Multiple Turnover Research Articles

Subcellular Compartment‐Specific Amplified Imaging of Metal Ions via Ribosomal RNA‐Regulated DNAzyme Sensors

AbstractAlthough DNAzyme sensors have been widely developed for imaging metal ions, their application in specific subcellular compartments remains challenging due to low spatial controllability. Here we present a locally activatable, DNAzyme‐based sensing technology that enables subcellular compartment‐specific imaging of metal ions through ribosomal RNA (rRNA) regulated signal amplification. The system leverages a subcellularly encoded rRNA to locally activate DNAzyme‐based sensors, and further drives signal amplification via multiple turnover cleavage of molecular beacons, to significantly enhance sensitivity and spatial precision for metal‐ion imaging in specific organelles (e.g. mitochondria) or membraneless compartments (e.g. cytosol). Furthermore, we demonstrate that the system allows in situ monitoring of subcellular dynamics of mitochondrial Zn2+ during ischemia and the drug intervention. This study expands the DNAzyme toolbox for investigating the role of subcellular metal‐ion dynamics in disease processes.

Subcellular Compartment-Specific Amplified Imaging of Metal Ions via Ribosomal RNA-Regulated DNAzyme Sensors.

Although DNAzyme sensors have been widely developed for imaging metal ions, their application in specific subcellular compartments remains challenging due to low spatial controllability. Here we present a locally activatable, DNAzyme-based sensing technology that enables subcellular compartment-specific imaging of metal ions through ribosomal RNA (rRNA) regulated signal amplification. The system leverages a subcellularly encoded rRNA to locally activate DNAzyme-based sensors, and further drives signal amplification via multiple turnover cleavage of molecular beacons, to significantly enhance sensitivity and spatial precision for metal-ion imaging in specific organelles (e.g. mitochondria) or membraneless compartments (e.g. cytosol). Furthermore, we demonstrate that the system allows in situ monitoring of subcellular dynamics of mitochondrial Zn2+ during ischemia and the drug intervention. This study expands the DNAzyme toolbox for investigating the role of subcellular metal-ion dynamics in disease processes.

Alternative substrate kinetics of SARS-CoV-2 Nsp15 endonuclease reveals a specificity landscape dominated by RNA structure.

Coronavirus endoribonuclease Nsp15 contributes to the evasion of host innate immunity by suppressing levels of viral dsRNA. Nsp15 cleaves both ssRNA and dsRNA in vitro with a strong preference for unpaired or bulged U residues, and its activity is stimulated by divalent ions. Here, we systematically quantified effects of RNA sequence and structure context that define its specificity. The results show that sequence preference for U↓A/G, observed previously, contributes only ca. 2-fold to kcat/Km. In contrast, dsRNA structure flanking a bulged U residue increases kcat/Km by an order of magnitude compared to ssRNA while base pairing in dsRNA essentially blocks cleavage. Despite enormous differences in multiple turnover kinetics, the effect of RNA structure on the cleavage step is minimal. Surprisingly, although divalent ion activation of Nsp15 is widely considered to be important for its biological function, the effect on kcat/Km is only ∼2-fold and independent of RNA structure. These results reveal a specificity landscape dominated by RNA structure and provide a quantitative framework for identifying interactions that underlie specificity, determining mechanisms of inhibition and resistance and defining targets important for coronavirus biology.

Capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for highly sensitive visual detection of HPV DNA

The rapid and specific identification and sensitive detection of human papillomavirus (HPV) infection is critical for preventing cervical cancer, particularly in resource-limited regions. In this work, we hope to propose a capillarity-powered and CRISPR/Cas12a-responsive DNA hydrogel distance sensor for point-of-care (POC) DNA testing. Using the thermal reversibility of DNA hydrogel and capillarity, the novel DNA hydrogel distance sensor can be rapidly and simply constructed by loading an ultra-thin CRISPR/Cas12a-responsive DNA-crosslinked hydrogel film at the end of the capillary tube. The target DNA-specific recombinase polymerase reaction (RPA) amplicons activate the trans-cleavage activity of the Cas12a enzyme, cleaving the crosslinked DNA in hydrogel film, and causing an increase of hydrogel's permeability. As a result, a sample solution containing target DNA travels into the capillary tube at a longer distance compared to the negative samples. Reading the solution traveling distance in capillary tubes, the novel sensor realizes target DNA detection without any special equipment. Benefiting from the exponential target amplification of RPA and multiple turnover response of trans-cleavage of CRISPR/Cas12a, the developed sensor can visually and specifically detect as low as 1 aM HPV 16 DNA within 30 min. These outstanding features, including exceptional sensitivity and specificity, simple and portable design, mild measurement conditions, quick turnaround time, and user-friendly read-out, make the novel distance sensor a promising option for POC diagnostic applications.

Probing ligands to reaction centers to limit the photocycle in photosynthetic bacterium Rubrivivax gelatinosus

Light-induced electron flow between reaction center and cytochrome bc1 complexes is mediated by quinones and electron donors in purple photosynthetic bacteria. Upon high-intensity excitation, the contribution of the cytochrome bc1 complex is limited kinetically and the electron supply should be provided by the pool of reduced electron donors. The kinetic limitation of electron shuttle between reaction center and cytochrome bc1 complex and its consequences on the photocycle were studied by tracking the redox changes of the primary electron donor (BChl dimer) via absorption change and the opening of the closed reaction center via relaxation of the bacteriochlorophyll fluorescence in intact cells of wild type and pufC mutant strains of Rubrivivax gelatinosus. The results were simulated by a minimum model of reversible binding of different ligands (internal and external electron donors and inhibitors) to donor and acceptor sides of the reaction center. The calculated binding and kinetic parameters revealed that control of the rate of the photocycle is primarily due to 1) the light intensity, 2) the size and redox state of the donor pool, and 3) the unbinding rates of the oxidized donor and inhibitor from the reaction center. The similar kinetics of strains WT and pufC lacking the tetraheme cytochrome subunit attached to the reaction center raise the issue of the physiological importance of this subunit discussed from different points of view. SignificanceA crucial factor for the efficacy of electron donors in photosynthetic photocycle is not just the substantial size of the pool and large binding affinity (small dissociation constant KD = koff/kon) to the RC, but also the mean residence time (koff)−1 in the binding pocket. This is an important parameter that regulates the time of re-activation of the RC during multiple turnovers. The determination of koff has proven challenging and was performed by simulation of widespread experimental data on the kinetics of P+ and relaxation of fluorescence. This work is a step towards better understanding the complex pathways of electron transfer in proteins and simulation-based design of more effective electron transfer components in natural and artificial systems.

Apollon: a deoxyribozyme that generates a yellow product.

Colorimetric assays in which the color of a solution changes in the presence of an input provide a simple and inexpensive way to monitor experimental readouts. In this study we used in vitro selection to identify a self-phosphorylating kinase deoxyribozyme that produces a colorimetric signal by converting the colorless substrate pNPP into the yellow product pNP. The minimized catalytic core, sequence requirements, secondary structure, and buffer requirements of this deoxyribozyme, which we named Apollon, were characterized using a variety of techniques including reselection experiments, high-throughput sequencing, comparative analysis, biochemical activity assays, and NMR. A bimolecular version of Apollon catalyzed multiple turnover phosphorylation and amplified the colorimetric signal. Engineered versions of Apollon could detect oligonucleotides with specific sequences as well as several different types of nucleases in homogenous assays that can be performed in a single tube without the need for washes or purifications. We anticipate that Apollon will be particularly useful to reduce costs in high-throughput screens and for applications in which specialized equipment is not available.

A CoII-Hydroxide Complex That Converts Directly to a CoII-Acetamide during Catalytic Nitrile Hydration.

In exploring structural and functional mimics of nitrile hydratases, we report the synthesis of the pseudo-trigonal bipyramidal CoII complexes (K)[CoII(DMF)(LPh)] (1(DMF)), (NMe4)2[CoII(OAc)(LPh)] (1(OAc)), and (NMe4)2[CoII(OH)(LPh)] (1(OH)) (LPh = 2,2',2''-nitrilo-tris-(N-phenylacetamide; DMF = N,N-dimethylformamide; -OAc = acetate)). The complexes were characterized using NMR, FT-IR, ESI-MS, electronic absorption spectroscopy, and X-ray crystallography, showing the LPh ligand to bind in a tetradentate tripodal fashion alongside the respective ancillary donor. One of the complexes, 1(OH), is an unusual structural and functional mimic of the Co active site in Co nitrile hydratases. 1(OH) reacted with acetonitrile to yield the CoII-acetamide complex (NMe4)2[CoII(NHC(O)CH3)(LPh)], 2, which was also thoroughly characterized. In the presence of excess hydroxide, 1(OH) was found to catalyze quantitative conversion of the added hydroxide into acetamide. Despite the differences in Co oxidation state in nitrile hydratases and 1(OH) (CoIII versus CoII, respectively), 1(OH) was nonetheless an effective nitrile hydration catalyst, selectively producing acetamide over multiple turnovers.

Comparison of Human Long-Term Liver Models for Clearance Prediction of Slowly Metabolized Compounds.

The accurate prediction of human clearance is an important task during drug development. The proportion of low clearance compounds has increased in drug development pipelines across the industry since such compounds may be dosed in lower amounts and at lower frequency. These type of compounds present new challenges to in vitro systems used for clearance extrapolation. In this study, we compared the accuracy of clearance predictions of suspension culture to four different long-term stable in vitro liver models, including HepaRG sandwich culture, the Hµrel stochastic co-culture, the Hepatopac micropatterned co-culture (MPCC), and a micro-array spheroid culture. Hepatocytes in long-term stable systems remained viable and active over several days of incubation. Although intrinsic clearance values were generally high in suspension culture, clearance of low turnover compounds could frequently not be determined using this method. Metabolic activity and intrinsic clearance values from HepaRG cultures were low and, consequently, many compounds with low turnover did not show significant decline despite long incubation times. Similarly, stochastic co-cultures occasionally failed to show significant turnover for multiple low and medium turnover compounds. Among the different methods, MPCCs and spheroids provided the most consistent measurements. Notably, all culture methods resulted in underprediction of clearance; this could, however, be compensated for by regression correction. Combined, the results indicate that spheroid culture as well as the MPCC system provide adequate in vitro tools for human extrapolation for compounds with low metabolic turnover. SIGNIFICANCE STATEMENT: In this study, we compared suspension cultures, HepaRG sandwich cultures, the Hµrel liver stochastic co-cultures, the Hepatopac micropatterned co-cultures (MPCC), and micro-array spheroid cultures for low clearance determination and prediction. Overall, HepaRG and suspension cultures showed modest value for the low determination and prediction of clearance compounds. The micro-array spheroid culture resulted in the most robust clearance measurements, whereas using the MPCC resulted in the most accurate prediction for low clearance compounds.

Impact of Multiple Job Demands on Chinese University Teachers’ Turnover Intentions

Teacher turnover presents a significant challenge in education. Despite recognizing the importance of examining turnover intention to address this issue, exploring the interplay between multiple job demands and turnover intention is lacking within the framework of the job demands-resources theory. To fill this gap, the present study theoretically examined the relationship among personal resources (career adaptability), job burnout, turnover intention, and various job demands (teaching–research and work–family conflicts). Data were collected through an online survey of 875 Chinese university teachers, and the conceptual model was estimated using maximum likelihood estimation. The results revealed that work–family and teaching–research conflicts directly predict teachers’ turnover intentions and that job burnout mediates between multiple job demands and turnover intentions. Furthermore, career adaptability moderates the mediating role of burnout. These findings offer innovative ideas for mitigating and preventing faculty turnover intentions.

EPR studies of ferredoxin in spinach and cyanobacterial thylakoids related to photosystem I-driven NADP+ reduction.

Photosynthetic light-dependent reactions occur in thylakoid membranes where embedded proteins capture light energy and convert it to chemical energy in the form of ATP and NADPH for use in carbon fixation. One of these integral membrane proteins is Photosystem I (PSI). PSI catalyzes light-driven transmembrane electron transfer from plastocyanin (Pc) to oxidized ferredoxin (Fd). Electrons from reduced Fd are used by the enzyme ferredoxin-NADP+ reductase (FNR) for the reduction of NADP+ to NADPH. Fd and Pc are both small soluble proteins whereas the larger FNR enzyme is associated with the membrane. To investigate electron shuttling between these diffusible and embedded proteins, thylakoid photoreduction of NADP+ was studied. As isolated, both spinach and cyanobacterial thylakoids generate NADPH upon illumination without extraneous addition of Fd. These findings indicate that isolated thylakoids either (i) retain a "pool" of Fd which diffuses between PSI and membrane bound FNR or (ii) that a fraction of PSI is associated with Fd, with the membrane environment facilitating PSI-Fd-FNR interactions which enable multiple turnovers of the complex with a single Fd. To explore the functional association of Fd with PSI in thylakoids, electron paramagnetic resonance (EPR) spectroscopic methodologies were developed to distinguish the signals for the reduced Fe-S clusters of PSI and Fd. Temperature-dependent EPR studies show that the EPR signals of the terminal [4Fe-4S] cluster of PSI can be distinguished from the [2Fe-2S] cluster of Fd at > 30K. At 50K, the cw X-band EPR spectra of cyanobacterial and spinach thylakoids reduced with dithionite exhibit EPR signals of a [2Fe-2S] cluster with g-values gx = 2.05, gy = 1.96, and gz = 1.89, confirming that Fd is present in thylakoid preparations capable of NADP+ photoreduction. Quantitation of the EPR signals of P700+ and dithionite reduced Fd reveal that Fd is present at a ratio of ~ 1 Fd per PSI monomer in both spinach and cyanobacterial thylakoids. Light-driven electron transfer from PSI to Fd in thylakoids confirms Fd is functionally associated (< 0.4 Fd/PSI) with the acceptor end of PSI in isolated cyanobacterial thylakoids. These EPR experiments provide a benchmark for future spectroscopic characterization of Fd interactions involved in multistep relay of electrons following PSI charge separation in the context of photosynthetic thylakoid microenvironments.

Exquisite skeletons of a new transitional plesiosaur fill gap in the evolutionary history of plesiosauroids

Plesiosaurs are Mesozoic reptiles fully adapted to an aquatic lifestyle. Throughout their evolutionary history exceeding 140 million years plesiosaurs dispersed globally, achieved substantial diversity, occupied a variety of ecological niches, and experienced multiple faunal turnovers. Of those, the Early/Middle Jurassic transition event (∼175–171 Mya) has recently became of increased interest because it apparently profoundly affected all three major lineages of plesiosaurs. Once dominant Rhomaleosauridae started to vanish, while Pliosauridae and Plesiosauroidea diversified and gave rise to several clades that flourished for tens of millions of years. Here, we report exquisite, three-dimensionally preserved skeletons of a new plesiosaur from the Lower Jurassic of Germany. Franconiasaurus brevispinus gen. et sp. nov. lived during the late Toarcian (∼175 Mya), near the onset of the Early–Middle Jurassic turnover. Franconiasaurus displays an intriguing mixture of features, combining characters almost uniformly distributed among early plesiosaurs with those typically observed in later-diverging members of the clade. Phylogenetic analyses firmly place Franconiasaurus as the sister taxon to Cryptoclidia, bridging an evolutionary gap between early plesiosauroids, such as Plesiosaurus-like forms and microcleidids, and later-diverging representatives of the clade, such as cryptoclidids, leptocleidians, and elasmosaurids.

ITC-based kinetics assay for NIS synthetases.

NIS Synthetases are a widely distributed, novel superfamily of enzymes critical to stealth siderophore production-small molecules increasingly associated with virulence. Study of these enzymes for inhibition or utilization in biosynthesis of new antibiotics has been hindered by multiple kinetics assays utilizing different limiting reporters or relying on product dissociation as a precursor to signal. We present a label free, continuous readout assay optimized for NIS Synthetase systems utilizing an isothermal titration calorimetry instrument. This assay has been tested in an iterative system comparing multiple turnovers on a single substrate to a single bond formation event and is able to delineate these complex kinetics well. The ITC-based kinetic assay is the first label-free assay for the NIS field, which may allow for more detailed kinetic comparisons in the future, and may also have broader use for iterative enzymes in general.

Protocols for Myosin and Actin-Myosin Assays Using Rapid, Stopped-Flow Kinetics.

Fast transient kinetics using stopped-flow fluorimetry is now a powerful method for defining the ATPase cycle of myosin and its subfragments and has foundwide use in defining the difference between myosin isoforms, myosins carrying disease linked mutations, and the effect of small molecules on the ATPase cycle. Here the protocols for completing the classical assays of myosin and actin.myosin using the stopped-flow are described. The assays include ATP and ADP binding to myosin and actin.myosin, displacement of ADP from myosin and actin.myosin, and the cleavage of ATP to ADP and phosphate on myosin. Single and multiple turnover assays are also described.

Roadmap of electrons from donor side to the reaction center of photosynthetic purple bacteria with mutated cytochromes

In photosynthetic bacteria, the absorbed light drives the canonical cyclic electron transfer between the reaction center and the cytochrome bc1 complexes via the pools of mobile electron carriers. If kinetic or structural barriers hinder the participation of the bc1 complex in the cyclic flow of electrons, then the pools of mobile redox agents must supply the electrons for the multiple turnovers of the reaction center. These conditions were achieved by continuous high light excitation of intact cells of bacterial strains Rba. sphaeroides and Rvx. gelatinosus with depleted donor side cytochromes c2 (cycA) and tetraheme cytochrome subunit (pufC), respectively. The gradual oxidation by ferricyanide further reduced the availability of electron donors to pufC. Electron transfer through the reaction center was tracked by absorption change and by induction and relaxation of the fluorescence of the bacteriochlorophyll dimer. The rate constants of the electron transfer (~ 3 × 103 s‒1) from the mobile donors of Rvx. gelatinosus bound either to the RC (pufC) or to the tetraheme subunit (wild type) were similar. The electrons transferred through the reaction center dimer were supplied entirely by the donor pool; their number amounted to about 5 in wild type Rvx. gelatinosus and decreased to 1 in pufC oxidized by ferricyanide. Fluorescence yield was measured as a function of the oxidized fraction of the dimer and its complex shape reveals the contribution of two competing processes: the migration of the excitation energy among the photosynthetic units and the availability of electron donors to the oxidized dimer. The experimental results were simulated and rationalized by a simple kinetic model of the two-electron cycling of the acceptor side combined with aperiodic one-electron redox function of the donor side.

Cleaving Folded RNA with DNAzyme Agents.

Cleavage of biological mRNA by DNAzymes (Dz) has been proposed as a variation of oligonucleotide gene therapy (OGT). The design of Dz-based OGT agents includes computational prediction of two RNA-binding arms with low affinity (melting temperatures (Tm ) close to the reaction temperature of 37 °C) to avoid product inhibition and maintain high specificity. However, RNA cleavage might be limited by the RNA binding step especially if the RNA is folded in secondary structures. This calls for the need for two high-affinity RNA-binding arms. In this study, we optimized 10-23 Dz-based OGT agents for cleavage of three RNA targets with different folding energies under multiple turnover conditions in 2 mM Mg2+ at 37 °C. Unexpectedly, one optimized Dz had each RNA-binding arm with a Tm ≥60 °C, without suffering from product inhibition or low selectivity. This phenomenon was explained by the folding of the RNA cleavage products into stable secondary structures. This result suggests that Dz with long (high affinity) RNA-binding arms should not be excluded from the candidate pool for OGT agents. Rather, analysis of the cleavage products' folding should be included in Dz selection algorithms. The Dz optimization workflow should include testing with folded rather than linear RNA substrates.

Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development

RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.

Interaction of CYP3A4 with caffeine: First insights into multiple substrate binding

Human cytochrome P450 3A4 (CYP3A4) is a major drug-metabolizing enzyme that shows extreme substrate promiscuity. Moreover, its large and malleable active site can simultaneously accommodate several substrate molecules of the same or different nature, which may lead to cooperative binding and allosteric behavior. Due to difficulty of crystallization of CYP3A4-substrate complexes, it remains unknown how multiple substrates can arrange in the active site. We determined crystal structures of CYP3A4 bound to three and six molecules of caffeine, a psychoactive alkaloid serving as a substrate and modulator of CYP3A4. In the ternary complex, one caffeine binds to the active site suitably for C8-hydroxylation, most preferable for CYP3A4. In the senary complex, three caffeine molecules stack parallel to the heme with the proximal ligand poised for 3-N-demethylation. However, the caffeine stack forms extensive hydrophobic interactions that could preclude product dissociation and multiple turnovers. In both complexes, caffeine is also bound in the substrate channel and on the outer surface known as a peripheral site. At all sites, aromatic stacking with the caffeine ring(s) is likely a dominant interaction, while direct and water-mediated polar contacts provide additional stabilization for the substrate-bound complexes. Protein-ligand interactions via the active site R212, intrachannel T224, and peripheral F219 were experimentally confirmed, and the latter two residues were identified as important for caffeine association. Collectively, the structural, spectral, and mutagenesis data provide valuable insights on the ligand binding mechanism and help better understand how purine-based pharmaceuticals and other aromatic compounds could interact with CYP3A4 and mediate drug-drug interactions.

Revisiting the role of electron donors in lytic polysaccharide monooxygenase biochemistry.

The plant cell wall is rich in carbohydrates and many fungi and bacteria have evolved to take advantage of this carbon source. These carbohydrates are largely locked away in polysaccharides and so these organisms deploy a range of enzymes that can liberate individual sugars from these challenging substrates. Glycoside hydrolases (GHs) are the enzymes that are largely responsible for bringing about this sugar release; however, 12 years ago, a family of enzymes known as lytic polysaccharide monooxygenases (LPMOs) were also shown to be of key importance in this process. LPMOs are copper-dependent oxidative enzymes that can introduce chain breaks within polysaccharide chains. Initial work demonstrated that they could activate O2 to attack the substrate through a reaction that most likely required multiple electrons to be delivered to the enzyme. More recently, it has emerged that LPMO kinetics are significantly improved if H2O2 is supplied to the enzyme as a cosubstrate instead of O2. Only a single electron is required to activate an LPMO and H2O2 cosubstrate and the enzyme has been shown to catalyse multiple turnovers following the initial one-electron reduction of the copper, which is not possible if O2 is used. This has led to further studies of the roles of the electron donor in LPMO biochemistry, and this review aims to highlight recent findings in this area and consider how ongoing research could impact our understanding of the interplay between redox processes in nature.

Dynamics of Target DNA Binding and Cleavage by Staphylococcus aureus Cas9 as Revealed by High-Speed Atomic Force Microscopy.

Programmable DNA binding and cleavage by CRISPR-Cas9 has revolutionized the life sciences. However, the off-target cleavage observed in DNA sequences with some homology to the target still represents a major limitation for a more widespread use of Cas9 in biology and medicine. For this reason, complete understanding of the dynamics of DNA binding, interrogation and cleavage by Cas9 is crucial to improve the efficiency of genome editing. Here, we use high-speed atomic force microscopy (HS-AFM) to investigate Staphylococcus aureus Cas9 (SaCas9) and its dynamics of DNA binding and cleavage. Upon binding to single-guide RNA (sgRNA), SaCas9 forms a close bilobed structure that transiently and flexibly adopts also an open configuration. The SaCas9-mediated DNA cleavage is characterized by release of cleaved DNA and immediate dissociation, confirming that SaCas9 operates as a multiple turnover endonuclease. According to present knowledge, the process of searching for target DNA is mainly governed by three-dimensional diffusion. Independent HS-AFM experiments show a potential long-range attractive interaction between SaCas9-sgRNA and its target DNA. The interaction precedes the formation of the stable ternary complex and is observed exclusively in the vicinity of the protospacer-adjacent motif (PAM), up to distances of several nanometers. The direct visualization of the process by sequential topographic images suggests that SaCas9-sgRNA binds to the target sequence first, while the following binding of the PAM is accompanied by local DNA bending and formation of the stable complex. Collectively, our HS-AFM data reveal a potential and unexpected behavior of SaCas9 during the search for DNA targets.

On the Origin of the Rate Enhancement by a R‐(+)‐1‐(1‐Naphthyl)‐Ethylamine‐Modified Pd(111) Model Catalyst for Methyl Pyruvate Hydrogenation

AbstractChiral modifiers of heterogeneous catalysts can function as activity promotors to minimize the influence of unmodified sites on the enantiomeric excess to obtain highly enantioselective catalysts. However, the origin on this effect is not well understood. It is investigated using a model catalyst of R‐(+)‐1‐(1‐naphthyl)‐ethylamine (R‐1‐NEA)/Pd(111) for the hydrogenation of methyl pyruvate (MP) to methyl lactate (ML). The activity of the model catalyst remains constant for multiple turnovers. No rate enhancement is found for R‐1‐NEA coverages below ∼0.5 monolayer (ML), but a significant increase is found at R‐1‐NEA coverages of ∼0.75 ML, with a rate approximately twice that of the unmodified catalyst. This is investigated using infrared spectroscopy to distinguish between MP monomers and dimers. MP titration experiments with hydrogen show a half‐order hydrogen pressure dependence, with the monomer reacting at twice the rate as the dimer. It is found that the dimer is the most abundant species on clean Pd(111), but the ratio of monomers to dimers increases as the R‐1‐NEA coverage increases due to surface crowding. The monomeric species is also found to be more stable on the crowded surface than on clean Pd(111); the chiral modifier also serves to stabilize the reactant. Finally, this model nicely explains the unusual 1‐NEA‐covergae dependence of the reactivity.