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Atomic force microscopy and differential pulse voltammetry were used to characterize the interaction of small highly structured ribozymes with two carbon electrode surfaces. The ribozymes spontaneously self-assemble in two-dimensional networks that cover the entire HOPG surface uniformly. The full-length ribozyme was adsorbed to a lesser extent than a truncated RNA sequence, presumably due to the formation of a more compact overall structure. All four nucleobases composing the ribozyme could be detected by anodic voltammetry on glassy carbon electrodes, and no signals corresponding to free nucleobases were found, indicating the integrity of the ribozyme molecules. [Mg.sup.2+] cations significantly reduced the adsorption of ribozymes to the surfaces, in agreement with the stabilization of this ribozyme's compact, stable, and tightly folded tertiary structure by [Mg.sup.2+] ions that could prevent the hydrophobic bases from interacting with the HOPG surface. Treatment with [Pb.sup.2+] ions, on the other hand, resulted in an increased adsorption of the RNA due to well-known hydrolytic cleavage. The observed dependence of anodic peak currents on different folding states of RNA may provide an attractive method to electrochemically monitor structural changes associated with RNA folding, binding, and catalysis.

Polo-like kinase (Plk4) is a serine/threonine-protein kinase that is essential for biogenesis of the centriole organelle and is enriched at centrioles. Herein, we introduce Cen-TCO, a chemical probe based on the Plk4 inhibitor centrinone, to image Plk4 and centrioles in live or fixed cultured human cells. Specifically, we established a bio-orthogonal two-step labeling system that enables the Cen-TCO-mediated imaging of Plk4 by STED super-resolution microscopy. Such direct labeling of Plk4 results in an increased resolution in STED imaging compared with using anti-Plk4 antibodies, underlining the importance of direct labeling strategies for super-resolution microscopy. We anticipate that Cen-TCO will become an important tool for investigating the biology of Plk4 and of centrioles., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Coenzyme A (CoA) is one of the central cofactors of metabolism, yet a method for measuring its concentration in living cells is missing. Here we introduce the first biosensor for measuring CoA levels in different organelles of mammalian cells. The semisynthetic biosensor is generated through the specific labeling of an engineered GFP-HaloTag fusion protein with a fluorescent ligand. Its readout is based on CoA-dependent changes in Förster resonance energy transfer efficiency between GFP and the fluorescent ligand. Using this biosensor, we probe the role of numerous proteins involved in CoA biosynthesis and transport in mammalian cells. On the basis of these studies, we propose a cellular map of CoA biosynthesis that suggests how pools of cytosolic and mitochondrial CoA are maintained., (© 2022. The Author(s).)

Fluorogenic labeling via bioorthogonal tetrazine chemistry has proven to be highly successful in fluorescence microscopy of living cells. To date, trans -cyclooctene (TCO) and bicyclonyne have been found to be the most useful substrates for live-cell labeling owing to their fast labeling kinetics, high biocompatibility, and bioorthogonality. Recent kinetic studies of fluorogenic click reactions with TCO derivatives showed a transient fluorogenic effect but could not explain the reaction sequence and the contributions of different intermediates. More recently, fluorescence quenching by potential intermediates has been investigated, suggesting their occurrence in the reaction sequence. However, in situ studies of the click reaction that directly relate these observations to the known reaction sequence are still missing. In this study, we developed a single-molecule fluorescence detection framework to investigate fluorogenic click reactions. In combination with data from ultra-performance liquid chromatography-tandem mass spectrometry, this explains the transient intensity increase by relating fluorescent intermediates to the known reaction sequence of TCO with fluorogenic tetrazine dyes. More specifically, we confirm that the reaction of TCO with tetrazine rapidly forms a fluorescent 4,5-dihydropyridazine species that slowly tautomerizes to a weakly fluorescent 1,4-dihydropyridazine, explaining the observed drop in fluorescence intensity. On a much slower timescale of hours/days, the fluorescence intensity may be recovered by oxidation of the intermediate to a pyridazine. Our findings are of importance for quantitative applications in fluorescence microscopy and spectroscopy as the achieved peak intensity with TCO depends on the specific experimental settings. They clearly indicate the requirement for more robust benchmarking of click reactions with tetrazine dyes and the need for alternative dienophiles with fast reaction kinetics and stable fluorescence emission to further applications in advanced fluorescence microscopy., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Direct control of protein interactions by chemically induced protein proximity holds great potential for both cell and synthetic biology as well as therapeutic applications. Low toxicity, orthogonality and excellent cell permeability are important criteria for chemical inducers of proximity (CIPs), in particular for in vivo applications. Here, we present the use of the agrochemical mandipropamid (Mandi) as a highly efficient CIP in cell culture systems and living organisms. Mandi specifically induces complex formation between a sixfold mutant of the plant hormone receptor pyrabactin resistance 1 (PYR1) and abscisic acid insensitive (ABI). It is orthogonal to other plant hormone-based CIPs and rapamycin-based CIP systems. We demonstrate the applicability of the Mandi system for rapid and efficient protein translocation in mammalian cells and zebrafish embryos, protein network shuttling and manipulation of endogenous proteins., (© 2021. The Author(s).)

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labeling via bio-orthogonal tetrazine chemistry combines minimal label size with rapid labeling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and the fluorophore. Two synthetic routes give access to a series of cell-permeable and -impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as the dominant excited-state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multicolor and super-resolution STED, and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

High-resolution live-cell imaging is necessary to study complex biological phenomena. Modern fluorescence microscopy methods are increasingly combined with complementary, label-free techniques to put the fluorescence information into the cellular context. The most common high-resolution imaging approaches used in combination with fluorescence imaging are electron microscopy and atomic-force microscopy (AFM), originally developed for solid-state material characterization. AFM routinely resolves atomic steps, however on soft biological samples, the forces between the tip and the sample deform the fragile membrane, thereby distorting the otherwise high axial resolution of the technique. Here we present scanning ion-conductance microscopy (SICM) as an alternative approach for topographical imaging of soft biological samples, preserving high axial resolution on cells. SICM is complemented with live-cell compatible super-resolution optical fluctuation imaging (SOFI). To demonstrate the capabilities of our method we show correlative 3D cellular maps with SOFI implementation in both 2D and 3D with self-blinking dyes for two-color high-order SOFI imaging. Finally, we employ correlative SICM/SOFI microscopy for visualizing actin dynamics in live COS-7 cells with subdiffraction-resolution., (© 2021. The Author(s).)

We describe the development of a proximity-induced bio-orthogonal inverse electron demand Diels-Alder reaction that exploits the high-affinity interaction between a dienophile-modified RhoBAST aptamer and its tetramethyl rhodamine methyltetrazine substrate. We applied this concept for covalent RNA labeling in proof-of-principle experiments.

Bioorthogonal chemistry holds great potential to generate difficult-to-access protein-protein conjugate architectures. Current applications are hampered by challenging protein expression systems, slow conjugation chemistry, use of undesirable catalysts, or often do not result in quantitative product formation. Here we present a highly efficient technology for protein functionalization with commonly used bioorthogonal motifs for Diels-Alder cycloaddition with inverse electron demand (DA inv ). With the aim of precisely generating branched protein chimeras, we systematically assessed the reactivity, stability and side product formation of various bioorthogonal chemistries directly at the protein level. We demonstrate the efficiency and versatility of our conjugation platform using different functional proteins and the therapeutic antibody trastuzumab. This technology enables fast and routine access to tailored and hitherto inaccessible protein chimeras useful for a variety of scientific disciplines. We expect our work to substantially enhance antibody applications such as immunodetection and protein toxin-based targeted cancer therapies., (© 2020 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The cadherin-catenin complex at adherens junctions (AJs) is essential for the formation of cell-cell adhesion and epithelium integrity; however, studying the dynamic regulation of AJs at high spatio-temporal resolution remains challenging. Here we present an optochemical tool which allows reconstitution of AJs by chemical dimerization of the force bearing structures and their precise light-induced dissociation. For the dimerization, we reconstitute acto-myosin connection of a tailless E-cadherin by two ways: direct recruitment of α-catenin, and linking its cytosolic tail to the transmembrane domain. Our approach enables a specific ON-OFF switch for mechanical coupling between cells that can be controlled spatially on subcellular or tissue scale via photocleavage. The combination with cell migration analysis and traction force microscopy shows a wide-range of applicability and confirms the mechanical contribution of the reconstituted AJs. Remarkably, in vivo our tool is able to control structural and functional integrity of the epidermal layer in developing Xenopus embryos.

Recent developments in fluorescence microscopy call for novel small-molecule-based labels with multiple functionalities to satisfy different experimental requirements. A current limitation in the advancement of live-cell single-molecule localization microscopy is the high excitation power required to induce blinking. This is in marked contrast to the minimal phototoxicity required in live-cell experiments. At the same time, quality of super-resolution imaging depends on high label specificity, making removal of excess dye essential. Approaching both hurdles, we present the design and synthesis of a small-molecule label comprising both fluorogenic and self-blinking features. Bioorthogonal click chemistry ensures fast and highly selective attachment onto a variety of biomolecular targets. Along with spectroscopic characterization, we demonstrate that the probe improves quality and conditions for regular and single-molecule localization microscopy on live-cell samples., (© 2019 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

Light controlled tools are highly attractive for the modulation and manipulation of biological processes. As an external trigger light can be applied with high temporal and special control to various samples. In the recent years a number of optochemical and -genetic tools have been developed to translate the input of light into molecular changes that result in specific biological responses. Here we present a highly efficient system for light-induced protein dimerization in live cells using photocaged derivatives of the plant hormone gibberellic acid (GA3). We provide a precise protocol for a simple one-step synthesis of the photocaged CIP and its application for protein translocation in living cells., (© 2020 Elsevier Inc. All rights reserved.)

Diels-Alder reactions with inverse electron demand (DA inv ) have emerged as an indispensable tool for bioorthogonal labeling and the manipulation of biomolecules. In this context, reactions between tetrazines and strained dienophiles have received attention because of high reaction rates. Current methods for the DA inv -mediated functionalization of proteins suffer from slow reactivity, impaired stability, isomerization, or elimination of the incorporated strained dienophiles. We report here a versatile platform for the posttranslational, highly selective, and quantitative modification of proteins with stable dienes. New synthetic access to minimal size tetrazine and triazine derivatives enabled us to synthesize tailored diene substrates for the lipoic acid protein ligase A (LplA) from Escherichia coli, which we employ for the rapid, mild, and quantitative bioconjugation of proteins by DA inv . The presented method benefits from the minimal tag size for LplA recognition and can be applied to proteins from any source organism. We demonstrate its broad suitability by site-specific in vitro protein labeling and live cell labeling for fluorescence microscopy. With this work we expand the scope of DA inv bioorthogonal chemistry for site-specific protein labeling, providing additional experimental flexibility for preparing well-defined bioconjugates and addressing biological questions in complex biological environments.

Guanylate-binding protein (GBP) 5 is an interferon (IFN)-inducible cellular factor reducing HIV-1 infectivity by an incompletely understood mechanism. Here, we show that this activity is shared by GBP2, but not by other members of the human GBP family. GBP2/5 decrease the activity of the cellular proprotein convertase furin, which mediates conversion of the HIV-1 envelope protein (Env) precursor gp160 into mature gp120 and gp41. Because this process primes HIV-1 Env for membrane fusion, viral particles produced in the presence of GBP2/5 are poorly infectious due to increased incorporation of non-functional gp160. Furin activity is critical for the processing of envelope glycoproteins of many viral pathogens. Consistently, GBP2/5 also inhibit Zika, measles, and influenza A virus replication and decrease infectivity of viral particles carrying glycoproteins of Marburg and murine leukemia viruses. Collectively, our results show that GPB2/5 exert broad antiviral activity by suppressing the activity of the virus-dependency factor furin., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Bioorthogonal chemistry techniques enable the selective and targeted manipulation of living systems. In order to yield universally applicable techniques, it is of great importance for bioorthogonal reactions to take place rapidly, selectively, and with the formation of only benign side products. One of the reactions that match these criteria well is the inverse electron demand Diels-Alder reaction (DA inv ) between tetrazines and strained dienophiles. However, even this prime technique comes with the disadvantage of its reactants having limited stability under physiological conditions. In our protocol, an unreactive and therefore stable DA inv diene/dienophile pair reacts rapidly using DNA hybridization as secondary rate-accelerating process. Due to the fluorogenicity of the presented tetrazine rhodamine conjugate, this method enables the selective screening and evaluation of reactant pairs for proximity-mediated bioorthogonal chemistry.

The HIV type 1 pathogenicity factor Nef enhances viral replication by modulating multiple host cell pathways, including tuning the activation state of infected CD4 T lymphocytes to optimize virus spread. For this, Nef inhibits anterograde transport of the Src family kinase (SFK) Lck toward the plasma membrane (PM). This leads to retargeting of the kinase to the trans-Golgi network, whereas the intracellular transport of a related SFK, Fyn, is unaffected by Nef. The 18-amino acid Src homology 4 (SH4) domain membrane anchor of Lck is necessary and sufficient for Nef-mediated retargeting, but other details of this process are not known. The goal of this study was therefore to identify characteristics of SH4 domains responsive to Nef and the transport machinery used. Screening a panel of SFK SH4 domains revealed two groups that were sensitive or insensitive for trans-Golgi network retargeting by Nef as well as the importance of the amino acid at position 8 for determining Nef sensitivity. Anterograde transport of Nef-sensitive domains was characterized by slower delivery to the PM and initial targeting to Golgi membranes, where transport was arrested in the presence of Nef. For Nef-sensitive SH4 domains, ectopic expression of the lipoprotein binding chaperone Unc119a or the GTPase Arl3 or reduction of their endogenous expression phenocopied the effect of Nef. Together, these results suggest that, analogous to K-Ras, Nef-sensitive SH4 domains are transported to the PM by a cycle of solubilization and membrane insertion and that intrinsic properties define SH4 domains as cargo of this Nef-sensitive lipoprotein binding chaperone-GTPase transport cycle., (© 2018 Chase et al.)

Discovering the genetic changes underlying species differences is a central goal in evolutionary genetics. However, hybrid crosses between species in mammals often suffer from hybrid sterility, greatly complicating genetic mapping of trait variation across species. Here, we describe a simple, robust, and transgene-free technique to generate "in vitro crosses" in hybrid mouse embryonic stem (ES) cells by inducing random mitotic cross-overs with the drug ML216, which inhibits the DNA helicase Bloom syndrome (BLM). Starting with an interspecific F1 hybrid ES cell line between the Mus musculus laboratory mouse and Mus spretus (∼1.5 million years of divergence), we mapped the genetic basis of drug resistance to the antimetabolite tioguanine to a single region containing hypoxanthine-guanine phosphoribosyltransferase ( Hprt ) in as few as 21 d through "flow mapping" by coupling in vitro crosses with fluorescence-activated cell sorting (FACS). We also show how our platform can enable direct study of developmental variation by rederiving embryos with contribution from the recombinant ES cell lines. We demonstrate how in vitro crosses can overcome major bottlenecks in mouse complex trait genetics and address fundamental questions in evolutionary biology that are otherwise intractable through traditional breeding due to high cost, small litter sizes, and/or hybrid sterility. In doing so, we describe an experimental platform toward studying evolutionary systems biology in mouse and potentially in human and other mammals, including cross-species hybrids., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Here, we describe a two-step protocol for selective protein labeling based on enzyme-mediated peptide labeling utilizing lipoic acid ligase (LplA) and bioorthogonal chemistry. The method can be applied to purified proteins, protein in cell lysates, as well as living cells. In a first step a W37V mutant of the lipoic acid ligase (LplA W37V ) from Escherichia coli is utilized to ligate a synthetic chemical handle site-specifically to a lysine residue in a 13 amino acid peptide motif-a short sequence that can be genetically expressed as a fusion with any protein of interest. In a second step, a molecular probe can be attached to the chemical handle in a bioorthogonal Diels-Alder reaction with inverse electron demand (DA inv ). This method is a complementary approach to protein labeling using genetic code expansion and circumvents larger protein tags while maintaining label specificity, providing experimental flexibility and straightforwardness.

Bioorthogonal reactions have emerged as a versatile tool in life sciences. The inverse electron demand Diels-Alder reaction (DA inv ) stands out due to the availability of reactants with very fast kinetics. However, highly reactive dienophiles suffer the disadvantage of being less stable and prone to side reactions. Herein, we evaluate the extent of acceleration of rather unreactive but highly stable dienophiles by DNA-templated proximity. To this end, we developed a modular synthetic route for a novel bifunctional fluorogenic tetrazine rhodamine probe that we used to determine the reaction kinetics of various dienophiles in a fluorescence assay. Under proximity-driven conditions the reaction was found to be several orders of magnitude faster, and we observed almost no background reaction when proximity was not induced. This fundamental study identifies a minimally sized fluorogenic tetrazine dienophile reactant pair that has potential to be generally used for the visualization of biomolecular interactions with temporal and spatial resolution in living systems., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chemical fixation of living cells for microscopy is commonly achieved by crosslinking of intracellular proteins with dialdehydes prior to examination. We herein report a photocleavable protecting group for glutaraldehyde that results in a light-triggered and membrane-permeable fixative, which is nontoxic prior to photocleavage. Lipophilic ester groups allow for diffusion across the cell membrane and intracellular accumulation after enzymatic hydrolysis. Irradiation with UV light releases glutaraldehyde. The in situ generated fixative crosslinks intracellular proteins and preserves and stabilizes the cell so that it is ready for microscopy. In contrast to conventional glutaraldehyde fixation, tissue autofluorescence does not increase after fixation. Caged glutaraldehyde may in future enable functional experiments on living cells under a light microscope in which events of interest can be stopped in spatially confined volumes at defined time points. Samples with individually stopped events could then later be analyzed in ultrastructural studies., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Fluorogenic probes for bioorthogonal labeling chemistry are highly beneficial to reduce background signal in fluorescence microscopy imaging. 1,2,4,5-Tetrazines are known substrates for the bioorthogonal inverse electron demand Diels-Alder reaction (DA inv ) and tetrazine substituted fluorophores can exhibit fluorogenic properties. Herein, we report the synthesis of a palette of novel fluorogenic tetrazine dyes derived from widely-used fluorophores that cover the entire emission range from green to far-red. We demonstrate the power of the new fluorogenic probes in fixed and live cell labeling experiments and present the first example of intracellular live cell protein imaging using tetrazine-based probes under no-wash conditions.

We present new fluorophore-conjugates for dual-color photoactivation and super-resolution imaging inside live mammalian cells. These custom-designed, photo-caged Q-rhodamines and fluoresceins are cell-permeable, bright and localize specifically to intracellular targets. We utilized established orthogonal protein labeling strategies to precisely attach the photoactivatable fluorophores to proteins with subsequent activation of fluorescence by irradiation with UV light. That way, diffusive cytosolic proteins, histone proteins as well as filigree mitochondrial networks and focal adhesion proteins were visualized inside living cells. We applied the new photoactivatable probes in inverse fluorescence recovery after photo-bleaching (iFRAP) experiments, gaining real-time access to protein dynamics from live biological settings with resolution in space and time. Finally, we used the caged Q-rhodamine for photo-activated localization microscopy (PALM) on both fixed and live mammalian cells, where the superior molecular brightness and photo-stability directly resulted in improved localization precisions for different protein targets.

SERINC3 (serine incorporator 3) and SERINC5 are recently identified host cell inhibitors of HIV-1 particle infectivity that are counteracted by the viral pathogenesis factor Nef. Here we confirm that HIV-1 Nef, but not HIV-1 Vpu, antagonizes the particle infectivity restriction of SERINC5. SERINC5 antagonism occurred in parallel with other Nef activities, including cell surface receptor downregulation, trans-Golgi network targeting of Lck, and inhibition of host cell actin dynamics. Interaction motifs with host cell endocytic machinery and the Nef-associated kinase complex, as well as CD4 cytoplasmic tail/HIV-1 protease, were identified as essential Nef determinants for SERINC5 antagonism. Characterization of antagonism-deficient Nef mutants revealed that counteraction of SERINC5 occurs in the absence of retargeting of the restriction factor to intracellular compartments and reduction of SERINC5 cell surface density is insufficient for antagonism. Consistent with virion incorporation of SERINC5 being a prerequisite for its antiviral activity, the infectivity of HIV-1 particles produced in the absence of a SERINC5 antagonist decreased with increasing amounts of virion SERINC5. At low levels of SERINC5 expression, enhancement of virion infectivity by Nef was associated with reduced virion incorporation of SERINC5 and antagonism-defective Nef mutants failed to exclude SERINC5 from virions. However, at elevated levels of SERINC5 expression, Nef maintained infectious HIV particles, despite significant virion incorporation of the restriction factor. These results suggest that in addition to virion exclusion, Nef employs a cryptic mechanism to antagonize virion-associated SERINC5. The involvement of common determinants suggests that the antagonism of Nef to SERINC5 and the downregulation of cell surface CD4 by Nef involve related molecular mechanisms., Importance: HIV-1 Nef critically determines virus spread and disease progression in infected individuals by incompletely defined mechanisms. SERINC3 and SERINC5 were recently identified as potent inhibitors of HIV particle infectivity whose antiviral activity is antagonized by HIV-1 Nef. To address the mechanism of SERINC5 antagonism, we identified four molecular determinants of Nef antagonism that are all linked to the mechanism by which Nef downregulates cell surface CD4. Functional characterization of these mutants revealed that endosomal targeting and cell surface downregulation of SERINC5 are dispensable and insufficient for antagonism, respectively. In contrast, virion exclusion and antagonism of SERINC5 were correlated; however, Nef was also able to enhance the infectivity of virions that incorporated robust levels of SERINC5. These results suggest that the antagonism of HIV-1 Nef to SERINC5 restriction of virion infectivity is mediated by a dual mechanism that is related to CD4 downregulation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

A small synthetic calcium sensor that can be site-specifically coupled to proteins in living cells by utilizing the bio-orthogonal HaloTag labeling strategy is presented. We synthesized an iodo-derivatized BAPTA chelator with a tetramethyl rhodamine fluorophore that allows further modification by Sonogashira cross-coupling. The presented calcium sensitive dye shows a 200-fold increase in fluorescence upon calcium binding. The derivatization with an aliphatic linker bearing a terminal haloalkane-function by Sonogashira cross-coupling allows the localization of the calcium sensor to Halo fusion proteins which we successfully demonstrate in in vitro and in vivo experiments. The herein reported highly sensitive tetramethyl rhodamine based calcium indicator, which can be selectively localized to proteins, is a powerful tool to determine changes in calcium levels inside living cells with spatiotemporal resolution.

Inverse-electron-demand Diels-Alder cycloaddition (DAinv ) between strained alkenes and tetrazines is a highly bio-orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two-step labeling protocol for the site-specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplA(W37V) ), followed by the covalent attachment of tetrazine-modified fluorophores to the norbornene moiety through the bio-orthogonal DAinv  . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13-residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two-step labeling strategy to label proteins in cell lysates in a site-specific manner and performed cell-surface labeling on living cells., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We developed a highly efficient system for light-induced protein dimerization in live cells using photo-caged derivatives of the phytohormone gibberellic acid (GA3 ). We demonstrate the application of the photo-activatable chemical inducer of dimerization (CID) for the control of protein translocation with high spatiotemporal precision using light as an external trigger. Furthermore, we present a new two-photon (2P)-sensitive caging group, whose exceptionally high two-photon cross section allows the use of infrared light to efficiently unleash the active GA3 for inducing protein dimerization in living cells., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Labeling proteins in their natural settings with fluorescent proteins or protein tags often leads to problems. Despite the high specificity, these methods influence the natural functions due to the rather large size of the proteins used. Here we present a two-step labeling procedure for the attachment of various fluorescent probes to a small peptide sequence (13 amino acids) using enzyme-mediated peptide labeling in combination with palladium-catalyzed Sonogashira cross-coupling. We identified p-iodophenyl derivatives from a small library that can be covalently attached to a lysine residue within a specific 13-amino-acid peptide sequence by Escherichia coli lipoic acid ligase A (LplA). The derivatization with p-iodophenyl subsequently served as a reactive handle for bioorthogonal transition metal-catalyzed Sonogashira cross-coupling with alkyne-functionalized fluorophores on both the peptide as well as on the protein level. Our two-step labeling strategy combines high selectivity of enzyme-mediated labeling with the chemoselectivity of palladium-catalyzed Sonogashira cross-coupling.

1,2,4,5-Tetrazine fluorophore derivatives with structurally rigid molecular designs were synthesized using Sonogashira and Stille cross-coupling as well as copper-catalyzed azide-alkyne cycloaddition. The synthesized bichromophoric systems exhibit low fluorescence quantum yields due to quenching by the tetrazine. The extent of fluorescence quenching observed for those systems was shown to depend on the distance between the fluorophore and the tetrazine. The decreased fluorescence is "turned on" by conversion of the tetrazine in the inverse electron demand Diels-Alder cycloaddition. Time resolved spectroscopy indicated resonance energy transfer between BODIPY and the tetrazine as the underlying quenching mechanism. The synthesized conjugates were successfully applied in protein labeling experiments.

The micro-viscosity and molecular crowding experienced by specific proteins can regulate their dynamics and function within live cells. Taking advantage of the emerging TMP-tag technology, we present the design, synthesis and application of a hybrid genetic-chemical molecular rotor probe whose fluorescence lifetime can report protein-specific micro-environments in live cells.

The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to messenger RNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multiwavelength fluorescence microscopy to follow assembly of single spliceosomes in real time in whole-cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather, commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

The spatiotemporal resolution of subdiffraction fluorescence imaging has been limited by the difficulty of labeling proteins in cells with suitable fluorophores. Here we report a chemical tag that allows proteins to be labeled with an organic fluorophore with high photon flux and fast photoswitching performance in live cells. This label allowed us to image the dynamics of human histone H2B protein in living cells at approximately 20 nm resolution.

The active site of a Diels-Alderase ribozyme is located in solution by photoaffinity cross-linking using a productlike azidobenzyl probe. Two key nucleotides are identified that contact the Diels-Alder product in a conformation-dependent fashion. The design of such probes does not require knowledge of the three-dimensional structure of the ribozyme, and the technique yields both static and dynamic structural information. This work establishes photoaffinity cross-linking as an empirical approach that is applied here for the first time to an artificial ribozyme.

Poly(p-xylylene) (PPX) was deposited by chemical vapor deposition (CVD) on stainless steel substrates. These PPX films were coated by solution casting of poly(lactide)-poly(ethylene oxide)-poly(lactide) triblock copolymers (PLA-PEO-PLA) loaded with 14C-labeled paclitaxel. Adhesion of PLA-PEO-PLA on PPX substrate coatings was measured using the blister test method. Excellent adhesion of the block copolymers on PPX substrates was found. Stress behavior and film integrity of PLA-PEO-PLA was compared to pure PLA on unexpanded and expanded stent bodies and was found to be superior for the block copolymers. The release of paclitaxel from the biodegradable coatings was studied under physiological conditions using the scintillation counter method. Burst release of paclitaxel was observed from PLA-PEO-PLA layers regardless of composition, but an increase in paclitaxel loading was observed with increasing content of PEO.

The synthesis of highly structured small RNAs containing nonstandard nucleotides is of high interest for structural and functional investigations. A general approach is the joining, by T4 DNA ligase-mediated splinted ligation, of two or more RNA fragments, each of which may contain its own set of modified nucleotides. The RNA fragments hybridize with a complementary DNA splint to form a ternary ligation-competent-complex (LCC), which is then turned over by the DNA ligase. We studied the formation of the LCC and its precursors using size exclusion chromatography combined with a fluorescence detector. The spatial proximity of two cyanine-dye-labeled RNA fragments in LCCs was detected by monitoring FRET. An observed correlation of LCC formation and ligation yields suggests the use of long splints to stabilize LCCs. Splint oligos of increasing length, which in general appear to reduce the number of different hybridization intermediate species found in a reaction mixture, were applied to the synthesis by T4-DNA-ligation of two highly structured target molecules, one a 73 mer tRNA, the other a 49 mer synthetic ribozyme. A stable LCC could be isolated and turned over with>95% ligation efficiency. In conclusion, the use of long splints presents a generally applicable means to overcome the low propensity of highly structured RNAs for hybridization, and thus to significantly improve ligation efficiencies.

The majority of structural efforts addressing RNA's catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a lambda-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

Oligomers are thought to accelerate the hydrolytic degradation of devices prepared from poly(lactide-co-glycolide), PLGA, due to their increased number of carboxylic end groups. To experimentally verify this hypothesis, two D,L-lactic acid oligomers having molecular weights close to their critical limit of solubility were synthesized and incorporated into PLGA films in three concentrations (0, 10, and 30% w/w). All films were translucent, rather flexible and initially amorphous. With increasing oligomer concentration the glass transition temperature (T(g)) and the molecular weight of films decreased prior to erosion. The degradation studies show that initial mass loss and water absorption are increased in oligomer-containing films as a function of average molecular weight and oligomer concentration. However, the incorporation of oligomers does not accelerate the degradation of films. By contrast, oligomer-containing films show extended lag phase until onset of polymer erosion. This was shown to be related to crystallization. Moreover, it was found that crystallization occurs earlier in oligomer-containing films and that the degree of crystallization is related to the average molecular weight of the oligomer. These findings bring new insight into the role of oligomers in the degradation process and can be used to explain why erosion in massive polymer devices occurs from the center to the surface.