Site‐Specific Labeling of MicroRNA Precursors: A Structure–Activity Relationship Study

Functionalized oligoribonucleotides are essential tools in RNA chemical biology. Various synthetic routes have been developed over recent years to conjugate functional groups to oligoribonucleotides. However, the presence of the functional group on the oligoribonucleotide backbone can lead to partial or total loss of biological function. The limited knowledge concerning the positioning of functional groups therefore represents a hurdle for the development of oligoribonucleotide chemical tools. Here we describe a systematic investigation of site‐specific labeling of pre‐miRNAs to identify positions for the incorporation of functional groups, in order not to hinder their processing into active mature miRNAs.     

SCF-ChemBio: Chemical Biology Tour de France

Chemical biology is a steadily growing field that has traditionally struggled to clearly define its boundaries in a short sentence. However, it can be stated that through the development of chemical and physicochemical tools, concepts and methods, chemical biology aims to address or stimulate biological questions at the molecular level in living organisms. Chemical biologists design and develop molecular tools that can probe or modulate biological processes, in order to understand their function, and sometimes to modify it for specific applications, but also to observe and analyze these tools in complex biological environments. Essentially positioned as a fundamental approach, chemical biology often remains very close to potential applications as it builds molecular objects capable of reacting to a significant biological stimulus. Chemical biology therefore finds natural development in fields such as health for the design of drugs and diagnostic systems or the environment for applications in crop science and ecology.     

Molecular mechanisms of RNA-triggered gene silencing machineries

Gene silencing by RNA triggers is an ancient, evolutionarily conserved, and widespread phenomenon. This process, known as RNA interference (RNAi), occurs when double-stranded RNA helices induce cleavage of their complementary mRNAs. Because these RNA molecules can be introduced exogenously as small interfering RNAs (siRNAs), RNAi has become an everyday experimental tool in laboratory research. In addition, the number of RNA-based therapeutics that are currently in clinical trials for a variety of human diseases demonstrate the therapeutic potential of RNAi. In this Account, we focus on our current understanding of the structure and function of various classes of RNAi triggers and how this knowledge has contributed to our understanding of the biogenesis and catalytic functions of siRNA and microRNA in mammalian cells. Mechanistic studies to understand the structure and function of small RNAs that induce RNAi have illuminated broad functions of the ancient RNAi machinery in animals and plants. In addition, such studies have provided insight to identify endogenous physiological gene silencing RNA triggers that engage functional machineries similar to siRNAs. Several endogenous small RNA species have been identified: small noncoding RNAs (microRNAs), piwi-interacting RNAs (piRNAs), and endogenous siRNAs (endo-siRNAs). microRNAs are the most widespread class of small RNAs in mammalian cells. Despite their importance in biology and medicine, the molecular and cellular mechanisms of microRNA biogenesis and function are not fully understood. We provide an overview of the current understanding of how these molecules are synthesized within cells and how they act on gene targets. Interesting questions remain both for understanding the effects of modifications and editing on microRNAs and the interactions between microRNAs and other cellular RNAs such as long noncoding RNAs.     

Exploring RNA structural codes with SHAPE chemistry

RNA is the central conduit for gene expression. This role depends on an ability to encode information at two levels: in its linear sequence and in the complex structures RNA can form by folding back on itself. Understanding the global structure-function interrelationships mediated by RNA remains a great challenge in molecular and structural biology. In this Account, we discuss evolving work in our laboratory focused on creating facile, generic, quantitative, accurate, and highly informative approaches for understanding RNA structure in biologically important environments. The core innovation derives from our discovery that the nucleophilic reactivity of the ribose 2'-hydroxyl in RNA is gated by local nucleotide flexibility. The 2'-hydroxyl is reactive at conformationally flexible positions but is unreactive at nucleotides constrained by base pairing. Sites of modification in RNA can be detected efficiently either using primer extension or by protection from exoribonucleolytic degradation. This technology is now called SHAPE, for selective 2'-hydroxyl acylation analyzed by primer extension (or protection from exoribonuclease). SHAPE reactivities are largely independent of nucleotide identity but correlate closely with model-free measurements of molecular order. The simple SHAPE reaction is thus a robust, nucleotide-resolution, biophysical measurement of RNA structure. SHAPE can be used to provide an experimental correction to RNA folding algorithms and, in favorable cases, yield kilobase-scale secondary structure predictions with high accuracies. SHAPE chemistry is based on very simple reactive carbonyl centers that can be varied to yield slow- and fast-reacting reagents. Differential SHAPE reactivities can be used to detect specific RNA positions with slow local nucleotide dynamics. These positions, which are often in the C2'-endo conformation, have the potential to function as molecular timers that regulate RNA folding and function. In addition, fast-reacting SHAPE reagents can be used to visualize RNA structural biogenesis and RNA-protein assembly reactions in one second snapshots in very straightforward experiments. The application of SHAPE to challenging problems in biology has revealed surprises in well-studied systems. New regions have been identified that are likely to have critical functional roles on the basis of their high levels of RNA structure. For example, SHAPE analysis of large RNAs, such as authentic viral RNA genomes, suggests that RNA structure organizes regulatory motifs and regulates splicing, protein folding, genome recombination, and ribonucleoprotein assembly. SHAPE has also revealed limitations to the hierarchical model for RNA folding. Continued development and application of SHAPE technologies will advance our understanding of the many ways in which the genetic code is expressed through the underlying structure of RNA.     

Chemical tags for labeling proteins inside living cells

To build on the last century's tremendous strides in understanding the workings of individual proteins in the test tube, we now face the challenge of understanding how macromolecular machines, signaling pathways, and other biological networks operate in the complex environment of the living cell. The fluorescent proteins (FPs) revolutionized our ability to study protein function directly in the cell by enabling individual proteins to be selectively labeled through genetic encoding of a fluorescent tag. Although FPs continue to be invaluable tools for cell biology, they show limitations in the face of the increasingly sophisticated dynamic measurements of protein interactions now called for to unravel cellular mechanisms. Therefore, just as chemical methods for selectively labeling proteins in the test tube significantly impacted in vitro biophysics in the last century, chemical tagging technologies are now poised to provide a breakthrough to meet this century's challenge of understanding protein function in the living cell. With chemical tags, the protein of interest is attached to a polypeptide rather than an FP. The polypeptide is subsequently modified with an organic fluorophore or another probe. The FlAsH peptide tag was first reported in 1998. Since then, more refined protein tags, exemplified by the TMP- and SNAP-tag, have improved selectivity and enabled imaging of intracellular proteins with high signal-to-noise ratios. Further improvement is still required to achieve direct incorporation of powerful fluorophores, but enzyme-mediated chemical tags show promise for overcoming the difficulty of selectively labeling a short peptide tag. In this Account, we focus on the development and application of chemical tags for studying protein function within living cells. Thus, in our overview of different chemical tagging strategies and technologies, we emphasize the challenge of rendering the labeling reaction sufficiently selective and the fluorophore probe sufficiently well behaved to image intracellular proteins with high signal-to-noise ratios. We highlight recent applications in which the chemical tags have enabled sophisticated biophysical measurements that would be difficult or even impossible with FPs. Finally, we conclude by looking forward to (i) the development of high-photon-output chemical tags compatible with living cells to enable high-resolution imaging, (ii) the realization of the potential of the chemical tags to significantly reduce tag size, and (iii) the exploitation of the modular chemical tag label to go beyond fluorescent imaging.     

Single-Molecule FRET of Membrane Transport Proteins

Uncovering the structure and function of biomolecules is a fundamental goal in structural biology. Membrane-embedded transport proteins are ubiquitous in all kingdoms of life. Despite structural flexibility, their mechanisms are typically studied by ensemble biochemical methods or by static high-resolution structures, which complicate a detailed understanding of their dynamics. Here, we review the recent progress of single molecule Förster Resonance Energy Transfer (smFRET) in determining mechanisms and timescales of substrate transport across membranes. These studies do not only demonstrate the versatility and suitability of state-of-the-art smFRET tools for studying membrane transport proteins but they also highlight the importance of membrane mimicking environments in preserving the function of these proteins. The current achievements advance our understanding of transport mechanisms and have the potential to facilitate future progress in drug design.     

DNA-Targeted Metallodrugs: An Untapped Source of Artificial Gene Editing Technology

DNA binding metal complexes are synonymous with anticancer drug discovery. Given the array of structural and chemical reactivity properties available through careful design, metal complexes have been directed to bind nucleic acid structures through covalent or noncovalent binding modes. Several recognition modes – including crosslinking, intercalation, and oxidation – are central to the clinical success of broad-spectrum anticancer metallodrugs. However, recent progress in nucleic acid click chemistry coupled with advancement in our understanding of metal complex–nucleic acid interactions has opened up new avenues in genetic engineering and targeted therapies. Several of these applications are enabled by the hybridisation of oligonucleotide or polyamine probes to discrete metal complexes, which facilitate site-specific reactivity at the nucleic acid interface under the guidance of the probe. This Review focuses on recent advancements in hybrid design and, by way of an introduction to this topic, we provide a detailed overview of nucleic acid structures and metal complex–nucleic acid interactions. Our aim is to provide readers with an insight on the rational design of metal complexes with DNA recognition properties and an understanding of how the sequence-specific targeting of these interactions can be achieved for gene engineering applications.     

Structure Determination of the Top‐Loop of the Conserved 3′‐Terminal Secondary Structure in the Genome of Flaviviruses

To what extent small differences in RNA sequences (mutations) can have a profound impact on biology remains an intriguing question. This effect can be studied by using untranslated RNA regions as a model. We have studied the influence of mutations on the structure of an RNA hairpin that occurs in the 3′‐untranslated region (UTR) of Flaviviridae, and is known to have a large impact on the vector dependency of flaviviruses. Three related RNA sequences were studied by NMR spectroscopy. The selected sequences represent each one of the three clusters in the flavivirus genes (mosquito‐borne, tick‐borne, and no‐known‐vector viruses). A new strategy was used to obtain chemical shift signatures of carbonyl atoms in unlabeled uridine nucleobases to characterize their involvement in hydrogen bonding. Clear differences occur in the structures and stacking pattern of the three RNA hairpins. The observed differences cannot be predicted based on sequence analysis. A different biology can be correlated with a different RNA tertiary structure. The underlying biological mechanism, however, remains to be studied.     

Lessons in Organic Fluorescent Probe Discovery

Fluorescent probes have gained profound use in biotechnology, drug discovery, medical diagnostics, molecular and cell biology. The development of methods for the translation of fluorophores into fluorescent probes continues to be a robust field for medicinal chemists and chemical biologists, alike. Access to new experimental designs has enabled molecular diversification and led to the identification of new approaches to probe discovery. This review provides a synopsis of the recent lessons in modern fluorescent probe discovery.     

The molecular interactions that stabilize RNA tertiary structure: RNA motifs, patterns, and networks

RNA molecules adopt specific three-dimensional structures critical to their function. Many essential metabolic processes, including protein synthesis and RNA splicing, are carried out by RNA molecules with elaborate tertiary structures (e.g. 3QIQ, right). Indeed, the ribosome and self-splicing introns are complex RNA machines. But even the coding regions in messenger RNAs and viral RNAs are flanked by highly structured untranslated regions, which provide regulatory information necessary for gene expression. RNA tertiary structure is defined as the three-dimensional arrangement of RNA building blocks, which include helical duplexes, triple-stranded structures, and other components that are held together through connections collectively termed RNA tertiary interactions. The structural diversity of these interactions is now a subject of intense investigation, involving the techniques of NMR, X-ray crystallography, chemical genetics, and phylogenetic analysis. At the same time, many investigators are using biophysical techniques to elucidate the driving forces for tertiary structure formation and the mechanisms for its stabilization. RNA tertiary folding is promoted by maximization of base stacking, much like the hydrophobic effect that drives protein folding. RNA folding also requires electrostatic stabilization, both through charge screening and site binding of metals, and it is enhanced by desolvation of the phosphate backbone. In this Account, we provide an overview of the features that specify and stabilize RNA tertiary structure. A major determinant for overall tertiary RNA architecture is local conformation in secondary-structure junctions, which are regions from which two or more duplexes project. At junctions and other structures, such as pseudoknots and kissing loops, adjacent helices stack on one another, and these coaxial stacks play a major role in dictating the overall architectural form of an RNA molecule. In addition to RNA junction topology, a second determinant for RNA tertiary structure is the formation of sequence-specific interactions. Networks of triple helices, tetraloop-receptor interactions, and other sequence-specific contacts establish the framework for the overall tertiary fold. The third determinant of tertiary structure is the formation of stabilizing stacking and backbone interactions, and many are not sequence specific. For example, ribose zippers allow 2'-hydroxyl groups on different RNA strands to form networks of interdigitated hydrogen bonds, serving to seal strands together and thereby stabilize adjacent substructures. These motifs often require monovalent and divalent cations, which can interact diffusely or through chelation to specific RNA functional groups. As we learn more about the components of RNA tertiary structure, we will be able to predict the structures of RNA molecules from their sequences, thereby obtaining key information about biological function. Understanding and predicting RNA structure is particularly important given the recent discovery that although most of our genome is transcribed into RNA molecules, few of them have a known function. The prevalence of RNA viruses and pathogens with RNA genomes makes RNA drug discovery an active area of research. Finally, knowledge of RNA structure will facilitate the engineering of supramolecular RNA structures, which can be used as nanomechanical components for new materials. But all of this promise depends on a better understanding of the RNA parts list, and how the pieces fit together.     

Recent Advances in MRI Studies of Chemical Reactors: Ultrafast Imaging of Multiphase Flows

NMR has long been established as an in situ technique for studying the solid-state structure of catalysts and the chemical processes occurring during catalytic reactions. Increasingly, pulsed field gradient (PFG) NMR and magnetic resonance imaging (MRI) are being exploited in chemical reaction engineering to measure molecular diffusion, dispersion and flow hydrodynamics within reactors. By bringing together NMR spectroscopy, PFG NMR and MRI, we are now able to probe catalysts and catalytic processes from the angstrom-to-centimeter scale. This article briefly reviews current activities in the field of MRI studies applied to catalysts and catalytic reactors. State-of-the-art measurements, which can already be used in real reactor design studies, are illustrated with examples of single-phase flow with and without chemical reaction in a fixed-bed reactor. The ability to obtain high spatial resolution (< 200μm) in images of the internal structure and flow field within reactors is demonstrated, and the potential uses of these data in reactor design and understanding bed fouling phenomena are discussed. In particular, MRI has produced the first detailed measurements of the extent of heterogeneity in the flow field within fixed-bed reactors. The example of a fixed-bed esterification process is used to show how NMR spectroscopy and MRI techniques can be combined to provide spatially resolved information on both hydrodynamics and chemical conversion within a process unit. The emerging area of ultrafast MRI is then highlighted as an area of particular interest. Recent advances have demonstrated that it is possible to record 2D images over timescales of ～100ms in the magnetically heterogeneous environments typical of heterogeneous chemical reactors. These advances open up opportunities to image many unsteady state processes for the first time. Examples are given of real-time visualization of bubble-train flow in a ceramic monolith and exploring the stability of the gas–liquid distribution as a function of liquid flow rate in a trickle-bed reactor.     

The Chemical Biology-Medicinal Chemistry Continuum: EFMC′s Vision

The E uropean F ederation for M edicinal chemistry and C hemical biology (EFMC) is a federation of learned societies. It groups organizations of European scientists working in a dynamic field spanning chemical biology and medicinal chemistry. New ideas, tools, and technologies emerging from a wide array of scientific disciplines continuously energize this rapidly evolving area. Medicinal chemistry is the design, synthesis, and optimization of biologically active molecules aimed at discovering new drug candidates – a mission that in many ways overlaps with the scope of chemical biology. Chemical biology is by now a mature field of science for which a more precise definition of what it encompasses, in the frame of EFMC, is timely. This article discusses chemical biology as currently understood by EFMC, including all activities dealing with the design and synthesis of biologically active chemical tools and their use to probe, characterize, or influence biological systems.     

Activity-based probes for the study of proteases: recent advances and developments

Proteases are important targets for the treatment of human disease. Several protease inhibitors have failed in clinical trials due to a lack of in vivo specificity, indicating the need for studies of protease function and inhibition in complex, disease-related models. The tight post-translational regulation of protease activity complicates protease analysis by traditional proteomics methods. Activity-based protein profiling is a powerful technique that can resolve this issue. It uses small-molecule tools-activity-based probes-to label and analyze active enzymes in lysates, cells, and whole animals. Over the last twelve years, a wide variety of protease activity-based probes have been developed. These synthetic efforts have enabled techniques ranging from real-time in vivo imaging of protease activity to high-throughput screening of uncharacterized proteases. This Review introduces the general principles of activity-based protein profiling and describes the recent advancements in probe design and analysis techniques, which have increased the knowledge of protease biology and will aid future protease drug discovery.     

The Chirality of Aggregated Yariv Reagents Correlates with Their AGP-Binding Ability**

Yariv reagents are glycosylated triphenylazo dyes that bind to arabinogalactan proteins (AGPs), proteoglycans found in plant cell walls that are integral for plant growth and development. Yariv reagents are widely utilized as imaging, purification, and quantification tools for AGPs and represent the only small molecule probe for interrogating AGP function. The ability of Yariv reagents to bind to AGPs is dependent on the structure of the terminal glycoside on the dye. The reason for this selectivity has not been understood until the present work. Using circular dichroism spectroscopy, we show that the Yariv reagents form supramolecular aggregates with helical chirality. More significantly, the ability of the Yariv reagent to bind AGPs is correlated with this helical chirality. This finding paves the way towards developing a more detailed understanding of the nature of the Yariv-AGP complex, and the design of AGP-binding reagents with higher affinities and selectivities.     

Tecto‐GIRz: Engineered Group I Ribozyme the Catalytic Ability of Which Can Be Controlled by Self‐Dimerization

RNA is a promising biomaterial for self‐assembly of nano‐sized structures with a wide range of applications in nanotechnology and synthetic biology. Several RNA‐based nanostructures have been reported, but most are unrelated to intracellular RNA, which possesses modular structures that are sufficiently large and complex to serve as catalysts to promote sophisticated chemical reactions. In this study, we designed dimeric RNA structures based on the Tetrahymena group I ribozyme. The resulting dimeric RNAs (tecto group I ribozyme; tecto‐GIRz) exhibit catalytic ability that depended on controlled dimerization, by which a pair of ribozymes can be activated to perform cleavage and splicing reactions of two distinct substrates. Modular redesign of complex RNA structures affords large ribozymes for use as modules in RNA nanotechnology and RNA synthetic biology.     

Nucleosomes and Epigenetics from a Chemical Perspective

Nucleosomes, which are the fundamental building blocks of chromatin, are highly dynamic, they play vital roles in the formation of higher-order chromatin structures and orchestrate gene regulation. Nucleosome structures, histone modifications, nucleosome-binding proteins, and their functions are being gradually unravelled with the development of epigenetics. With the continuous development of research approaches such as cryo-EM, FRET and next-generation sequencing for genome-wide analysis of nucleosomes, the understanding of nucleosomes is getting wider and deeper. Herein, we review recent progress in research on nucleosomes and epigenetics, from nucleosome structure to chromatin formation, with a focus on chemical aspects. Basic knowledge of the nucleosome (nucleosome structure, nucleosome position sequence, nucleosome assembly and remodeling), epigenetic modifications, chromatin structure, chemical biology methods and nucleosome, observation nucleosome by AFM, phase separation and nucleosomes are described in this review.