Single-molecule Reactions Research Articles

Single-molecule determination of chemical equilibrium of DNA intercalation by electrical conductance.

We investigated a single-molecule reaction of DNA intercalation as an example of a bimolecular association reaction. Single-molecule conductance values of the product and reactant molecules adsorbed on an Au surface were measured to identify and quantify these molecules. The binding isotherm was constructed, and the association constant of the reaction was determined on a single-molecule basis.

A Single-Molecule Observation of Dichloroaurate(I) Binding to an Engineered Mycobacterium smegmatis porin A (MspA) Nanopore.

Gold(I) compounds are known to bind sulfur-containing proteins, forming the basis in the design of gold(I)-based drugs. However, the intrinsic molecular mechanism of the chemical reaction is easily hidden when monitored in ensemble. We have previously demonstrated that Mycobacterium smegmatis porin A (MspA) can be engineered (MspA-M) to contain a specialized nanoreactor to probe chemical reactions involving tetrachloroaurate(III). Here, we provide further investigations of coordination interactions between dichloroaurate(I) and MspA-M. Gold compounds of different coordination geometry and valence states are as well probed and evaluated, demonstrating the generality of MspA-M. With single-molecule evidence, MspA-M demonstrates a preference for dichloroaurate(I) than tetrachloroaurate(III), an observation in a single molecule that has never been reported. By counting the maximum number of simultaneous ion bindings, the narrowly confined pore restriction also efficiently distinguishes dichloroaurate(I) and tetrachloroaurate(III) according to their differences in geometry or size. The above demonstration complemented a previous study by demonstrating other possible gold-based single-molecule chemical reactions observable by MspA. These observations bring insights in the understanding of gold-based coordination chemistry in a nanoscale.

STM investigations of single-molecule chemical reactions

STM investigations of single-molecule chemical reactions

Single-molecule enzymatic reaction dynamics and mechanisms of GPX3 and TRXh9 from Arabidopsis thaliana.

Glutathione peroxidases (GPXs) regulate the levels of reactive oxygen species in cells and tissues. During the redox cycling, the plant GPX is regenerated by thioredoxins (TRXs) as reductant rather than glutathione as the electron donor. However, the direct experimental observation on the interaction dynamics between GPXs and TRXs has not been reported, and the redox mechanism is unclear. In this work, the protein interactions between oxidized AtGPX3 and reduced AtTRXh9 have been studied using single-molecule fluorescence resonance energy transfer (smFRET). The obtained results indicate there are four processes in these two protein interaction, including biological recognition, binding, intermediate and unbinding state. Two enzymatic reaction intermediate states have been identified in the dissociation of AtGPX3-AtTRXh9 complex from binding to unbinding state, suggesting two types of interaction pathways and intermediate complexes. In particular, the dynamical study reveals that the redox reaction between oxidized AtGPX3 and reduced AtTRXh9 is realized through the forming and breaking of disulfide bonds via the active sites of Cys4 and Cys57 in AtTRXh9. These findings are of significant for deep understanding the redox reaction and mechanism between GPXs and TRXs enzymes, and studying other protein dynamics at single-molecule level.

Real-time detection of single-molecule reaction by plasmon-enhanced spectroscopy.

Determining structural transformations of single molecules (SMs) is an important fundamental scientific endeavor. Optical spectroscopies are the dominant tools used to unravel the physical and chemical features of individual molecules and have substantially contributed to surface science and biotechnology. In particular, Raman spectroscopy can identify reaction intermediates and reveal underlying reaction mechanisms; however, SM Raman experiments are subject to intrinsically weak signal intensities and considerable signal attenuation within the spectral dispersion systems of the spectrometer. Here, to monitor the structural transformation of an SM on the millisecond time scale, a plasmonic nanocavity substrate has been used to enable Raman vibrational and fluorescence spectral signals to be simultaneously collected and correlated, which thus allows a detection of photo-induced bond cleavage between the xanthene and phenyl group of a single rhodamine B isothiocyanate molecule in real time. This technique provides a novel method for investigating light-matter interactions and chemical reactions at the SM level.

Development of a single-stranded DNA-binding protein fluorescent fusion toolbox.

Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

(Invited) Electrochemistry Confined Nanopore: Design and Application in Single Molecule Sensing

Single-molecule sensing is key to deep understanding of elemental biological processes and complex bioreactions. Nanopore-based single-molecule method, a unique technique based on the modulation in ionic current, provides a high temporal-spatial resolution for single molecule sensing. To achieve a high selectivity and sensitivity of biological nanopore, the modification of specific group presenting in the whole region of Aerolysin nanopore was successfully designed, and every single molecule forms a whole sensing interface.1,2 As a reasonable model, a single membrane protein which is assembled from several monomers is regarded as a single molecule interface Therefore, the side chain of every single amino acid groups could be designed respectively by single site mutation.3 The single molecule interface has been achieved in biological nanopore sensors. A single membrane protein molecule could be regarded as a single molecule interface. Single molecule interface facilities the design of single sensing zone which ensures high spatial- and temporal resolution towards single molecule analysis. The single-site modification along the single molecule interface manipulates each site of interface for the precise responses which meet the multiple sensing requirement. More importantly, single molecule interface provides a confinement for the single molecule reaction which offers the possibility to study reaction kinetics in high temporal resolution. These advances provide a new concept in designing advanced sensors. Moreover, by combining with other single molecule methods such as fluorescence or plasmonic technology, it is possible to construct an integrated single molecule interface for multi-element readouts, which is expected to further provide the rich structural information of a single molecule. Figure 1. Single-molecule music. A single-molecule interface likes a tuba. As each molecule flows through the single-molecule interface, one can control the ‘button’ along the ‘tuba’ to organize a beautiful rhythm for single-molecule music. Keywords: Nanopore; Aerolysin nanopore, Single biomolecule interface, Site Mutation, Single molecule sensing.

Nanopore Confined Space Toward Precision Asymmetric Synthesis of Single Molecules

Asymmetric synthesis converts the compound into unequal proportions of compounds that differs in the dissymmetry of their structures at the affected center. One of the challenges in asymmetric synthesis is the controllable stepwise adding of reactive group one-by-one into the affected center of the compound which is essential to achieve the unequal proportions during the asymmetric synthesis. The nanopore-based interface provides the possibility to achieve the single-molecule asymmetric synthesis, in which a single pore-forming protein molecule unequally forms the covalent bonds with the reactive group. However, the confined effects for modulating of this nanopore-based asymmetric synthesis are still unclear. In order to study both the steric effect and the substituent effect of the reactant for a single molecule synthesis inside an K238C aerolysin (AeL) nanoreactor, we selected three isomers of mercaptobenzoic acid (MBA) namely as 2-mercaptobenzoic acid (2-MBA), 3-mercaptobenzoic acid (3-MBA) and 4-mercaptobenzoic acid (4-MBA) as the reactant. The result shows that geometric isomers of MBA molecule can respond selectively to the multiple active sites of K238C AeL, leading to the asymmetric synthesis of single molecules. Consistent with the steric geometry of MBA molecules, the dominating number of multiple-disulfide-bond formations between MBA and seven cysteines at K238C AeL site is 5, 1, and 1 for 2-MBA, 3-MBA and 4-MBA, respectively. Moreover, both the steric effects and nucleophilic effects provide the 2-MBA the strongest affinity towards the K238C AeL. The further voltage dependent experiments suggest that a high applied voltage could induce a fast association and dissociation of the disulfide bonds. The results demonstrated that the asymmetric single-molecule reaction could be controlled through the modulation of the steric effects, substation effect and the applied voltage. Further, it is possible to achieve precious single-molecule synthesis with the array of aerolysin nanoreactors.

Quantifying the flux as the driving force for nonequilibrium dynamics and thermodynamics in non-Michaelis–Menten enzyme kinetics

The driving force for active physical and biological systems is determined by both the underlying landscape and nonequilibrium curl flux. While landscape can be experimentally quantified from the histograms of the collected real-time trajectories of the observables, quantifying the experimental flux remains challenging. In this work, we studied the single-molecule enzyme dynamics of horseradish peroxidase with dihydrorhodamine 123 and hydrogen peroxide (H2O2) as substrates. Surprisingly, significant deviations in the kinetics from the conventional Michaelis-Menten reaction rate were observed. Instead of a linear relationship between the inverse of the enzyme kinetic rate and the inverse of substrate concentration, a nonlinear relationship between the two emerged. We identified nonequilibrium flux as the origin of such non-Michaelis-Menten enzyme rate behavior. Furthermore, we quantified the nonequilibrium flux from experimentally obtained fluorescence correlation spectroscopy data and showed this flux to led to the deviations from the Michaelis-Menten kinetics. We also identified and quantified the nonequilibrium thermodynamic driving forces as the chemical potential and entropy production for such non-Michaelis-Menten kinetics. Moreover, through isothermal titration calorimetry measurements, we identified and quantified the origin of both nonequilibrium dynamic and thermodynamic driving forces as the heat absorbed (energy input) into the enzyme reaction system. Furthermore, we showed that the nonequilibrium driving forces led to time irreversibility through the difference between the forward and backward directions in time and high-order correlations were associated with the deviations from Michaelis-Menten kinetics. This study provided a general framework for experimentally quantifying the dynamic and thermodynamic driving forces for nonequilibrium systems.

Single-Molecule Surface-Enhanced Raman Scattering as a Probe of Single-Molecule Surface Reactions: Promises and Current Challenges.

The initial observations of surface-enhanced Raman scattering (SERS) from individual molecules (single-molecule SERS, SMSERS) have triggered ever more detailed mechanistic studies on the SERS process. The studies not only reveal the existence of extremely enhanced and confined fields at the gaps of Ag or Au nanoparticles but also reveal that the spatial, spectral, and temporal behaviors of the SMSERS signal critically depend on many factors, including plasmon resonances of nanostructures, diffusion (lateral and orientational) of molecules, molecular electronic resonances, and metal-molecule charge transfers. SMSERS spectra, with their molecular vibrational fingerprints, should in principle provide molecule-specific information on individual molecules in a way that any other existing single-molecule detection method (such as the ones based on fluorescence, mechanical forces, or electrical currents) cannot. Therefore, by following the spectro-temporal evolution of SMSERS signals of reacting molecules, one should be able to follow chemical reaction events of individual molecules without any additional labels. Despite such potential, however, real applications of SMSERS for single-molecule chemistry and analytical chemistry are scarce. In this Account, we discuss whether and how we can use SMSERS to monitor single-molecule chemical kinetics. The central problem lies in the experimental challenges of separately characterizing and controlling various sources of fluctuations and spatial variations in such a way that we can extract only the chemically relevant information from time-varying SMSERS signals. This Account is organized as follows. First, we outline the standard theory of SMSERS, providing an essential guide for identifying sources of spatial heterogeneity and temporal fluctuations in SMSERS signals. Second, we show how single-molecule reaction events of surface-immobilized reactants manifest themselves in experimental SMSERS trajectories. Comparison of the reactive SMSERS data (magnitudes and frequencies of discrete transitions) and the predictions of SMSERS models also allow us to assess how faithfully the SMSERS models represent reality. Third, we show how SMSERS spectral features can be used to discover new reaction intermediates and to interrogate metal-molecule electronic interactions. Finally, we propose possible improvements in experimental design (including nanogap structures and molecular systems) to make SMSERS applicable to a broader range of chemical reactions occurring under ambient conditions. The specific examples discussed in this Account are centered around the single-molecule photochemistry of 4-nitrobenzenethiol on metals, but the conclusions drawn from each example are generally applicable to any reaction system involving small organic molecules.

Systems NMR: single-sample quantification of RNA, proteins and metabolites for biomolecular network analysis.

Cellular behavior is controlled by the interplay of diverse biomolecules. Most experimental methods, however, can monitor only a single molecule class or reaction type at a time. We developed an in vitro Nuclear Magnetic Resonance spectroscopy (NMR) approach, which permitted dynamic quantification of an entire “heterotypic” network – simultaneously monitoring three distinct molecule classes (metabolites, proteins, RNA) and all elementary reaction types (bimolecular interactions, catalysis, unimolecular changes). Focusing on an 8-reaction co-transcriptional RNA folding network, in a single sample we recorded over 35 time-points with over 170 observables each, and accurately determined 5 core reaction constants in multiplex. This reconstruction revealed unexpected cross-talk between the different reactions. We further observed dynamic phase-separation in a system of five distinct RNA binding domains in the course of the RNA transcription reaction. Our Systems NMR approach provides a deeper understanding of biological network dynamics by combining the dynamic resolution of biochemical assays and the multiplexing ability of “omics”.

Nanopore-based sensing interface for single molecule electrochemistry

Nanopore techniques are experiencing a gallop since it walked out the notebook and show its charm on the science arena. The nanoscale pore offers a single-molecule resolution with a label-free and high-selective manner for the research of molecular structures, molecular dynamics, single-molecule reactions and for a variety of applications in biophysics and bionanotechnology. In this review, we introduce the construction of three types of nanopore platforms along with the latest progress in DNA sensing, structure and dynamics analysis of peptides/proteins, and the detection of redox reactions with new sensing mechanisms. Then, we depict nanopore data processing methods which provide an insight of data mining under the background of big data. We could fully expect the great impact of nanopore techniques on not only for DNA sequencing and sensing applications, but also in protein sequencing and clinical diagnostics.

Abstract 3780: Spatially resolved transcriptional patterns of PARP inhibitor-resistant high grade serous ovarian cancer PDX tissue

Abstract Ovarian cancer is the leading cause of mortality among gynecological malignancies; 70% of patients who initially respond to therapy eventually relapse and die. Within highly heterogeneous tumors, resistance originates from a small number of cells that express beneficial genes and receive favorable cues from their microenvironment. Understanding the mechanisms of drug resistance therefore requires the knowledge of which cell types compose the tumor, which genes they express, and where they are located respective to other cells and tissue landmarks. To this end, we have explored cell type and transcriptional heterogeneity in homologous recombination deficient patient-derived xenograft (PDX) models of high grade serous ovarian cancer (HGSOC) treated with the PARP inhibitor talazoparib. We are using single-cell RNA sequencing (scRNA-seq) to identify the discrete cell types present in drug naïve and drug resistant PDX tissue and to characterize the transcriptional changes driving the resistant phenotype. We are using single-molecule hybridization chain reaction (smHCR) to fluorescently label mRNAs and diSPIM light sheet microscopy to visualize fluorescently labeled mRNAs within large volumes of PDX tissue. We are currently developing computational strategies to deconvolve the 3D spatial organization of PDX cell types and to understand where and when resistance genes are expressed. This integrated scRNA seq and smHCR microscopy framework has allowed us to identify rare cell populations within HGSOC PDXs and to visualize their 3D localization relative to other cell types and tissue features, and it has the potential to elucidate how transcription of key genes supports transition to a resistant state. The insights gained from single cell transcriptional and 3D spatial profiles has the potential to greatly advance our understanding of how the 3D tumor tissue microenvironment allows and encourages rare cells with pre-resistant transcriptional programs to escape PARP inhibitor treatment. Further, this research has the potential to be predictive of disease progression and to suggest additional therapeutic targets that could be exploited as part of novel and/or personalized combination therapy regimens. An additional advantage is that this approach could be generalizable to many types of cancer to characterize the interplay between tissue organization and diverse processes such as tumor evolution, metastasis, and drug resistance. Citation Format: Benjamin King, Elke Van Oudenhove, Selim Misirlioglu, Ernesto Arostegui Fernandez, Douglas Levine, Timothee Lionnet. Spatially resolved transcriptional patterns of PARP inhibitor-resistant high grade serous ovarian cancer PDX tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3780.

A Single-Molecule Chemical Reaction Studied by High-Resolution Atomic Force Microscopy and Scanning Tunneling Microscopy Induced Light Emission.

Atomic force microscopy (AFM) as well as scanning tunneling microscopy induced light emission (STM-LE) are, each on their own, powerful tools used to investigate a large variety of properties of single molecules adsorbed on a surface. However, accessing both structural information by AFM as well as optical information by STM-LE on the same molecule so far remains elusive. We present a combined high-resolution AFM and STM-LE study on single metal-oxide phthalocyanines. Using atomic manipulation, the molecules can be deliberately reduced. We demonstrate structure elucidation and adsorption geometry determination of single molecules with atomic resolution combined with optical characterization by STM-LE and the possibility of investigating the change in a molecule’s exciton emission intensity by a chemical reaction.

Electric field–induced selective catalysis of single-molecule reaction

Oriented external electric fields (OEEFs) offer a unique chance to tune catalytic selectivity by orienting the alignment of the electric field along the axis of the activated bond for a specific chemical reaction; however, they remain a key experimental challenge. Here, we experimentally and theoretically investigated the OEEF-induced selective catalysis in a two-step cascade reaction of the Diels-Alder addition followed by an aromatization process. Characterized by the mechanically controllable break junction (MCBJ) technique in the nanogap and confirmed by nuclear magnetic resonance (NMR) in bottles, OEEFs are found to selectively catalyze the aromatization reaction by one order of magnitude owing to the alignment of the electric field on the reaction axis. Meanwhile, the Diels-Alder reaction remained unchanged since its reaction axis is orthogonal to the electric fields. This orientation-selective catalytic effect of OEEFs reveals that chemical reactions can be selectively manipulated through the elegant alignment between the electric fields and the reaction axis.

Revealing the Dynamics of Single-Molecule Reactions in a Single-Molecule Nanoreactor

The dynamics of bond breaking and formation at the single-molecule level are of both fundamental importance and practical significance. Tremendous efforts have been made over the last decade toward the visualization of such transient phenomenon, with quite a few amazing single-molecule techniques being designed and developed [1-3]. However, it remains a major challenge to realize the truly single-molecule (cascade) reactions largely due to the stochastic nature of, e.g. molecular motions. Inspired by the enzymatic biosynthesis, the authors consider that a nanoscale confined space, or nanoreactor, must be essential to the highly ordered single-molecule reactions. Moreover, an ideal nanoreactor also needs to possess a well defined structure that is of comparable size to the individual reactant molecules. Hence, the rationally-mutated biological nanopores may be applied as a new brand of single-molecule nanoreactors, where the entry of reactants and exit of products are voltage-driven. Herein, a mutant areolysin was adopted to study the unique confinement induced selectivity of nanopore reactor. In addition to regio- or stereo-selectivity, it is hypothesized that the mismatch between the bond formation kinetics of reactant molecules and their translocation speeds can offer an extra level of control on reaction selectivity. To prove this concept, the reaction dynamics of three constitutional isomers, 2-, 3-, and 4-mercaptobenzoic acid, at seven identical reaction sites (i.e. at the same position on each monomer) were examined in this work. Surprisingly, those tiny structural variations of three isomers could be easily distinguished by not only the different current blockades caused by each tethered molecule, but also the most frequent and prolonged current levels as well. This observation shed light on the transient dynamics of single-molecule reactions within a single-molecule nanoreactor, providing a crucial first step to the ultimate objective of 100% yield of targeted product at ambient conditions. Scheme 1 Illustration of the single-molecule nanoreactor presented in this work, the mutant aerolysin nanopore. Keywords: Single-Molecule Reactions, Single-Molecule Nanoreactor, Mutant Aerolysin Nanopore, Confinement Induced Selectivity, Electroanalytical Chemistry

(Invited) High-Precision Analyses of True Single-Molecule Chemistry using Nanopore Methods

Measurement of single-molecule reactions can elucidate microscopic mechanisms that are often hidden from ensemble analysis. This presentation report the acid–base titration of a single DNA duplex confined within the wild-type α-hemolysin (α-HL) nanopore for up to 3 hours. Analysis of the pH-dependent kinetics of >100,000 base flipping events of a single GC base pair using a hidden Markov kinetic model demonstrates that protonation/deprotonation occurs while the base pair is in the intra-helical state. Our experiments correspond to the longest single-molecule chemical reaction kinetic measurements performed using a nanopore.

Theoretical study of the conditional non-monotonic off rate dependence of catalytic reaction rates in single enzymes in the presence of conformational fluctuations

In ordinary Michaelis–Menten kinetics of enzyme-catalyzed reactions, the enzymatic velocity decreases with the increase in the off dissociation rate constant of the enzyme-substrate complex. In this work, we study the role of the off dissociation rate constants in single-molecule enzymatic reactions in which both the free enzyme and the enzyme-substrate complex undergo slow conformational fluctuations. By applying our theoretical formalism, we calculate the enzymatic velocity and show that there can be an increase in the reaction velocity with the off rate constant dependence which is not very intuitive. From our analytical calculations, we can establish a region of the parameter space and subsequently explore a range of substrate concentration where the enzymatic velocity is accelerated with an increase in the off rate constant. Further, we study the role of the substrate unbinding rate constants on the randomness parameter, which is a measure of the dynamic disorder present in the system.

Near-Field Spectral Response of Optically Excited Scanning Tunneling Microscope Junctions Probed by Single-Molecule Action Spectroscopy.

The near-field spectral response of metallic nanocavities is a key characteristic in plasmon-assisted photophysical and photochemical processes. Here, we show that the near-field spectral response of an optically excited plasmonic scanning tunneling microscope (STM) junction can be probed by single-molecule reactions that serve as a nanoscale sensor detecting the local field intensity. Near-field action spectroscopy for the cis ↔ cis tautomerization of porphycene on a Cu(110) surface reveals that the field enhancement in the STM junction largely depends on microscopic structures not only on the tip apex, but also on its shaft. Using nanofabrication of Au tips with focused ion beam, we show that the spectral response is strongly modulated through the interference between the localized surface plasmon in the junction and propagating surface plasmon polariton generated on the shaft. Furthermore, it is demonstrated that the near-field spectral response can be manipulated by precisely shaping the tip shaft.

Toward Precision Measurement and Manipulation of Single-Molecule Reactions by a Confined Space.

All chemical reactions can be divided into a series of single molecule reactions (SMRs), the elementary steps that involve only isomerization of, dissociation from, and addition to an individual molecule. Analyzing SMRs is of paramount importance to identify the intrinsic molecular mechanism of a complex chemical reaction, which is otherwise implausible to reveal in an ensemble fashion, owing to the significant static and dynamic heterogeneity of real-world chemical systems. The single-molecule measurement and manipulation methods developed recently are playing an increasingly irreplaceable role to detect and recognize short-lived intermediates, visualize their transient existence, and determinate the kinetics and dynamics of single bond breaking and formation. Notably, none of the above SMRs characterizations can be realized without the aid of a confined space. Therefore, this Review aims to highlight the recent progress in the development of confined space enabled single-molecule sensing, imaging, and tuning methods to study chemical reactions. Future prospects of SMRs research are also included, including a push toward the physical limit on transduction of information to signals and vice versa, transmission and recording of signals, computational modeling and simulation, and rational design of a confined space for precise SMRs.