Photoactivatable rhodamine spiroamides and spirocyclic diazoketones emerged recently as synthetic markers applicable in multicolor super-resolution microscopy. However, their applicability in single molecule localization microscopy (SMLM) is often limited by aggregation, unspecific adhesion, and low reactivity caused by insufficient solubility and precipitation from aqueous solutions. We report here two synthetic modifications increasing the polarity of compact polycyclic and hydrophobic labels decorated with a reactive group: attachment of 3-sulfo-l-alanyl-beta-alanine dipeptide (a "universal hydrophilizer") or allylic hydroxylation in photosensitive rhodamine diazoketones (and spiroamides). The super-resolution images of tubulin and keratin filaments in fixed and living cells exemplify the performance of "blinking" spiroamides derived from N, N, N', N'-tetramethyl rhodamine.

The complex interplay between actin regulatory proteins facilitates the formation of diverse cellular actin structures. Formin homology proteins (formins) play an essential role in the formation of actin stress fibers and yeast actin cables, to which the major actin depolymerizing factor cofilin barely associates. In vitro, F-actin decorated with cofilin exhibits a marked increase in the filament twist. On the other hand, a mammalian formin mDia1 rotates along the long-pitch actin helix during processive actin elongation (helical rotation). Helical rotation may impose torsional force on F-actin in the opposite direction of the cofilin-induced twisting. Here, we show that helical rotation of mDia1 converts F-actin resistant to cofilin both in vivo and in vitro. F-actin assembled by mDia1 without rotational freedom became more resistant to the severing and binding activities of cofilin than freely rotatable F-actin. Electron micrographic analysis revealed untwisting of the long-pitch helix of F-actin elongating from mDia1 on tethering of both mDia1 and the pointed end side of the filament. In cells, single molecules of mDia1ΔC63, an activated mutant containing N-terminal regulatory domains, showed tethering to cell structures more frequently than autoinhibited wild-type mDia1 and mDia1 devoid of N-terminal domains. Overexpression of mDia1ΔC63 induced the formation of F-actin, which has prolonged lifetime and accelerates dissociation of cofilin. Helical rotation of formins may thus serve as an F-actin stabilizing mechanism by which a barbed end-bound molecule can enhance the stability of a filament over a long range.

The transcriptome encompasses a range of species including messenger RNA, and other noncoding RNA such as rRNA, tRNA, and short and long noncoding RNAs. Due to the huge role played by mRNA in development and disease, several methods have been developed to sequence and characterize mRNA, with RNA sequencing (RNA-Seq) emerging as the current method of choice particularly for large high-throughput studies. Short-read RNA-Seq which involves sequencing of short cDNA fragments and computationally assembling them to reconstruct the transcriptome, or aligning them to a reference is the most widely used approach. However, due to inherent limitations of this approach in de novo transcriptome assembly and isoform quantification, long-read RNA-Seq approaches, which also happen to be single molecule sequencing approaches, are increasingly becoming the standard for de novo transcriptome assembly and isoform quantification. In this chapter, we review the technical aspects of the current methods of RNA-Seq, both short and long-read approaches, and data analysis methods available. We discuss recent advances in single-cell RNA-Seq and direct RNA-Seq approaches, which perhaps will dominate the future of RNA-Seq.

The NanoString nCounter Analysis System uses a digital fluorescent barcode technology that allows for direct multiplexed measurement of gene expression (mRNA), DNA, and protein. The technology uses molecular barcodes and single-molecule imaging to detect and count unique mRNA and protein targets in a single reaction. nCounter-based detection is enzyme-free (no amplification of mRNA is required), fully automated, and allows simultaneous detection of up to 770 mRNA and 30 protein targets from multiple sample types. Target counting is fully digital with quantitative data output. Here we describe preparation of solid tumor lysate samples for use in the nCounter Analysis System.

Recombinant adeno-associated virus (rAAV)-based gene therapy has entered a phase of clinical translation and commercialization. Despite this progress, vector integrity following production is often overlooked. Compromised vectors may negatively impact therapeutic efficacy and safety. Using single molecule, real-time (SMRT) sequencing, we can comprehensively profile packaged genomes as a single intact molecule and directly assess vector integrity without extensive preparation. We have exploited this methodology to profile all heterogeneic populations of self-complementary AAV genomes via bioinformatics pipelines and have coined this approach AAV-genome population sequencing (AAV-GPseq). The approach can reveal the relative distribution of truncated genomes versus full-length genomes in vector preparations. Preparations that seemingly show high genome homogeneity by gel electrophoresis are revealed to consist of less than 50% full-length species. With AAV-GPseq, we can also detect many reverse-packaged genomes that encompass sequences originating from plasmid backbone, as well as sequences from packaging and helper plasmids. Finally, we detect host-cell genomic sequences that are chimeric with inverted terminal repeat (ITR)-containing vector sequences. We show that vector populations can contain between 1.3% and 2.3% of this type of undesirable genome. These discoveries redefine quality control standards for viral vector preparations and highlight the degree of foreign products in rAAV-based therapeutic vectors.

The investigation of liquid crystal (LC) mixtures is of great interest in tailoring material properties for specific applications. The recent discovery of the twist-bend nematic phase (NTB) has sparked great interest in the scientific community, not only from a fundamental viewpoint, but also due to its potential for innovative applications. Here we report on the unexpected phase behaviour of a binary mixture of twist-bend nematogens. A binary phase diagram for mixtures of imino-linked cyanobiphenyl (CBI) dimer and imino-linked benzoyloxy-benzylidene (BB) dimer shows two distinct domains. While mixtures containing less than 35 mol % of BB possess a wide temperature range twist-bend nematic phase, the mixtures containing 55-80 mol % of BB exhibit a smectic phase despite that both pure compounds display a Iso-N-NTB-Cr phase sequence. The phase diagram shows that the addition of BB of up to 30 mol % significantly extends the temperature range of the NTB phase, maintaining the temperature range of the nematic phase. The periodicity, obtained by atomic force microscopy (AFM) imaging, is in the range of 6-7 nm. The induction of the smectic phase in the mixtures containing 55-80 mol % of BB was confirmed using polarising optical microscopy (POM), differential scanning calorimetry (DSC) and X-ray diffraction. The origin of the intercalated smectic phase was unravelled by combined spectroscopic and computational methods and can be traced to conformational disorder of the terminal chains. These results show the importance of understanding the phase behaviour of binary mixtures, not only in targeting a wide temperature range but also in controlling the self-organizing processes.

N6-Methyladenine (m6dA) has been discovered as a novel form of DNA methylation prevalent in eukaryotes; however, methods for high-resolution mapping of m6dA events are still lacking. Single-molecule real-time (SMRT) sequencing has enabled the detection of m6dA events at single-nucleotide resolution in prokaryotic genomes, but its application to detecting m6dA in eukaryotic genomes has not been rigorously examined. Herein, we identified unique characteristics of eukaryotic m6dA methylomes that fundamentally differ from those of prokaryotes. Based on these differences, we describe the first approach for mapping m6dA events using SMRT sequencing specifically designed for the study of eukaryotic genomes and provide appropriate strategies for designing experiments and carrying out sequencing in future studies. We apply the novel approach to study two eukaryotic genomes. For green algae, we construct the first complete genome-wide map of m6dA at single-nucleotide and single-molecule resolution. For human lymphoblastoid cells (hLCLs), it was necessary to integrate SMRT sequencing data with independent sequencing data. The joint analyses suggest putative m6dA events are enriched in the promoters of young full-length LINE-1 elements (L1s), but call for validation by additional methods. These analyses demonstrate a general method for rigorous mapping and characterization of m6dA events in eukaryotic genomes.

Two-dimensional (2D) layered metal dichalcogenides can form spiral nanostructures by a screw-dislocation-driven mechanism, which leads to changes in crystal symmetry and layer stackings that introduce attractive physical properties different from their bulk and few-layer nanostructures. However, controllable growth of spirals is challenging and their growth mechanisms are poorly understood. Here, we report the controllable growth of WS2 spiral nanoplates with different stackings by a vapor phase deposition route and investigate their formation mechanisms by combining atomic force microscopy with second harmonic generation imaging. Previously not observed "spiral arm" features could be explained as covered dislocation spiral steps, and the number of spiral arms correlates with the number of screw dislocations initiated at the bottom plane. The supersaturation-dependent growth can generate new screw dislocations from the existing layers, or even new layers templated by existing screw dislocations. Different number of dislocations and orientation of new layers result in distinct morphologies, different layer stackings, and more complex nanostructures, such as triangular spiral nanoplates with hexagonal spiral pattern on top. This work provides the understanding and control of dislocation-driven growth of 2D nanostructures. These spiral nanostructures offer diverse candidates for probing the physical properties of layered materials and exploring new applications in functional nanoelectronic and optoelectronic devices.

Background: The accurate sequencing and assembly of very large, often polyploid, genomes remains a challenging task, limiting long-range sequence information and phased sequence variation for applications such as plant breeding. The 15-Gb hexaploid bread wheat (Triticum aestivum) genome has been particularly challenging to sequence, and several different approaches have recently generated long-range assemblies. Mapping and understanding the types of assembly errors are important for optimising future sequencing and assembly approaches and for comparative genomics. Results: Here we use a Fosill 38-kb jumping library to assess medium and longer-range order of different publicly available wheat genome assemblies. Modifications to the Fosill protocol generated longer Illumina sequences and enabled comprehensive genome coverage. Analyses of two independent Bacterial Artificial Chromosome (BAC)-based chromosome-scale assemblies, two independent Illumina whole genome shotgun assemblies, and a hybrid Single Molecule Real Time (SMRT-PacBio) and short read (Illumina) assembly were carried out. We revealed a surprising scale and variety of discrepancies using Fosill mate-pair mapping and validated several of each class. In addition, Fosill mate-pairs were used to scaffold a whole genome Illumina assembly, leading to a 3-fold increase in N50 values. Conclusions: Our analyses, using an independent means to validate different wheat genome assemblies, show that whole genome shotgun assemblies based solely on Illumina sequences are significantly more accurate by all measures compared to BAC-based chromosome-scale assemblies and hybrid SMRT-Illumina approaches. Although current whole genome assemblies are reasonably accurate and useful, additional improvements will be needed to generate complete assemblies of wheat genomes using open-source, computationally efficient, and cost-effective methods.

In Gram-positive bacteria, T-box riboswitches control gene expression to maintain the cellular pools of aminoacylated tRNAs essential for protein biosynthesis. Co-transcriptional binding of an uncharged tRNA to the riboswitch stabilizes an antiterminator, allowing transcription read-through, whereas an aminoacylated tRNA does not. Recent structural studies have resolved two contact points between tRNA and Stem-I in the 5' half of the T-box riboswitch, but little is known about the mechanism empowering transcriptional control by a small, distal aminoacyl modification. Using single-molecule fluorescence microscopy, we have probed the kinetic and structural underpinnings of tRNA binding to a glycyl T-box riboswitch. We observe a two-step mechanism where fast, dynamic recruitment of tRNA by Stem-I is followed by ultra-stable anchoring by the downstream antiterminator, but only without aminoacylation. Our results support a hierarchical sensing mechanism wherein dynamic global binding of the tRNA body is followed by localized readout of its aminoacylation status by snap-lock-based trapping.

Ion hydration and transport at interfaces are relevant to a wide range of applied fields and natural processes1-5. Interfacial effects are particularly profound in confined geometries such as nanometre-sized channels6-8, where the mechanisms of ion transport in bulk solutions may not apply9,10. To correlate atomic structure with the transport properties of hydrated ions, both the interfacial inhomogeneity and the complex competing interactions among ions, water and surfaces require detailed molecular-level characterization. Here we constructed individual sodium ion (Na+) hydrates on a NaCl(001) surface by progressively attaching single water molecules (one to five) to the Na+ ion using a combined scanning tunnelling microscopy and noncontact atomic force microscopy system. We found that the Na+ ion hydrated with three water molecules diffuses orders of magnitude more quickly than other ion hydrates. Ab initio calculations revealed that such high ion mobility arises from the existence of a metastable state, in which the three water molecules around the Na+ ion can rotate collectively with a rather small energy barrier. This scenario would apply even at room temperature according to our classical molecular dynamics simulations. Our work suggests that anomalously high diffusion rates for specific hydration numbers of ions are generally determined by the degree of symmetry match between the hydrates and the surface lattice.

The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.

The angular velocity profile of the 120° F1-ATPase power stroke was resolved as a function of temperature from 16.3 to 44.6 °C using a ΔμATP = -31.25 kBT at a time resolution of 10 μs. Angular velocities during the first 60° of the power stroke (phase 1) varied inversely with temperature, resulting in negative activation energies with a parabolic dependence. This is direct evidence that phase 1 rotation derives from elastic energy (spring constant, κ = 50 kBT·rad-2). Phase 2 of the power stroke had an enthalpic component indicating that additional energy input occurred to enable the γ-subunit to overcome energy stored by the spring after rotating beyond its 34° equilibrium position. The correlation between the probability distribution of ATP binding to the empty catalytic site and the negative Ea values of the power stroke during phase 1 suggests that this additional energy is derived from the binding of ATP to the empty catalytic site. A second torsion spring (κ = 150 kBT·rad-2; equilibrium position, 90°) was also evident that mitigated the enthalpic cost of phase 2 rotation. The maximum ΔGǂ was 22.6 kBT, and maximum efficiency was 72%. An elastic coupling mechanism is proposed that uses the coiled-coil domain of the γ-subunit rotor as a torsion spring during phase 1, and then as a crankshaft driven by ATP-binding-dependent conformational changes during phase 2 to drive the power stroke.

Ribonucleic acid (RNA) is capable of hosting a variety of chemically diverse modifications, in both naturally-occurring post-transcriptional modifications and artificial chemical modifications used to expand the functionality of RNA. However, few studies have addressed how base modifications affect RNA polymerase and reverse transcriptase activity and fidelity. Here, we describe the fidelity of RNA synthesis and reverse transcription of modified ribonucleotides using an assay based on Pacific Biosciences Single Molecule Real-Time sequencing. Several modified bases, including methylated (m6A, m5C and m5U), hydroxymethylated (hm5U) and isomeric bases (pseudouridine), were examined. By comparing each modified base to the equivalent unmodified RNA base, we can determine how the modification affected cumulative RNA polymerase and reverse transcriptase fidelity. 5-hydroxymethyluridine and N6-methyladenosine both increased the combined error rate of T7 RNA polymerase and reverse transcriptases, while pseudouridine specifically increased the error rate of RNA synthesis by T7 RNA polymerase. In addition, we examined the frequency, mutational spectrum and sequence context of reverse transcription errors on DNA templates from an analysis of second strand DNA synthesis.

Prostate-associated gene 4 (PAGE4) is an intrinsically disordered protein implicated in prostate cancer. Thestress-response kinase homeodomain-interacting protein kinase 1 (HIPK1) phosphorylates two residues in PAGE4, serine 9 and threonine 51. Phosphorylation of these two residues facilitates the interaction of PAGE4 with activator protein-1 (AP-1) transcription factor complex to potentiate AP-1's activity. In contrast, hyperphosphorylation of PAGE4 by CDC-like kinase 2 (CLK2) attenuates this interaction with AP-1. Small-angleX-ray scattering and single-molecule fluorescence resonance energy transfer measurements have shown that PAGE4 expands upon hyperphosphorylation and that this expansion is localized to its N-terminal half. To understand the interactions underlying this structural transition, we performed molecular dynamics simulations using Atomistic AWSEM, a multi-scale molecular model that combines atomistic and coarse-grained simulation approaches. Our simulations show that electrostatic interactions drive transient formation of an N-terminal loop, the destabilization of which accounts for the dramatic change in size upon hyperphosphorylation. Phosphorylation also changes the preference of secondary structure formation of the PAGE4 ensemble, which leads to a transition between states that display different degrees of disorder. Finally, we construct a mechanism-based mathematical model that allows us to capture the interactions ofdifferent phosphoforms of PAGE4 with AP-1 and its downstream target, the androgen receptor (AR)-a key therapeutic target in prostate cancer. Our model predicts intracellular oscillatory dynamics of HIPK1-PAGE4, CLK2-PAGE4, and AR activity, indicating phenotypic heterogeneity in an isogenic cell population. Thus, conformational switching of PAGE4 may potentially affect the efficiency of therapeutically targeting AR activity.

In search of magneto-optic materials, the mononuclear compounds LnIII (depma)(NO3 )3 (hmpa)2 (Ln=Dy, Gd) were synthesized. The anthracene moieties undergo [4+4] dimerization when irradiated at 365 nm without loss of crystallinity. The Dy compound switches from a single-ion to a single-molecule magnet with doubling of the spin reversal barrier energy and from yellow-green to blue-white emission. The dimerization is reversed by heating at 100 °C or partially on light irradiating at 254 nm. The results suggest that lanthanide phosphonates with anthracene are promising smart materials displaying synergistic magneto-optic property.

Background: Bombax ceiba L. (the red silk cotton tree) is a large deciduous tree that is distributed in tropical and sub-tropical Asia as well as northern Australia. It has great economic and ecological importance, with several applications in industry and traditional medicine in many Asian countries. To facilitate further utilization of this plant resource, we present here the draft genome sequence for B. ceiba. Findings: We assembled a relatively intact genome of B. ceiba by using PacBio single-molecule sequencing and BioNano optical mapping technologies. The final draft genome is approximately 895 Mb long, with contig and scaffold N50 sizes of 1.0 Mb and 2.06 Mb, respectively. Conclusions: The high-quality draft genome assembly of B. ceiba will be a valuable resource enabling further genetic improvement and more effective use of this tree species.

In this study, we introduce an efficient data sorting algorithm, including filters for noisy signals, conductance mapping for analyzing the most dominant conductance group and sub-population groups. The capacity of our data analysis process has also been corroborated on real experimental data sets of Au-1,6-hexanedithiol-Au and Au-1,8-octanedithiol-Au molecular junctions. The fully automated and unsupervised program requires less than one minute on a standard PC to sort the data and generate histograms. The resulting one-dimensional and two-dimensional log histograms give conductance values in good agreement with previous studies. Our algorithm is a straightforward, fast and user-friendly tool for single molecule charge transport data analysis. We also analyze the data in a form of a conductance map which can offer evidence for diversity in molecular conductance. The code for automatic data analysis is openly available, well-documented and ready to use, thereby offering a useful new tool for single molecule electronics.

Diverse biological systems utilize fluctuations ("noise") in gene expression to drive lineage-commitment decisions. However, once a commitment is made, noise becomes detrimental to reliable function, and the mechanisms enabling post-commitment noise suppression are unclear. Here, we find that architectural constraints on noise suppression are overcome to stabilize fate commitment. Using single-molecule and time-lapse imaging, we find that-after a noise-driven event-human immunodeficiency virus (HIV) strongly attenuates expression noise through a non-transcriptional negative-feedback circuit. Feedback is established through a serial cascade of post-transcriptional splicing, whereby proteins generated from spliced mRNAs auto-deplete their own precursor unspliced mRNAs. Strikingly, this auto-depletion circuitry minimizes noise to stabilize HIV's commitment decision, and a noise-suppression molecule promotes stabilization. This feedback mechanism for noise suppression suggests a functional role for delayed splicing in other systems and may represent a generalizable architecture of diverse homeostatic signaling circuits.

Protein diffusion is crucial for understanding the formation of protein complexes in vivo and has been the subject of many fluorescence microscopy studies in cells; however, such microscopy efforts are often limited by low sensitivity and resolution. During the past decade, these limitations have been addressed by new super-resolution imaging methods, most of which rely on single-particle tracking and single-molecule detection; these methods are revolutionizing our understanding of molecular diffusion inside bacterial cells by directly visualizing the motion of proteins and the effects of the local and global environment on diffusion. Here we review key methods that made such experiments possible, with particular emphasis on versions of single-molecule tracking based on photo-activated fluorescent proteins. We also discuss studies that provide estimates of the time a diffusing protein takes to locate a target site, as well as studies that examined the stoichiometries of diffusing species, the effect of stable and weak interactions on diffusion, and the constraints of large macromolecular structures on the ability of proteins and their complexes to access the entire cytoplasm.

Resin biostability is of critical importance to the durability of methacrylate-based dental resin restorations. Current methods for evaluating biostability take considerable time, from weeks to months, and provide no short-time kinetics of resin degradation. The objective of this study is to develop a more sensitive method to assess resin biostability over short-time spans (hours to days) that will enhance our understanding of biostability and its resin chemistry. Ultra-flat resin films of equimolar urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) are produced through photo-curing between two flat surfaces. Next, metal-covered enclaves and bare-resin channels are generated using stencil lithography to create both degradable and protected (internal reference) regions simultaneously in a single specimen. Resins having three different degrees of vinyl conversion (DC) are compared, and changes of surface roughness and step height in the two regions are monitored by atomic force microscopy (AFM) before and after incubated in enzyme solutions and saline controls. Specimen biostability is ranked based on the topological profile changes when viewed in cross-section before and after enzymatic challenges. In addition, a model is proposed to quantify specimen enzymatic degradation. Based on this model, enzymatic degradation is detected as early as 4 h, and a surge of enzymatic degradation is detected between 4 h and 8 h. The correlation between the DC of resin network and the surge in degradation is discussed. In summary, this new method is effective in ranking biostability and quantifying enzymatic degradation while also reducing labor, time and cost, which lends itself well to materials development and evaluation of dental resins.STATEMENT OF SIGNIFICANCE: We report, for the first time, the short-time kinetics of enzymatic degradation of methacrylate dental resins. A nanotechnology based method is developed to accelerate the evaluation of resin biostability. This new method reduces experimental time from weeks to one or two days, which will significantly reduce the costs of labor and enzymes. It also introduces a corresponding parameter (ΔH) and a three-cause model for ranking biostability, which confirms the correlation of chemical structure (DC) and material performance and opens new opportunities for studying the resin biostability and its impact on dental applications. Overall, this is a new tool for evaluating resin biostability and developing new materials.

Understanding the mechanisms behind amyloid protein aggregation in diseases, such as Parkinson's and Alzheimer's disease, is often hampered by the reproducibility of in vitro assays. Yet, understanding the basic mechanisms of protein misfolding is essential for the development of novel therapeutic strategies. We show here, that for the amyloid protein α-synuclein (aSyn), a protein involved in Parkinson's disease (PD), chromatographic buffers and storage conditions can significantly interfere with the overall structure of the protein and thus affect protein aggregation kinetics. We apply several biophysical and biochemical methods, including size exclusion chromatography (SEC), dynamic light scattering (DLS), and atomic force microscopy (AFM), to characterize the high molecular weight conformers formed during protein purification and storage. We further apply hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to characterize the monomeric form of aSyn and reveal a thus far unknown structural component of aSyn at the C-terminus of the protein. Furthermore, lyophilizing the protein greatly affected the overall structure of this monomeric conformer. We conclude from this study that structural polymorphism may occur under different storage conditions, but knowing the structure of the majority of the protein at the start of each experiment, as well as the factors that may influence it, may pave the way to an improved understanding of the mechanism leading to aSyn pathology in PD.

Understanding the fundamental properties of charge carriers on the surface of semiconductor photo(electro)catalysts is key to the rational design of efficient photo(electro)catalytic devices for sunlight-driven energy conversion. Here high spatial resolution information is always desirable because of the ubiquitous heterogeneity in semiconductor particles. In this Perspective, we review the latest advances in nanoscale imaging and quantitative analysis of charge carrier activities on individual semiconductor particles down to subparticle resolution, covering the approaches of single-molecule super-resolution fluorescence imaging, scanning electron microscopy, and photoluminescence microscopy. We further highlight direct, operando functional assessments of their performances toward the targeted photo(electro)catalytic processes through single- and subparticle photocurrent measurements. We also discuss the significance of establishing quantitative relations between the desired functions of photo(electro)catalysts and their surface charge carrier activities. These fundamental relations can provide guiding principles for rationally engineering photo(electro)catalytic systems, for example with cocatalysts, for a broad range of applications.

Aromatic C-H bond activation has attracted much attention due to its versatile applications in the synthesis of aryl-containing chemicals. The major challenge lies in the minimization of the activation barrier and maximization of the regioselectivity. Here, we report the highly selective activation of the central aromatic C-H bond in meta-aryne species anchored to a copper surface, which catalyzes the C-H bond dissociation. Two prototype molecules, i.e., 4',6'-dibromo- meta-terphenyl and 3',5'-dibromo- ortho-terphenyl, have been employed to perform C-C coupling reactions on Cu(111). The chemical structures of the resulting products have been clarified by a combination of scanning tunneling microscopy and noncontact atomic force microscopy. Both methods demonstrate a remarkable weakening of the targeted C-H bond. Density functional theory calculations reveal that this efficient C-H activation stems from the extraordinary chemisorption of the meta-aryne on the Cu(111) surface, resulting in the close proximity of the targeted C-H group to the Cu(111) surface and the absence of planarity of the phenyl ring. These effects lead to a lowering of the C-H dissociation barrier from 1.80 to 1.12 eV, in agreement with the experimental data.

Our ability to directly relate results from test-tube biochemical experiments to the kinetics in living cells is very limited. Here we present experimental and analytical tools to directly study the kinetics of fast biochemical reactions in live cells. Dye-labeled molecules are electroporated into bacterial cells and tracked using super-resolved single-molecule microscopy. Trajectories are analyzed by machine-learning algorithms to directly monitor transitions between bound and free states. In particular, we measure the dwell time of tRNAs on ribosomes, and hence achieve direct measurements of translation rates inside living cells at codon resolution. We find elongation rates with tRNAPhe that are in perfect agreement with previous indirect estimates, and once fMet-tRNAfMet has bound to the 30S ribosomal subunit, initiation of translation is surprisingly fast and does not limit the overall rate of protein synthesis. The experimental and analytical tools for direct kinetics measurements in live cells have applications far beyond bacterial protein synthesis.