of the HTFP assay to detect both low– and high–molecular weight binding partners is a major improvement on the standard homogeneous FP assay, while the lack of washing steps is a clear advantage compared to enzyme-linked immunoabsorbance assays. The authors suggest that this assay is flexible enough to be adapted to other applications, such as analysis of protein complexes or co-activators of enzymatic reactions.(See "Heterogeneous transition metal–based fluorescence polarization (HTFP) assay for probing protein interactions".)Dual Control for Double-strand BreaksDNA double-strand breaks (DSBs) occur in response to ionizing radiation, chemical damage, or mechanical stress; or as intermediates in biological recombination events. If unrepaired, these lesions can lead to apoptosis, senescence, or tumorigenesis. Physical and chemical methods exist for experimental induction of DSBs, but these techniques result in a variety of lesions that can impact cell physiology and confound the interpretation of experimental results. Rare-cutting homing endonucleases, such as I-SceI, I-CreI, or I-PpoI, can successfully cleave DNA without generating other lesions or inducing other adverse physiological effects, yet they cut at predetermined recognition sites that occur at low frequency in the mammalian genome. Bacterial restriction endonucleases (REs) cut sites that occur more frequently, allowing RE activity to more closely mimic the stochastic distribution of naturally occurring DSBs. But use of bacterial REs has been limited by the requirement for electroporation or other treatments to introduce the protein or plasmid into the cell and the inability to control RE activity once inside. A. Maslov, M. Metrikin, and J. Vijg at the Albert Einstein College of Medicine (Bronx, NY) created an adenoviral vector carrying a CMV promoter directing reverse tetracycline-controlled transactivator (rtTA) expression and a tet-inducible promoter controlling the SacI RE gene. The mouse genome used in the study contains approximately 130,000 potential SacI restriction sites, ensuring stochastic distribution of DSBs resulting from the use of the vector. To completely suppress basal expression of the enzyme, the authors combine the use of the PTight TRE-based promoter and fuse SacI to a mutated estrogen receptor to prevent migration of the endonuclease into the nucleus in the absence of the drug tamoxifen. This dual activation system enables complete suppression of activity in the absence of both doxycycline and tamoxifen and tight control over SacI activity, which was not previously possible using either approach alone. Induction of SacI expression in the presence of tamoxifen led to elevated levels of SacI-induced DSBs in the genomic DNA of transduced cells that could be controlled by the duration and dose of doxycycline.(See "A dual-activation, adenoviral-based system for the controlled induction of DNA double-strand breaks by the restriction endonuclease SacI".)Sound Mixing by Mixing with SoundThe rapid mixing of liquids in volumes greater than 10 microliters can be easily achieved through turbulence (e.g., by mixing a container). But for volumes of less than 10 microliters, turbulence is absent, which poses a problem since thorough mixing by molecular diffusion can take hours. To facilitate rapid mixing in microscale volumes, various micromixing techniques requiring specialized devices have been developed. One technique is acoustic microstreaming, where sound waves propagating through a liquid can induce flow when a small obstacle such as a bubble is present in the liquid or when acoustic frequencies of more than 100 MHz are used on open wells. However, the general applicability of this method is limited since the former requires a specially-designed chamber and the introduction of bubbles into the liquid, while the latter depends on special piezoceramic speakers. In this issue, K. Petkovic-Duran, R. Manasseh, and colleagues at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) and the University of Melbourne (Melbourne, Australia) have developed a simplified acoustic microstreaming method that does not require special equipment and will be easily adaptable for use in standard multiwell plates. Their method is based on the observation that a drop of liquid in a small well will have a meniscus with a small radius of curvature, creating a locally large gradient in the sound field generated in the liquid by standard acoustic speakers that results in microstreaming. The authors examined the mixing of a droplet of dye solution into a drop of glycerol solution in a 4-mm diameter microwell of an acrylic plate mounted directly onto two speakers. Without applying sound, it took more than an hour for uniform mixing to be achieved, whereas the application of sound at particular frequency bands (in the hundreds of Hz range) instantly caused streaming. Two frequency bands caused different streaming patterns— vortex or dipole—that were each suitable for chaotic mixing, with the former significantly reducing the mixing time to 5 min. Alternating these two patterns was even better, decreasing the mixing time by an additional 30–40%.(See "Chaotic micromixing in open wells using audio-frequency acoustic microstreaming".)A Proper ReferenceVariations in mitochondrial DNA (mtDNA) copy number are associated with aging, cancer, and numerous other human pathologies. Although essential to studies in these fields, consistent recovery and quantification of mtDNA remains problematic. Quantification is usually achieved with quantitative real-time PCR (qPCR) normalized by comparison with nuclear DNA (nDNA). Recovery of the DNA can be erratic since mtDNA and nDNA differ in quantity, molecular weight, and base composition, which cause them to behave differently during extraction. Also, changes in the nuclear ploidy of the cells of interest could introduce further inaccuracy to the mtDNA copy number estimate. In this issue, M. Myers, R. Mittelstaedt, and R. Heflich at the National Center for Toxicological Research (Jefferson, AR) report variations in the ratios of mtDNA to nDNA, even following efficient organic DNA extraction protocols. Although the extraction efficiency of nDNA remained relatively constant in replicate DNA extractions, qPCR analysis estimated up to a 9-fold difference in the mtDNA copy number from replicate extractions when using nDNA as a reference. In an effort to circumvent this problem, the authors tested ΦX174 DNA as an exogenous reference for quantifying mtDNA. DNA from the ΦX174 bacteriophage is a double-stranded, circular genome of similar size to mtDNA that extracted with efficiency similar to that obtained with mtDNA. For analysis of mtDNA copy number, the authors recommend introducing a known quantity of ΦX174 DNA relative to the tissue weight into the tissue digestion solution early in the extraction procedure. They showed that this method overcame the problems of variation in DNA extraction efficiencies, ploidy between cells, and the dependence upon nDNA as a reference. Their study demonstrated that ΦX174 DNA is a more suitable reference for obtaining accurate quantification of mtDNA.(See "Using ΦX174 DNA as an exogenous reference for measuring mitochondrial DNA copy number".)FiguresReferencesRelatedDetails Vol. 47, No. 4 Follow us on social media for the latest updates Metrics Downloaded 165 times History Published online 25 April 2018 Published in print October 2009 Information© 2009 Author(s)PDF download