RESUMO
Antibacterial resistance is a major threat for human health. There is a need for new antibacterials to stay ahead of constantly-evolving resistant bacteria. Nucleic acid therapeutics hold promise as powerful antibiotics, but issues with their delivery hamper their applicability. Here, we exploit the siderophore-mediated iron uptake pathway to efficiently transport antisense oligomers into bacteria. We appended a synthetic siderophore to antisense oligomers targeting the essential acpP gene in Escherichia coli. Siderophore-conjugated PNA and PMO antisense oligomers displayed potent antibacterial properties. Conjugates bearing a minimal siderophore consisting of a mono-catechol group showed equally effective. Targeting the lacZ transcript resulted in dose-dependent decreased ß-galactosidase production, demonstrating selective protein downregulation. Applying this concept to Acinetobacter baumannii also showed concentration-dependent growth inhibition. Whole-genome sequencing of resistant mutants and competition experiments with the endogenous siderophore verified selective uptake through the siderophore-mediated iron uptake pathway. Lastly, no toxicity towards mammalian cells was found. Collectively, we demonstrate for the first time that large nucleic acid therapeutics can be efficiently transported into bacteria using synthetic siderophore mimics.
Assuntos
Acinetobacter baumannii , Antibacterianos , Catecóis , Escherichia coli , Sideróforos , Sideróforos/química , Sideróforos/farmacologia , Catecóis/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Antibacterianos/farmacologia , Antibacterianos/química , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Testes de Sensibilidade Microbiana , Humanos , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismoRESUMO
Terminal deoxynucleotidyl Transferase (TdT) is a template-independent DNA polymerase that plays an essential role in the human adaptive immune system and is upregulated in several types of leukemia. It has therefore gained interest as a leukemia biomarker and potential therapeutic target. Herein, we describe a FRET-quenched fluorogenic probe based on a size-expanded deoxyadenosine that reports directly on TdT enzymatic activity. The probe enables real-time detection of primer extension and de novo synthesis activity of TdT and displays selectivity over other polymerase and phosphatase enzymes. Importantly, TdT activity and its response to treatment with a promiscuous polymerase inhibitor could be monitored in human T-lymphocyte cell extract and Jurkat cells using a simple fluorescence assay. Finally, employing the probe in a high-throughput assay resulted in the identification of a non-nucleoside TdT inhibitor.
Assuntos
DNA Nucleotidilexotransferase , Leucemia , Humanos , Nucleotídeos , DNA Polimerase Dirigida por DNA , BiomarcadoresRESUMO
Riboswitches are structural RNA elements that control gene expression. These naturally occurring RNA sensors are of continued interest as antibiotic targets, molecular sensors, and functional elements of synthetic circuits. Here, we describe affinity-based profiling of the flavin mononucleotide (FMN) riboswitch to characterize ligand binding and structural folding. We designed and synthesized photoreactive ligands and used them for photoaffinity labeling. We showed selective labeling of the FMN riboswitch and used this covalent interaction to quantitatively measure ligand binding, which we demonstrate with the naturally occurring antibiotic roseoflavin. We measured conditional riboswitch folding as a function of temperature and cation concentration. Furthermore, combining photoaffinity labeling with reverse transcription revealed ligand binding sites within the aptamer domain with single-nucleotide resolution. The photoaffinity probe was applied to cellular extracts of Bacillus subtilis to demonstrate conditional folding of the endogenous low-abundant ribD FMN riboswitch in biologically derived samples using quantitative PCR. Lastly, binding of the riboswitch-targeting antibiotic roseoflavin to the FMN riboswitch was measured in live bacteria using the photoaffinity probe.
Assuntos
Aptâmeros de Nucleotídeos , Riboswitch , Antibacterianos/metabolismo , Aptâmeros de Nucleotídeos/química , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Mononucleotídeo de Flavina/química , Ligantes , Conformação de Ácido Nucleico , RNA/metabolismoRESUMO
RNA-RNA interactions are essential for biology, but they can be difficult to study due to their transient nature. While crosslinking strategies can in principle be used to trap such interactions, virtually all existing strategies for crosslinking are poorly reversible, chemically modifying the RNA and hindering molecular analysis. We describe a soluble crosslinker design (BINARI) that reacts with RNA through acylation. We show that it efficiently crosslinks noncovalent RNA complexes with mimimal sequence bias and establish that the crosslink can be reversed by phosphine reduction of azide trigger groups, thereby liberating the individual RNA components for further analysis. The utility of the new approach is demonstrated by reversible protection against nuclease degradation and trapping transient RNA complexes of E. coli DsrA-rpoS derived bulge-loop interactions, which underlines the potential of BINARI crosslinkers to probe RNA regulatory networks.
Assuntos
Azidas/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , Escherichia coli/química , Fosfinas/metabolismo , RNA Bacteriano/metabolismo , Acilação , Azidas/química , Reagentes de Ligações Cruzadas/química , Escherichia coli/metabolismo , Fosfinas/química , RNA Bacteriano/químicaRESUMO
External photocontrol over RNA function has emerged as a useful tool for studying nucleic acid biology. Most current methods rely on fully synthetic nucleic acids with photocaged nucleobases, limiting application to relatively short synthetic RNAs. Here we report a method to gain photocontrol over RNA by postsynthetic acylation of 2'-hydroxyls with photoprotecting groups. One-step introduction of these groups efficiently blocks hybridization, which is restored after light exposure. Polyacylation (termed cloaking) enables control over a hammerhead ribozyme, illustrating optical control of RNA catalytic function. Use of the new approach on a transcribed 237 nt RNA aptamer demonstrates the utility of this method to switch on RNA folding in a cellular context, and underlines the potential for application in biological studies.
Assuntos
Processos Fotoquímicos , RNA/metabolismo , Acilação , Estrutura Molecular , RNA/químicaRESUMO
DNA polymerase θ (Polθ) is a unique A-family polymerase that is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Because Polθ is highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, it represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority. Here, we demonstrate that Polθ efficiently and selectively incorporates into DNA large benzo-expanded nucleotide analogs (dxAMP, dxGMP, dxTMP, dxAMP) which exhibit canonical base-pairing and enhanced base stacking. In contrast, functionally related Y-family translesion polymerases exhibit a severely reduced ability to incorporate dxNMPs, and all other human polymerases tested from the X, B and A families fail to incorporate them under the same conditions as Polθ. We further find that Polθ is inhibited after multiple dxGMP incorporation events, and that Polθ efficiency for dxGMP incorporation approaches that of native dGMP. These data demonstrate a unique function for Polθ in incorporating synthetic large-sized nucleotides and suggest the future possibility of the use of dxG nucleoside or related prodrug analogs as selective inhibitors of Polθ activity.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/genética , DNA/metabolismo , Humanos , Nucleotídeos/metabolismo , Ligação Proteica , DNA Polimerase tetaRESUMO
Nucleic acids detection is essential to the study of biological processes and to diagnosis of pathological states. Although PCR is highly effective in vitro, methods that can function without prior sample preparation, thermal cycling, or enzymes are of interest due to their simplicity. Most current non-PCR detection methods rely on linear signal amplification, which hinders the detection of small amounts of genetic material. To address this limitation, we tested a new strategy for attaining higher-order signal amplification, in which a target sequence templates a chemical ligation, and the product of this reaction is in turn detected with a second templated reaction. The method is nonenzymatic, isothermal, and fluorogenic, allowing the direct detection of nucleic acids in complex matrices. Using this approach, as little as 500 attomoles (10 pM) could be detected with single nucleotide resolution. In a test of selectivity, single nucleotide substitutions and deletions could successfully be detected, including a deletion that is associated with tetracycline resistance in Helicobacter pylori. Compatibility with biological matrices was demonstrated by the direct detection of rRNA in bacterial lysate. Imaging and detection of target sequences on a solid support further illustrates the potential of the new approach for high-throughput analysis.
Assuntos
Corantes Fluorescentes/química , RNA/análise , RNA/genética , Moldes Genéticos , Helicobacter pylori/genética , RNA/químicaRESUMO
Posttranscriptional modifications of RNA bases are not only found in many noncoding RNAs but have also recently been identified in coding (messenger) RNAs as well. They require complex and laborious methods to locate, and many still lack methods for localized detection. Here we test the ability of next-generation sequencing (NGS) to detect and distinguish between ten modified bases in synthetic RNAs. We compare ultradeep sequencing patterns of modified bases, including miscoding, insertions and deletions (indels), and truncations, to unmodified bases in the same contexts. The data show widely varied responses to modification, ranging from no response, to high levels of mutations, insertions, deletions, and truncations. The patterns are distinct for several of the modifications, and suggest the future use of ultradeep sequencing as a fingerprinting strategy for locating and identifying modifications in cellular RNAs.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Mutação , RNA/genética , Mutação INDEL , Modelos Moleculares , RNA Mensageiro/genéticaRESUMO
Photocleavable protecting groups (PPGs) are extensively used in chemical and biological sciences. In their application, advantage is taken of using light as an external, non-invasive stimulus, which can be delivered with very high spatiotemporal precision. More recently, orthogonally addressing multiple PPGs, in a single system and with different wavelengths of light, has been explored. This approach allows one to independently control multiple functionalities in an external, non-invasive fashion. In this tutorial review, we discuss the design principles for dynamic systems involving wavelength-selective deprotection, focusing on the choice and optimization of PPGs, synthetic methods for their introduction and strategies for combining multiple PPGs into one system. Finally, we illustrate the design principles with representative examples, aiming at providing the reader with an instructive overview on how the wavelength-selective cleavage of photoprotecting groups can be applied in materials science, organic synthesis and biological systems.
Assuntos
Luz , Modelos Químicos , Modelos Moleculares , Fotoquímica , Fotólise , Álcoois Benzílicos/química , Álcoois Benzílicos/efeitos da radiação , IsomerismoRESUMO
Photopharmacology aims to locally treat diseases and study biological processes with photoresponsive drugs. Herein, easy access to photoswitchable drugs is crucial, which is supported by simple and robust drug modifications. We investigated the possibility of creating drugs that can undergo remote activation and deactivation with light, by conjugating molecular photoswitches to the exterior of an existing drug in a single chemical step. This facile strategy allows the convenient introduction of various photochromic systems into a drug molecule, rendering it photoresponsive. To demonstrate the feasibility of this approach, two photoswitch-modified ciprofloxacin antibiotics were synthesized. Remarkably, for one of them a 50-fold increase in activity compared to the original ciprofloxacin was observed. Their antimicrobial activity could be spatiotemporally controlled with light, which was exemplified by bacterial patterning studies.
Assuntos
Antibacterianos/química , Ciprofloxacina/química , Fotoquímica/métodos , Antibacterianos/farmacologia , Ciprofloxacina/farmacologia , Escherichia coli/efeitos dos fármacos , Infecções por Escherichia coli/tratamento farmacológico , Humanos , LuzRESUMO
Cancer treatment suffers from limitations that have a major impact on the patient's quality of life and survival. In the case of chemotherapy, the systemic distribution of cytotoxic drugs reduces their efficacy and causes severe side effects due to nonselective toxicity. Photopharmacology allows a novel approach to address these problems because it employs external, local activation of chemotherapeutic agents by using light. The development of photoswitchable histone deacetylase (HDAC) inhibitors as potential antitumor agents is reported herein. Analogues of the clinically used chemotherapeutic agents vorinostat, panobinostat, and belinostat were designed with a photoswitchable azobenzene moiety incorporated into their structure. The most promising compound exhibits high inhibitory potency in the thermodynamically less stable cis form and a significantly lower activity for the trans form, both in terms of HDAC activity and proliferation of HeLa cells. This approach offers a clear prospect towards local photoactivation of HDAC inhibition to avoid severe side effects in chemotherapy.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/química , Indóis/química , Sulfonamidas/química , Antineoplásicos/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Luz , Panobinostat , Sulfonamidas/farmacologia , VorinostatRESUMO
Patterning of multiple bacterial strains in one system is achieved by employing a single photo-activated antibiotic. Varying the light-exposure time results in zones with mixed and single populations.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Luz , Micrococcus luteus/efeitos dos fármacos , Antibacterianos/química , Escherichia coli/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Micrococcus luteus/crescimento & desenvolvimento , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Pharmacotherapy is often severely hindered by issues related to poor drug selectivity, including side effects, environmental toxicity, and the emergence of resistance. Lack of selectivity is caused by the inability to control drug activity in time and space. Photopharmacology aims at solving this issue by incorporating photoswitchable groups into the molecular structure of bioactive compounds. These switching units allow for the use of light as an external control element for pharmacological activity, which can be delivered with very high spatiotemporal precision. This Perspective presents the reader with the current state and outlook on photopharmacology. In particular, the principles behind photoregulation of bioactivity, the challenges of molecular design, and the possible therapeutic scenarios are discussed.
Assuntos
Química Farmacêutica , LuzRESUMO
Proteasome inhibitors are widely used in cancer treatment as chemotherapeutic agents. However, their employment often results in severe side effects, due to their non-specific cytotoxicity towards healthy tissue. This problem might be overcome by using a photopharmacological approach, that is, by attaining external, dynamic, spatiotemporal photocontrol over the activity of a cytotoxic agent, achieved by the introduction of a photoswitchable moiety into its molecular structure. Here we describe the design, synthesis, and activity of photoswitchable proteasome inhibitors. Substantial differences in proteasome inhibitory activity in cell extracts were observed before and after irradiation with light. The presented results show potential for the development of chemotherapeutic agents that can be switched on and off with light, constituting a new strategy for spatiotemporally modulating proteasomal activity.
Assuntos
Compostos Azo/química , Compostos Azo/farmacologia , Luz , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Compostos Azo/síntese química , Sobrevivência Celular/efeitos dos fármacos , Desenho de Fármacos , Células HeLa , Humanos , Neoplasias/tratamento farmacológico , Inibidores de Proteassoma/síntese químicaRESUMO
Photocaged compounds are important tools for studying and regulating multiple processes, including biological functions. Reported herein is the use of the Passerini multicomponent reaction for modular preparation of photocaged carboxylic acids. The reaction is compatible with several functionalities and proceeds smoothly both in water and dichloromethane. The choice of aldehyde determines the wavelength used for deprotection and enables formation of orthogonally protected products. The isocyanide component can be used for introduction of reactive tags and photosensitizers, as well as for immobilization on a solid support.
Assuntos
Ácidos Carboxílicos/química , Fármacos Fotossensibilizantes/química , Cianetos/química , Luz , Cloreto de Metileno/química , Água/químicaRESUMO
Antibiotic resistance is an enormous problem that is accountable for over a million deaths annually, with numbers expected to significantly increase over the coming decades. Although some of the underlying causes leading up to antibiotic resistance are well understood, many of the molecular processes involved remain elusive. To better appreciate at a molecular level how resistance emerges, customized chemical biology tools can offer a solution. This Feature Article attempts to provide an overview of the wide variety of tools that have been developed over the last decade, by highlighting some of the more illustrative examples. These include the use of fluorescent, photoaffinity and activatable antibiotics and bacterial components to start to unravel the molecular mechanisms involved in resistance. The antibiotic crisis is an eminent global threat and requires the continuous development of creative chemical tools to dissect and ultimately counteract resistance.
Assuntos
Antibacterianos , Bactérias , Resistência Microbiana a Medicamentos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência BacterianaRESUMO
In recent years, RNA has emerged as a multifaceted biomolecule that is involved in virtually every function of the cell and is critical for human health. This has led to a substantial increase in research efforts to uncover the many chemical and biological aspects of RNA and target RNA for therapeutic purposes. In particular, analysis of RNA structures and interactions in cells has been critical for understanding their diverse functions and druggability. In the last 5 years, several chemical methods have been developed to achieve this goal, using chemical cross-linking combined with high-throughput sequencing and computational analysis. Applications of these methods resulted in important new insights into RNA functions in a variety of biological contexts. Given the rapid development of new chemical technologies, a thorough perspective on the past and future of this field is provided. In particular, the various RNA cross-linkers and their mechanisms, the computational analysis and challenges, and illustrative examples from recent literature are discussed.
RESUMO
The off-target toxicity of drugs targeted to proteins imparts substantial health and economic costs. Proteome interaction studies can reveal off-target effects with unintended proteins; however, little attention has been paid to intracellular RNAs as potential off-targets that may contribute to toxicity. To begin to assess this, we developed a reactivity-based RNA profiling methodology and applied it to uncover transcriptome interactions of a set of Food and Drug Administration-approved small-molecule drugs in vivo. We show that these protein-targeted drugs pervasively interact with the human transcriptome and can exert unintended biological effects on RNA functions. In addition, we show that many off-target interactions occur at RNA loci associated with protein binding and structural changes, allowing us to generate hypotheses to infer the biological consequences of RNA off-target binding. The results suggest that rigorous characterization of drugs' transcriptome interactions may help assess target specificity and potentially avoid toxicity and clinical failures.
RESUMO
Three-dimensional (3D) structures dictate the functions of RNA molecules in a wide variety of biological processes. However, direct determination of RNA 3D structures in vivo is difficult due to their large sizes, conformational heterogeneity, and dynamics. Here we present a method, Spatial 2'-Hydroxyl Acylation Reversible Crosslinking (SHARC), which uses chemical crosslinkers of defined lengths to measure distances between nucleotides in cellular RNA. Integrating crosslinking, exonuclease (exo) trimming, proximity ligation, and high throughput sequencing, SHARC enables transcriptome-wide tertiary structure contact maps at high accuracy and precision, revealing heterogeneous RNA structures and interactions. SHARC data provide constraints that improves Rosetta-based RNA 3D structure modeling at near-nanometer resolution. Integrating SHARC-exo with other crosslinking-based methods, we discover compact folding of the 7SK RNA, a critical regulator of transcriptional elongation. These results establish a strategy for measuring RNA 3D distances and alternative conformations in their native cellular context.
Assuntos
Modelos Moleculares , RNA/ultraestrutura , Acilação , Reagentes de Ligações Cruzadas/química , Células HEK293 , Células HeLa , Humanos , Conformação de Ácido Nucleico , RNA/química , RNA/isolamento & purificação , Dobramento de RNA , Elongação da Transcrição GenéticaRESUMO
Direct determination of RNA structures and interactions in living cells is critical for understanding their functions in normal physiology and disease states. Here, we present PARIS2, a dramatically improved method for RNA duplex determination in vivo with >4000-fold higher efficiency than previous methods. PARIS2 captures ribosome binding sites on mRNAs, reporting translation status on a transcriptome scale. Applying PARIS2 to the U8 snoRNA mutated in the neurological disorder LCC, we discover a network of dynamic RNA structures and interactions which are destabilized by patient mutations. We report the first whole genome structure of enterovirus D68, an RNA virus that causes polio-like symptoms, revealing highly dynamic conformations altered by antiviral drugs and different pathogenic strains. We also discover a replication-associated asymmetry on the (+) and (-) strands of the viral genome. This study establishes a powerful technology for efficient interrogation of the RNA structurome and interactome in human diseases.