Single-molecule Observation Research Articles

Probability of Double-Strand Breaks in Genome-Sized DNA by γ-Ray Decreases Markedly as the DNA Concentration Increases

DNA double-strand breaks (DSBs) present a serious threat to all living things and thus many quantitative studies have been carried out. However, there is no established hypothesis that accounts for the statistics of their production, in particular, the number of DSBs per base pair per unit Gy, P1, which is the most important parameter for evaluating the degree of risk posed by DSBs. In fact, the reported values scatter by three orders of magnitude [1,2]. This scattering is partly attributable to varying DNA concentrations. So, we evaluate DSBs caused by γ-ray with giant DNA (166 kbp) for a wide region of DNA concentrations using high sensitive single-molecule observation [3]. We find that P1 is inversely proportional to the concentration above a certain threshold. We give a theoretical interpretation in terms of attack of reactive species upon DNA molecules. Our theoretical model suggests the importance of the size of DNA and its characteristics as semiflexible polymers.[1] Sutherland B M et al., PNAS 97: 103-108 (2000).[2] Chen C-Z et al., Electrophoresis 10: 318-326 (1989).[3] Yoshikawa Y et al., Chem. Phys. Lett. 456: 80-83 (2008).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Single-molecule observation of the K+-induced switching of valinomycin within a template network

The K(+)-induced switching of valinomycin has been studied using a molecular template formed by an aromatic oligoamide macrocycle at the liquid/solid interface by scanning tunneling microscopy (STM). Individual valinomycin and its K(+) complex can be identified and resolved in the molecular template, and the high-resolution STM images of valinomycin and its K(+) complex show triangle-like and cyclic structural characteristics, respectively.

Single-Molecule Observation of Viral DNA Targeting by CRISPR/Cas Immune Systems

Bacteria and archaea maintain a history of viral infections by integrating small fragments of foreign DNA into specialized genomic loci called clustered regularly interspaced short palindromic repeats (CRISPRs). An adaptive immune response is triggered during subsequent infections by CRISPR-derived RNAs (crRNAs), which function together with CRISPR-associated (Cas) proteins to identify and destroy complementary viral DNA sequences. DNA targeting by crRNA/Cas ribonucleoprotein surveillance complexes proceeds via RNA-DNA base-pairing interactions and requires melting of the double-stranded substrate, as well as protein-mediated recognition of a proximal DNA sequence motif to discriminate self from non-self. How these complexes find short target sequences within the larger context of genomic DNA is unknown, and prior studies have been limited by their use of oligonucleotide substrates and dependence on bulk electrophoretic mobility shift assays that obscure all dynamics of the search process. To gain deeper insights into this critical step of CRISPR/Cas based immunity, we are using fluorescence microscopy to visualize single crRNA/Cas complexes on nanofabricated curtains of viral DNA in real-time. These experiments enable direct observation of the kinetics and position-dependence of DNA binding, and have revealed the mechanism by which phylogenetically distinct surveillance complexes use sequence information in the crRNA to locate complementary DNA targets. Ongoing work is aimed at understanding how this recognition event promotes destruction of viral DNA by dedicated Cas nucleases.

Single-Molecule Observation of the Induction of k-Turn RNA Structure on Binding L7Ae Protein

The k-turn is a commonly occurring structural motif that introduces a tight kink into duplex RNA. In free solution, it can exist in an extended form, or by folding into the kinked structure. Binding of proteins including the L7Ae family can induce the formation of the kinked geometry, raising the question of whether this occurs by passive selection of the kinked structure, or a more active process in which the protein manipulates the RNA structure. We have devised a single-molecule experiment whereby immobilized L7Ae protein binds Cy3-Cy5-labeled RNA from free solution. We find that all bound RNA is in the kinked geometry, with no evidence for transitions to an extended form at the millisecond timescale of the camera. Furthermore, real-time binding experiments provide no evidence for a more extended intermediate even at the earliest times, at a time resolution of 16 ms. The data support a passive conformational selection model by which the protein selects a fraction of RNA that is already in the kinked conformation, thereby drawing the equilibrium into this form.

What precision‐protein‐tuning and nano‐resolved single molecule sciences can do for each other

While innovations in modern microscopy, spectroscopy, and nanoscopy techniques have made single molecule observation a standard in many laboratories, the actual design of meaningful fluorescence reporter systems now hinders major scientific breakthroughs. Even though the field of chemical biology is supercharging the fluorescence toolbox, surprisingly few strategies exist that make the transition from model systems to biologically relevant applications. At the same time, the number of microscopy techniques is growing dramatically. We explain our view on how the impact of modern technologies is influenced not only by further hard- and software developments, but also by the availability and suitability of protein-engineering tools. We identify how the largely independent research fields of chemical biology and fluorescence nanoscopy can influence each other to synergistically drive future technology that can visualize the localization, structure, and dynamics of molecular function without constraints.

First steps to rafts?

Single-molecule observations reveal that lipid- and protein-based interactions jointly contribute to the interactions among glycosylphosphatidylinositol-anchored proteins in membranes. Understanding these interactions will help to refine long-evolving (and still debated) models of 'raft' domains in biological membranes.

Mechanical Coupling between Myosin Molecules Causes Differences between Ensemble and Single-Molecule Measurements

In contracting muscle, individual myosin molecules function as part of a large ensemble, hydrolyzing ATP to power the relative sliding of actin filaments. The technological advances that have enabled direct observation and manipulation of single molecules, including recent experiments that have explored myosin’s force-dependent properties, provide detailed insight into the kinetics of myosin’s mechanochemical interaction with actin. However, it has been difficult to reconcile these single-molecule observations with the behavior of myosin in an ensemble. Here, using a combination of simulations and theory, we show that the kinetic mechanism derived from single-molecule experiments describes ensemble behavior; but the connection between single molecule and ensemble is complex. In particular, even in the absence of external force, internal forces generated between myosin molecules in a large ensemble accelerate ADP release and increase how far actin moves during a single myosin attachment. These myosin-induced changes in strong binding lifetime and attachment distance cause measurable properties, such as actin speed in the motility assay, to vary depending on the number of myosin molecules interacting with an actin filament. This ensemble-size effect challenges the simple detachment limited model of motility, because even when motility speed is limited by ADP release, increasing attachment rate can increase motility speed.

Mass Spectrometric Screening of Ligands with Lower Off-Rate from a Clicked-Based Pooled Library

This paper describes a convenient screening method using ion trap electrospray ionization mass spectrometry to classify ligands to a target molecule in terms of kinetic parameters. We demonstrate this method in the screening of ligands to a hexahistidine tag from a pooled library synthesized by click chemistry. The ion trap mass spectrometry analysis revealed that higher stabilities of ligand-target complexes in the gas phase were related to lower dissociation rate constants, i.e., off-rates in solution. Finally, we prepared a fluorescent probe utilizing the ligand with lowest off-rate and succeeded in performing single molecule observations of hexahistidine-tagged myosin V walking on actin filaments.

From Single Molecule Fluctuations to Muscle Contraction: A Brownian Model of A.F. Huxley's Hypotheses

Muscular force generation in response to external stimuli is the result of thermally fluctuating, cyclical interactions between myosin and actin, which together form the actomyosin complex. Normally, these fluctuations are modelled using transition rate functions that are based on muscle fiber behaviour, in a phenomenological fashion. However, such a basis reduces the predictive power of these models. As an alternative, we propose a model which uses direct single molecule observations of actomyosin fluctuations reported in the literature. We precisely estimate the actomyosin potential bias and use diffusion theory to obtain a Brownian ratchet model that reproduces the complete cross-bridge cycle. The model is validated by simulating several macroscopic experimental conditions, while its interpretation is compatible with two different force-generating scenarios.

Molecular Mechanism of ATP Hydrolysis in F1-ATPase Revealed by Molecular Simulations and Single-Molecule Observations

Enzymatic hydrolysis of nucleotide triphosphate (NTP) plays a pivotal role in protein functions. In spite of its biological significance, however, the chemistry of the hydrolysis catalysis remains obscure because of the complex nature of the reaction. Here we report a study of the molecular mechanism of hydrolysis of adenosine triphosphate (ATP) in F(1)-ATPase, an ATP-driven rotary motor protein. Molecular simulations predicted and single-molecule observation experiments verified that the rate-determining step (RDS) is proton transfer (PT) from the lytic water molecule, which is strongly activated by a metaphosphate generated by a preceding P(γ)-O(β) bond dissociation (POD). Catalysis of the POD that triggers the chain activation of the PT is fulfilled by hydrogen bonds between Walker motif A and an arginine finger, which commonly exist in many NTPases. The reaction mechanism unveiled here indicates that the protein can regulate the enzymatic activity for the function in both the POD and PT steps despite the fact that the RDS is the PT step.

Phototriggered DNA Complexation and Compaction Using Poly(vinyl alcohol) Carrying a Malachite Green Moiety

Photoinduced DNA compaction was performed using the interaction of DNA with a photoresponsive random copolymer of poly(vinyl alcohol) carrying a malachite green moiety (PVAMG). Although PVAMG does not have any affinity for DNA under dark conditions, it undergoes photoionization upon exposure to UV light, consequently resulting in a cationic binding site for DNA. Electrophoresis results demonstrated that irradiation of PVAMG retarded the DNA bands due to their complexation, whereas the bands remained unchanged under dark conditions. The binding of PVAMG to DNA occurs at a cationic site/DNA phosphate ratio of approximately 0.036. Single-molecule observations of DNA by fluorescence microscopy revealed that irradiation of PVAMG induced a coil-globule transition in the DNA molecule. Complete compaction of DNA has been accomplished at a cationic site/DNA phosphate ratio >8.0, indicating that PVAMG offers an effective system to photochemically trigger DNA compaction.

Single-molecule observation of helix staggering, sliding, and coiled coil misfolding

The biological functions of coiled coils generally depend on efficient folding and perfect pairing of their α-helices. Dynamic changes in the helical registry that lead to staggered helices have only been proposed for a few special systems and not found in generic coiled coils. Here, we report our observations of multiple staggered helical structures of two canonical coiled coils. The partially folded structures are formed predominantly by coiled coil misfolding and occasionally by helix sliding. Using high-resolution optical tweezers, we characterized their energies and transition kinetics at a single-molecule level. The staggered states occur less than 2% of the time and about 0.1% of the time at zero force. We conclude that dynamic changes in helical registry may be a general property of coiled coils. Our findings should have broad and unique implications in functions and dysfunctions of proteins containing coiled coils.

Visualization of Subunit-Specific Delivery of Glutamate Receptors to Postsynaptic Membrane during Hippocampal Long-Term Potentiation

An increase in the number of AMPA-type glutamate receptors (AMPARs) is critical for long-term potentiation (LTP), synaptic plasticity regarded as a basal mechanism of learning and memory. However, when and how each type of AMPAR reaches the postsynaptic membrane remain unclear. We have developed experimental methods to form postsynaptic-like membrane (PSLM) on a glass surface to precisely visualize the location and movement of receptors. We observed fluorescence-labeled AMPAR subunits (GluA1-3) around PSLM with total internal reflection fluorescence microscopy. The increases of GluA1, 2, and 3 in PSLM showed different time courses after LTP induction. GluA1 increased first, and was exocytosed to the periphery of PSLM soon after LTP induction. GluA2 and GluA3 initially decreased, and then increased. Exocytosis of GluA2 and GluA3 occurred primarily in non-PSLM, and later than exocytosis of GluA1. Thus, GluA1-3 appear to increase in the postsynaptic membrane through distinct pathways during LTP.

High-throughput single-molecule analysis of DNA-protein interactions by tethered particle motion.

Tethered particle motion (TPM) monitors the variations in the effective length of a single DNA molecule by tracking the Brownian motion of a bead tethered to a support by the DNA molecule. Providing information about DNA conformations in real time, this technique enables a refined characterization of DNA–protein interactions. To increase the output of this powerful but time-consuming single-molecule assay, we have developed a biochip for the simultaneous acquisition of data from more than 500 single DNA molecules. The controlled positioning of individual DNA molecules is achieved by self-assembly on nanoscale arrays fabricated through a standard microcontact printing method. We demonstrate the capacity of our biochip to study biological processes by applying our method to explore the enzymatic activity of the T7 bacteriophage exonuclease. Our single molecule observations shed new light on its behaviour that had only been examined in bulk assays previously and, more specifically, on its processivity.

Sequence-Selective Single-Molecule Alkylation with a Pyrrole–Imidazole Polyamide Visualized in a DNA Nanoscaffold

We demonstrate a novel strategy for visualizing sequence-selective alkylation of target double-stranded DNA (dsDNA) using a synthetic pyrrole-imidazole (PI) polyamide in a designed DNA origami scaffold. Doubly functionalized PI polyamide was designed by introduction of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site in the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation by functionalized PI polyamide using atomic force microscopy (AFM), the target position in the dsDNA (∼200 base pairs) was alkylated and then visualized by labeling with streptavidin. Newly designed DNA origami scaffold named "five-well DNA frame" carrying five different dsDNA sequences in its cavities was used for the detailed analysis of the sequence-selectivity and alkylation. The 64-mer dsDNAs were introduced to five individual wells, in which target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences. The fully matched sequence was alkylated with 88% selectivity over other mismatched sequences. In addition, the PI polyamide failed to attach to the target sequence lacking the alkylation site after washing and streptavidin treatment. Therefore, the PI polyamide discriminated the one mismatched nucleotide at the single-molecule level, and alkylation anchored the PI polyamide to the target dsDNA.

Mechanism of Transcription Initiation at an Activator-Dependent Promoter Defined by Single-Molecule Observation

Understanding the pathway and kinetic mechanisms of transcription initiation is essential for quantitative understanding of gene regulation, but initiation is a multistep process, the features of which can be obscured in bulk analysis. We used a multiwavelength single-molecule fluorescence colocalization approach, CoSMoS, to define the initiation pathway at an activator-dependent bacterial σ(54) promoter that recapitulates characteristic features of eukaryotic promoters activated by enhancer binding proteins. The experiments kinetically characterize all major steps of the initiation process, revealing heretofore unknown features, including reversible formation of two closed complexes with greatly differing stabilities, multiple attempts for each successful formation of an open complex, and efficient release of σ(54) from the polymerase core at the start of transcript synthesis. Open complexes are committed to transcription, suggesting that regulation likely targets earlier steps in the mechanism. CoSMoS is a powerful, generally applicable method to elucidate the mechanisms of transcription and other multistep biochemical processes.

Probing the Electronic State of a Single Coronene Molecule by the Emission from Proximate Fluorophores

We measured electronic transitions of the 2D graphene-type molecule hexa-peri-hexabenzocoronene (HBC) at the single-molecule level. The large intersystem crossing rate and long triplet state lifetime in the range of seconds are prohibitive for direct single-molecule observation. By covalently coupling fluorescent acceptor molecules (perylenecarboximide, PMI) to HBC, efficient singlet energy transfer gives rise to strong PMI fluorescence. Confocal single-molecule fluorescence microscopy with two excitation colours matching the HBC and PMI transition frequencies, respectively, was conducted. Single HBC-6PMI molecules were readily observed via the PMI emission. It was found that after selective excitation of the HBC the PMI emission is interrupted by dark intervals whose length of several seconds is in agreement with the triplet state lifetime of HBC. Accordingly, the presence/absence of PMI emission permits to read out the spin state of a single HBC molecule. Moreover, due to spectral overlap, the HBC triplet state acts as an energy acceptor for PMI in the excited singlet state, thus leading to efficient singlet-triplet annihilation (STA) during its lifetime. Hence, intersystem crossing into the HBC triplet state serves as a collective fluorescence switch for individual multichromophores.

Effects of Covalent Modifications on the Structure and Assembly of Nucleosomes

We studied the structure of nucleosomes, the assembly of nucleosomes and how these properties are altered by DNA methylation and histone acetylation based on single molecule and ensemble fluorescence measurements. Our study revealed that a compact and rigid nucleosome structure is induced by CpG methylation in both internal and terminal regions of nucleosomal DNA. Real-time monitoring of nucleosome assembly with NAP1 revealed that there are at least 3 stable intermediate states during the assembly. The kinetic stabilities of the intermediate states are significantly elevated upon CpG methylation. These results suggest that CpG methylation stabilizes the nucleosome structure and inhibits the disassembly by destabilizing the transition states. We also characterized effects of histone acetylation by Piccolo NuA4 on the structure of a nucleosome and dinucleosomes. Upon the acetylation, we observed directional unwrapping of nucleosomal DNA that accompanies a topology change. We proposed a structural model for a dinucleosome in chromatin based on the structure of a dinucleosome spontaneously formed by two mononucleosomes in solution, which depends strongly on Mg2+ concentration and histone acetylation state. Mainly rendered by single molecule observations, these results suggest that structural changes of nucleosomes induced upon DNA methylation and histone acetylation may contribute to the regulation of genome activities.

OS3-1-1 壁面近傍における単鎖DNA拡散現象の一分子イメージング(OS3 マイクロ・ナノ生体医工学(1))

OS3-1-1 壁面近傍における単鎖DNA拡散現象の一分子イメージング(OS3 マイクロ・ナノ生体医工学(1))

Single Molecule Observation of MicroRNA Processing Enzymes at Action

MicroRNAs (miRNAs) regulate mRNA expression via RNA interference. MicroRNA is generated when nuclear RNase III Drosha cleaves a long primary transcript liberating a hairpin precursor miRNA (pre-miRNA). The pre-miRNA is processed into a ∼22 nt mature miRNA by cytoplasmic RNase III Dicer. In stem cells and certain cancer cells, the maturation process is interrupted when the pre-miRNA is hijacked by Lin28, a pluripotency factor, and the 3′ end of the RNA is uridylated by a noncanonical polyA polymerase, TUT4.With single molecule fluorescence microscopy, here we reveal the action mechanisms of human Dicer and TUT4 proteins at the molecular level. As it is difficult to prepare as large eukaryotic proteins as human Dicer and TUT4 proteins, we used a single molecule immunoprecipitation technique that we had recently developed [1].We, for the first time, observed Dicer's cleavage activity in real time using single molecule FRET and revealed stepwise processes of the cleavage action. Next, we investigated how Lin28 interfered with Dicer's activity and mediated TUT4's uridylation of pre-miRNA. Our real-time analysis suggests that Lin28 functions as a processivity factor of TUT4 and TUT4 utilizes a looping mechanism when it attaches uridines to a recruited pre-miRNA.Reference:[1] K. H. Yeom, I. Heo, J. Lee, S. Hohng, V. N. Kim, C. Joo, (2011) “Single Molecule Approach to Immunoprecipitated Protein Complexes: Insights into MiRNA Uridylation” EMBO Reports, 12, 690-696.