The introduction of exogenous DNA into Escherichia coli is a cornerstone of molecular biology. Herein, we investigate two new mechanisms for bacterial transformation involving either the use of microwave irradiation or a freeze–thaw protocol in liquid nitrogen. Ultimately, both methods afforded successful transfer of plasmid DNA into bacterial cells, with the freeze–thaw technique yielding efficiencies of ~105. More importantly, both techniques effectively eliminated the need for the preparation of competent cells.
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Microwave irradiation; Bacterial transformation; Molecular biology
The site-specific incorporation of unnatural amino acids (UAAs) into proteins in bacteria is made possible by the evolution of aminoacyl-tRNA synthetases that selectively recognize and aminoacylate the amino acid of interest. Recently we have discovered that some of the previously evolved aaRSs display a degree of polyspecificity and are capable of recognizing multiple UAAs. Herein we report the polyspecificity of an aaRS evolved to encode a comarin containing amino acid. This polyspecificity was then exploited to introduce several UAAs into the fluorophore of GFP, altering its photophysical properties.
Unnatural amino acids; Polyspecificity; Green Fluorescence Protein; Aminoacyl-tRNA Synthetase; Fluorescence modulation
Short interfering RNAs (siRNAs) and microRNAs (miRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules.
A light-activatable bacteriophage T7 RNA polymerase (T7RNAP) has been generated through the site-specific introduction of a photocaged tyrosine residue at the crucial position Tyr639 within the active site of the enzyme. The photocaged tyrosine disrupts polymerase activity by blocking the incoming nucleotide from reaching the active site of the enzyme. However, a brief irradiation with nonphototoxic UV light of 365 nm removes the ortho-nitrobenzyl caging group from Tyr639 and restores the RNA polymerase activity of T7RNAP. The complete orthogonality of T7RNAP to all endogenous RNA polymerases in pro- and eukaryotic systems allowed for the photochemical activation of gene expression in bacterial and mammalian cells. Specifically, E. coli cells were engineered to produce photocaged T7RNAP in the presence of a GFP reporter gene under the control of a T7 promoter. UV irradiation of these cells led to the spatiotemporal activation of GFP expression. In an analogous fashion, caged T7RNAP was transfected into human embryonic kidney (HEK293T) cells. Irradiation with UV light led to the activation of T7RNAP, thereby inducing RNA polymerization and expression of a luciferase reporter gene in tissue culture. The ability to achieve spatiotemporal regulation of orthogonal RNA synthesis enables the precise dissection and manipulation of a wide range of cellular events, including gene function.
amino acids; caged proteins; light activation; polymerases; RNA
Photochemical control of the polymerase chain reaction has been achieved through the incorporation of light-triggered nucleotides into DNA.
Recent investigations continue to emphasize the importance of glycosylation in various diseases including cancer. In this work, we present a step by step protocol describing a method for N-linked glycan profiling of plasma glycoproteins by nano-flow liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). A large experimental space was initially explored and is described herein. Three internal standards were spiked into the sample and provided normalization of plasma glycan abundance across different experimental conditions. Incubation methods, times and the effect of NP40 detergent on glycan abundance were explored. It was found that an 18-hour incubation with no detergent lead to the greatest ion abundance; however, data could be obtained in less than one day from raw plasma samples utilizing microwave irradiation or shorter incubation periods. The inter-sample precision of three different glycans was less than 5.5% (RSD) when the internal standards were added prior to the initial processing step. The high mass measurement accuracy (<3 ppm) afforded by the FT-ICR mass spectrometer provided confident identifications of several glycan species.
A ribozyme based gene control element enabled the spatio-temporal regulation of gene function in mammalian cell culture with light.
We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aaRSs against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the canonical twenty amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone, alkynyl, and hydrazino substituted amino acids. In an attempt to better understand this polyspecificity, the x-ray crystal structure of the aaRS/p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificitiy.
Unnatural amino acids; p-cyanophenylalanine; aminoacyl-tRNA synthetase; polyspecificity
Tyrosyl radicals (Y•s) are prevalent in biological catalysis and are formed under physiological conditions by the coupled loss of both a proton and an electron. Fluorotyrosines (FnYs, n=1–4) are promising tools for studying the mechanism of Y• formation and reactivity, as their pKas and peak potentials span four units and 300 mV, respectively, between pH 6–10. In this manuscript, we present the directed evolution of aminoacyl-tRNA synthetases (aaRS) for 2,3,5-trifluorotyrosine (2,3,5-F3Y) and demonstrate their ability to charge an orthogonal tRNA with a series of FnYs, while maintaining high specificity over Y. An evolved aaRS is then used to site-specifically incorporate FnYs into the two subunits (α2 and β2) of E. coli class Ia ribonucleotide reductase (RNR), an enzyme that employs stable and transient Y•s to mediate long-range, reversible radical hopping during catalysis. Each of four conserved Ys in RNR is replaced with FnY(s) and the resulting proteins isolated in good yields. FnYs incorporated at position 122 of β2, the site of a stable Y• in the wt RNR, generate long-lived FnY•s that are characterized by EPR spectroscopy. Furthermore, we demonstrate that the radical pathway in the mutant Y122(2,3,5)F3Y-β2 is energetically and/or conformationally modulated such that the enzyme retains its activity, but that a new on-pathway Y• can accumulate. The distinct EPR properties of the 2,3,5-F3Y• facilitate spectral subtractions that make detection and identification of new Y•s straightforward.
microRNA; inhibitors; cell based assay; medicinal chemistry; cancer
We developed a new system for light-induced protein dimerization in living cells using a novel photocaged analog of rapamycin (pRap) together with an engineered rapamycin binding domain (iFKBP). Using focal adhesion kinase as a target, we demonstrated successful light-mediated regulation of protein interaction and localization in living cells. Modification of this approach enabled light-triggered activation of a protein kinase and initiation of kinase-induced phenotypic changes in vivo.
Disruptions of anatomical left-right asymmetry result in life-threatening heterotaxic birth defects in vital organs. We performed a small molecule screen for left-right asymmetry phenotypes in Xenopus embryos and discovered a novel pyridine analog, heterotaxin, which disrupts both cardiovascular and digestive organ laterality and inhibits TGF-β-dependent left-right asymmetric gene expression. Heterotaxin analogs also perturb vascular development, melanogenesis, cell migration and adhesion, and indirectly inhibit the phosphorylation of an intracellular mediator of TGF-β signaling. This combined phenotypic profile identifies these compounds as a novel class of TGF-β signaling inhibitors. Notably, heterotaxin analogs also possess highly desirable anti-tumor properties, inhibiting epithelial-mesenchymal transition, angiogenesis and tumor cell proliferation in mammalian systems. Our results suggest that assessing multiple organ, tissue, cellular and molecular parameters in a whole organism context is a valuable strategy for identifying the mechanism of action of novel compounds.
heterotaxia; TGF-β; Smad2; left-right asymmetry; Xenopus; pyridine
The photochemical regulation of biological systems represents a very precise means of achieving high-resolution control over gene expression in both a spatial and a temporal fashion. DNAzymes are enzymatically active deoxyoligonucleotides that enable the site-specific cleavage of RNA, and have been used in a variety of in vitro applications. We have previously reported the photochemical activation of DNAzymes and antisense agents through the preparation of a caged DNA phosphoramidite and its site-specific incorporation into oligonucleotides. The presence of the caging group disrupts either DNA:RNA hybridization or catalytic activity, until removed via a brief irradiation with UV light. Here, we are expanding this concept by investigating the photochemical deactivation of DNAzymes and antisense agents. Moreover, we report the application of light-activated and light-deactivated antisense agents to the regulation of gene function in mammalian cells. This represents the first example of gene silencing antisense agents that can be turned on and turned off in mammalian tissue culture.
DNA cleavage; enzymes; caging; light; DNA
The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.
A new photocaged nucleoside was synthesized and incorporated into DNA using standard synthesis conditions. This approach enabled the disruption of specific H-bonds and allowed for the analysis of their contribution to the activity of a DNAzyme. Brief irradiation with non-photodamaging UV light led to rapid decaging and almost quantitative restoration of DNAzyme activity. The developed strategy has the potential to find widespread application in the light-induced regulation of oligonucleotide function.