RESUMO
The 5' cap of mRNA plays a critical role in mRNA processing, quality control and turnover. Enzymatic availability of the 5' cap governs translation and could be a tool to investigate cell fate decisions and protein functions or develop protein replacement therapies. We have previously reported on the chemical synthesis of 5' cap analogues with photocleavable groups for this purpose. However, the synthesis is complex and post-synthetic enzymatic installation may make the technique more applicable to biological researchers. Common 5' cap analogues, like the cap 0, are commercially available and routinely used for inâ vitro transcription. Here, we report a facile enzymatic approach to attach photocleavable groups site-specifically to the N2 position of m7 G of the 5' cap. By expanding the substrate scope of the methyltransferase variant GlaTgs V34A and using synthetic co-substrate analogues, we could enzymatically photocage the 5' cap and recover it after irradiation.
Assuntos
Metiltransferases , Processamento Pós-Transcricional do RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Metiltransferases/metabolismoRESUMO
Post-transcriptional regulation of gene expression occurs by multiple mechanisms, including subcellular localization of mRNA and alteration of the poly(A) tail length. These mechanisms play crucial roles in the dynamics of cell polarization and embryonic development. Furthermore, mRNAs are emerging therapeutics and chemical alterations to increase their translational efficiency are highly sought after. We show that yeast poly(A) polymerase can be used to install multiple azido-modified adenosine nucleotides to luciferase and eGFP-mRNAs. These mRNAs can be efficiently reacted in a bioorthogonal click reaction with fluorescent reporters without degradation and without sequence alterations in their coding or untranslated regions. Importantly, the modifications in the poly(A) tail impact positively on the translational efficiency of reporter-mRNAs in vitro and in cells. Therefore, covalent fluorescent labeling at the poly(A) tail presents a new way to increase the amount of reporter protein from exogenous mRNA and to label genetically unaltered and translationally active mRNAs.
Assuntos
Sobrevivência Celular , Fluorescência , Poli A/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Coloração e Rotulagem/métodos , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Células HeLa , Humanos , Poli A/química , RNA Mensageiro/químicaRESUMO
Selective modification of nucleobases with photolabile caging groups enables the study and control of processes and interactions of nucleic acids. Numerous positions on nucleobases have been targeted, but all involve formal substitution of a hydrogen atom with a photocaging group. Nature, however, also uses ring-nitrogen methylation, such as m7 G and m1 A, to change the electronic structure and properties of RNA and control biomolecular interactions essential for translation and turnover. We report that aryl ketones such as benzophenone and α-hydroxyalkyl ketone are photolabile caging groups if installed at the N7 position of guanosine or the N1 position of adenosine. Common photocaging groups derived from the ortho-nitrobenzyl moiety were not suitable. Both chemical and enzymatic methods for site-specific modification of N7G in nucleosides, dinucleotides, and RNA were developed, thereby opening the door to studying the molecular interactions of m7 G and m1 A with spatiotemporal control.
Assuntos
Benzofenonas/química , Guanosina/química , RNA/química , HumanosRESUMO
The 5'-cap is a hallmark of eukaryotic mRNAs and plays fundamental roles in RNA metabolism, ranging from quality control to export and translation. Modifying the 5'-cap may thus enable modulation of the underlying processes and investigation or tuning of several biological functions. A straightforward approach is presented for the efficient production of a range of N7-modified caps based on the highly promiscuous methyltransferase Ecm1. We show that these, as well as N(2) -modified 5'-caps, can be used to tune translation of the respective mRNAs both inâ vitro and in cells. Appropriate modifications allow subsequent bioorthogonal chemistry, as demonstrated by intracellular live-cell labeling of a target mRNA. The efficient and versatile N7 manipulation of the mRNA cap makes mRNAs amenable to both modulation of their biological function and intracellular labeling, and represents a valuable addition to the chemical biology toolbox.
Assuntos
Capuzes de RNA/química , RNA Mensageiro/química , Química Click , Encephalitozoon cuniculi/enzimologia , Eucariotos/genética , Proteínas Fúngicas/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Metiltransferases/metabolismo , Microscopia Confocal , Capuzes de RNA/metabolismo , RNA Mensageiro/metabolismo , S-Adenosilmetionina/análogos & derivadosRESUMO
BACKGROUND: For healthcare workers, surface disinfections are daily routine tasks. An assessment of the inhalation exposure to hazardous substances, in this case the disinfectant´s active ingredients, is necessary to ensure workers safety. However, deciding which exposure model is best for exposure assessment remains difficult. OBJECTIVE: The aim of the study was to evaluate the applicability of different exposure models for disinfection of small surfaces in healthcare settings. METHODS: Measurements of the air concentration of active ingredients in disinfectants (ethanol, formaldehyde, glutaraldehyde, hydrogen peroxide, peroxyacetic acid) together with other exposure parameters were recorded in a test chamber. The measurements were performed using personal and stationary air sampling. In addition, exposure modelling was performed using three deterministic models (unsteady 1-zone, ConsExpo and 2-component) and one modifying-factor model (Stoffenmanager®). Their estimates were compared with the measured values using various methods to assess model quality (like accuracy and level of conservatism). RESULTS: The deterministic models showed overestimation predominantly in the range of two- to fivefold relative to the measured data and high conservatism for all active ingredients of disinfectants with the exception of ethanol. With Stoffenmanager® an exposure distribution was estimated for ethanol, which was in good accordance with the measured data. IMPACT STATEMENT: To date, workplace exposure assessments often involve expensive and time consuming air measurements. Reliable exposure models can be used to assess occupational inhalation exposure to hazardous substances, in this case surface disinfectants. This study describes the applicability of three deterministic and one modifying-factor model for disinfection of small surfaces in healthcare settings, in direct comparison to measurements performed and will facilitate future exposure assessments at these workplaces.
Assuntos
Desinfetantes , Desinfecção , Exposição por Inalação , Exposição Ocupacional , Exposição Ocupacional/análise , Humanos , Exposição por Inalação/análise , Desinfetantes/análise , Desinfecção/métodos , Modelos Teóricos , Poluentes Ocupacionais do Ar/análise , Monitoramento Ambiental/métodosRESUMO
BACKGROUND: Several drugs for human use possess genotoxic properties as a necessary consequence of their intended therapeutic effect (e.g. antineoplastics). Health workers may be exposed to these chemicals in various occupational settings such as dose preparation and administration. To date, there are no quantitative risk assessment models to estimate the cancer risk of health workers due to the handling of genotoxic drugs. We therefore developed a quantitative risk assessment model to assess the cancer risk of occupational exposure to genotoxic drugs in healthcare settings based on the threshold of toxicological concern (TTC) concept. This model was used to evaluate the cancer risk of health workers due to the handling of genotoxic drugs in modern health care facilities. METHODS: We modified the threshold of toxicological concern (TTC) concept to fit the purpose of occupational cancer risk assessment. The risk model underlying ICH guideline M7 (R1): "assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals to limit potential carcinogenic risk" was used as a starting point for our model. We conducted a short review of studies on the occupational exposure of health workers to genotoxic drugs. These occupational exposure data were compared to the acceptable exposure levels resulting from our TTC based risk model. RESULTS: Based on the threshold of toxicological concern (TTC) concept, we defined an acceptable daily intake (ADI) of 4 µg/day as threshold of no concern for the exposure of health workers to genotoxic drugs. Regarding the dermal exposure of health workers to genotoxic drugs, we derived a corresponding acceptable surface contamination level (ASCL) of 20 ng/cm2. Both ADI and ASCL are usually not exceeded in modern healthcare settings. Current safety precautions provide sufficient protection to health workers. CONCLUSIONS: The application of our model indicates that workers in modern healthcare facilities are not at risk of developing work related cancer above widely accepted cancer risk levels due to the occupational exposure to genotoxic drugs. Hence, the present study may assist employers and public authorities to make informed decisions concerning the need for (further) protective measures and during risk communication to health workers.
RESUMO
A requirement for biochemical labeling strategies is a pronounced biocompatibility of the underlying reaction methodology. This protocol enables a systematic evaluation of the biocompatibility of (new) reaction methodologies that are potentially attractive for biochemical applications. The cellular environment for in vitro and in vivo applications is mimicked by the one-by-one addition of diverse bio-additives to the reaction. The influence of the bio-additives on the product yield, termed bio-robustness, is quantified by gas chromatography (GC) or NMR techniques, whereas qualitative analysis of the level of biomolecule preservation by ultra-HPLC-mass spectrometry (UHPLC-MS) or gel electrophoresis enables monitoring of the effects of the reaction conditions on the biomolecule stability, e.g., bio-additive modification or degradation. The 22 chosen bio-additives and the required controls can be completely evaluated within 5-7 working days, depending on reaction time, instrument and the general equipment availability of the lab. We illustrate this protocol by assessing the reaction biocompatibility of a copper-catalyzed N-arylation of sulfonamides. The hereby obtained results are compared to those for a reaction that is characterized by high reaction biocompatibility: the energy-transfer-enabled disulfide-ene reaction.
Assuntos
Materiais Biocompatíveis/análise , Teste de Materiais/métodos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Cromatografia Gasosa-Espectrometria de Massas , Células HeLa , Humanos , Espectrometria de MassasRESUMO
Methyltransferases are powerful tools for site-specific transfer of non-natural functional groups from synthetic analogs of their cosubstrate S-adenosyl-l-methionine (AdoMet). We present a new class of AdoMet analogs containing photo-caging (PC) groups in their side chain, enzymatic transfer of PC groups by a promiscuous DNA MTase as well as light-triggered removal from the target DNA. This strategy provides a new avenue to reversibly modulate the functionality of DNA at MTase target sites.
Assuntos
Biocatálise , Metilases de Modificação do DNA/metabolismo , DNA/metabolismo , Luz , S-Adenosilmetionina/metabolismo , DNA/química , Metilases de Modificação do DNA/química , Estrutura Molecular , Processos Fotoquímicos , Plasmídeos , S-Adenosilmetionina/químicaRESUMO
Sulfur-containing molecules participate in many essential biological processes. Of utmost importance is the methylthioether moiety, present in the proteinogenic amino acid methionine and installed in tRNA by radical-S-adenosylmethionine methylthiotransferases. Although the thiol-ene reaction for carbon-sulfur bond formation has found widespread applications in materials or medicinal science, a biocompatible chemo- and regioselective hydrothiolation of unactivated alkenes and alkynes remains elusive. Here, we describe the design of a general chemoselective anti-Markovnikov hydroalkyl/aryl thiolation of alkenes and alkynes-also allowing the biologically important hydromethylthiolation-by triplet-triplet energy transfer activation of disulfides. This fast disulfide-ene reaction shows extraordinary functional group tolerance and biocompatibility. Transient absorption spectroscopy was used to study the sensitization process in detail. The hereby gained mechanistic insights were successfully employed for optimization of the catalytic system. This photosensitized transformation should stimulate bioimaging applications and carbon-sulfur bond-forming late-stage functionalization chemistry, especially in the context of metabolic labelling.
Assuntos
Alcenos/química , Dissulfetos/química , Alcinos/química , Carbono/química , Catálise , Transferência de Energia , Irídio/química , Luz , Cadeias de Markov , Estereoisomerismo , Enxofre/químicaRESUMO
Dendrite pruning of Drosophila sensory neurons during metamorphosis is induced by the steroid hormone ecdysone through a transcriptional program. In addition, ecdysone activates the eukaryotic initiation factor 4E-binding protein (4E-BP) to inhibit cap-dependent translation initiation. To uncover how efficient translation of ecdysone targets is achieved under these conditions, we assessed the requirements for translation initiation factors during dendrite pruning. We found that the canonical cap-binding complex eIF4F is dispensable for dendrite pruning, but the eIF3 complex and the helicase eIF4A are required, indicating that differential translation initiation mechanisms are operating during dendrite pruning. eIF4A and eIF3 are stringently required for translation of the ecdysone target Mical, and this depends on the 5' UTR of Mical mRNA. Functional analyses indicate that eIF4A regulates eIF3-mRNA interactions in a helicase-dependent manner. We propose that an eIF3-eIF4A-dependent alternative initiation pathway bypasses 4E-BP to ensure adequate translation of ecdysone-induced genes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Ecdisona/genética , Fator de Iniciação 4E em Eucariotos/genética , Animais , Diferenciação CelularRESUMO
RNA molecules can play diverse roles in the cell owing to their secondary structure dynamics and various binding modes. Studying localization and dynamics of RNA in vitro or in cells requires tagging with suitable reporter molecules-fluorophores being the most prominent ones. Enzymatic RNA labeling approaches are currently emerging as valuable alternatives to purely chemical synthesis and to binding- or hybridization-based RNA-imaging approaches. Different classes of enzymes allow for cotranscriptional or posttranscriptional installation of small functional groups in RNA. The enzymatic step is typically combined with a second chemical step, providing flexibility regarding the reporter. The flourishing field of bioorthogonal chemistry propels this approach. We will present latest achievements and remaining challenges in the field of enzyme-mediated RNA tagging and emphasize efforts to achieve site-specificity and intracellular labeling.