Inducible Genetic Code Expansion in Eukaryotes.

Genetic code expansion (GCE) is a versatile tool to site-specifically incorporate a noncanonical amino acid (ncAA) into a protein, for example, to perform fluorescent labeling inside living cells. To this end, an orthogonal aminoacyl-tRNA-synthetase/tRNA (RS/tRNA) pair is used to insert the ncAA in response to an amber stop codon in the protein of interest. One of the drawbacks of this system is that, in order to achieve maximum efficiency, high levels of the orthogonal tRNA are required, and this could interfere with host cell functionality. To minimize the adverse effects on the host, we have developed an inducible GCE system that enables us to switch on tRNA or RS expression when needed. In particular, we tested different promotors in the context of the T-REx or Tet-On systems to control expression of the desired orthogonal tRNA and/or RS. We discuss our result with respect to the control of GCE components as well as efficiency. We found that only the T-REx system enables simultaneous control of tRNA and RS expression.

Fluorogenic Tetrazine-Siliconrhodamine Probe for the Labeling of Noncanonical Amino Acid Tagged Proteins.

Tetrazine-bearing fluorescent labels enable site-specific tagging of proteins that are genetically manipulated with dienophile modified noncanonical amino acids. The inverse electron demand Diels-Alder reaction between the tetrazine and the dienophile fulfills the criteria of bioorthogonality allowing fluorescent labeling schemes of live cells. Here, we describe the detailed synthetic and labeling protocols of a near infrared emitting siliconrhodamine-tetrazine probe suitable for super-resolution imaging of residue-specifically engineered proteins in mammalian cells.

Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements.

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy.

Super-resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine-based GCE system for click chemistry, combined with DNA-PAINT microscopy, enables the visualization of even low-abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue-specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.

New generation of bioorthogonally applicable fluorogenic dyes with visible excitations and large Stokes shifts.

Synthesis of a set of new, azide bearing, biorthogonally applicable fluorogenic dyes with large Stokes shifts is presented herein. To assess the fluorogenic performance of these new dyes we have labeled a genetically modulated, cyclooctyne-bearing protein in lysate medium. Studies showed that the labels produce specific signal with minimal background fluorescence. We also provide theoretical insights into the design of such fluorogenic labels.

Relative proximity of chromosome territories influences chromosome exchange partners in radiation-induced chromosome rearrangements in primary human bronchial epithelial cells.

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation.