Single-molecule Probes Research Articles

Correction: Corrigendum: Single Molecule Investigation of Ag+ Interactions with Single Cytosine-, Methylcytosine- and Hydroxymethylcytosine-Cytosine Mismatches in a Nanopore

Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair, and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications.

Single-Molecule Investigations of Morphology and Mass Transport Dynamics in Nanostructured Materials.

Nanostructured materials such as mesoporous metal oxides and phase-separated block copolymers form the basis for new monolith, membrane, and thin film technologies having applications in energy storage, chemical catalysis, and separations. Mass transport plays an integral role in governing the application-specific performance characteristics of many such materials. The majority of methods employed in their characterization provide only ensemble data, often masking the nanoscale, molecular-level details of materials morphology and mass transport. Single-molecule fluorescence methods offer direct routes to probing these characteristics on a single-molecule/single-nanostructure basis. This article provides a review of single-molecule studies focused on measurements of anisotropic diffusion, adsorption, partitioning, and confinement in nanostructured materials. Experimental methods covered include confocal and wide-field fluorescence microscopy. The results obtained promise to deepen our understanding of mass transport mechanisms in nanostructures, thus aiding in the realization of advanced materials systems.

Unbiased Clustering of Molecular Dynamics for Spatially Resolved Analysis of Chemically Heterogeneous Surfaces.

A technique is described for resolving and interpreting molecular interactions with a chemically heterogeneous surface. Using total internal reflection fluorescence microscopy, dynamic single molecule trajectories were accumulated simultaneously for fluorescently labeled fatty acid (interacting primarily via hydrophobic interactions) and dextran (interacting via hydrogen-bonding interactions) probe molecules at the interface between an aqueous solvent and a photopatterned solid surface with distinct regions of amine-terminated and poly(ethylene glycol) self-assembled monolayers. Using dynamic properties of the probe molecules (adsorption rate, surface diffusion coefficient, residence time), an unsupervised Gaussian mixture model algorithm was used to identify areas of the surface that were chemically related to each other, and the dynamic behaviors of the probe molecules were studied statistically on these distinct regions. The dynamic data were compared to data from homogeneous surfaces of known chemistry to provide a chemical identification of each location on the surface. Spatial maps were also constructed, allowing for spatial visualization of surface chemistry on a hydrophilic substrate. This work enables the direct study of interactions between single-molecule probes and distinct surface chemistries, even in the presence of spatial heterogeneity, without human bias, assumptions about surface structure, or model-dependent analysis.

Single Molecule Investigation of Glycine-Chlorite Interaction by Cross-Correlated Scanning Probe Microscopy and Quantum Mechanics Simulations.

In this work, we studied the interaction of glycine with the (001) surface of chlorite mineral at a single molecule level by cross-correlating scanning probe microscopy (SPM) and ab initio quantum mechanics (QM) investigations. Chlorite mineral is particularly interesting and peculiar for the interaction with organic molecules because it presents an alternated stacking of brucite-like (hydrophobic) and talc-like (hydrophilic) layers of different polarities. Brucite-like is positive, whereas talc-like is negative. The experimental atomic force microscopy (AFM) observations show that glycine is stably and selectively adsorbed on the brucite-like layer, organized in monolayers with different patterns. The sizes of single molecules of glycine measured by AFM are in agreement with those calculated by QM. Glycine molecules were found to align both at the edges and on the terraces of the brucitic surface. QM simulations confirmed the AFM observations that glycine molecule is adsorbed with high adsorption energy preferentially with its plane parallel to the (001) brucite-like surface. QM also provided the geometry conformation of the molecule and the bonding scheme between glycine and brucite surface. This kind of data can be very helpful both to biotechnological applications of this substrate and to depict some important processes that might have been occurred in prebiotic environments.

Correction: A Single Molecule Investigation of the Photostability of Quantum Dots

Correction: A Single Molecule Investigation of the Photostability of Quantum Dots

Revealing and resolving degeneracies in stretching exponents in temporally heterogeneous environments.

Supercooled liquids are proposed to be dynamically heterogeneous, with regions exhibiting relaxation time scales that vary in space and time. Measurement of the distribution of such time scales could be an important test of various proposed theories of vitrification. Single molecule fluorescence experiments attempt to uncover this distribution, typically by embedding single molecule probes into these systems and monitoring their individual rotational relaxations from a computed autocorrelation function (ACF). These ACFs may exhibit stretched exponential decays, with the value of the stretching exponent assumed to report the set of dynamical environments explored by the probe. Here, we use simulated trajectories of rotation to investigate how the time scale of dynamic exchange relative to underlying relaxation time scales in the system affects probe ability to report the distribution relaxation of time scales present. We find that dynamically heterogeneous regions must persist for approximately 50 times the median relaxation time scale for a single molecule to accurately report the full distribution of time scales it has experienced. In systems with faster dynamic exchange, single molecule ACFs average over successive environments, limiting the reported heterogeneity of the system. This leads to degeneracies in stretching exponent for systems with different underlying relaxation time distributions. We show that monitoring single molecule median stretching exponent as a function of trajectory length or simultaneously measuring median stretching exponent and measured relaxation time distribution at a given trajectory length can resolve these degeneracies, revealing the underlying set of relaxation times as well as median exchange time.

RNA dances to center stage.

RNA dances to center stage.

Visualization and thermodynamic encoding of single-molecule partition function projections.

Ensemble averaging of molecular states is fundamental for the experimental determination of thermodynamic quantities. A special case occurs for single-molecule investigations under equilibrium conditions, for which free energy, entropy and enthalpy at finite temperatures are challenging to determine with ensemble averaging alone. Here we report a method to directly record time-averaged equilibrium probability distributions by confining an individual molecule to a nanoscopic pore of a two-dimensional metal-organic nanomesh, using temperature-controlled scanning tunnelling microscopy. We associate these distributions with partition function projections to assess real-space-projected thermodynamic quantities, aided by computational modelling. The presented molecular dynamics-based analysis is able to reproduce experimentally observed projected microstates with high accuracy. By an in silico customized energy landscape, we demonstrate that distinct probability distributions can be encrypted at different temperatures. Such modulation provides means to encode and decode information into position-temperature space.

Study of PNA–DNA hybridization by AFM-based single-molecule force spectroscopy

Abstract The PNA–DNA interaction was probed with single-molecule AFM force measurements. The PNA sequence with only six thymine bases, p(T)6, could form stable hybrids with the complementary DNA sequence of d(A)6. Rupture of the p(T)6–d(A)6 hybrids required forces of around 148 pN, which was larger than the forces to unbind short DNA duplex that were usually below 100 pN. Fitting into the rupture force–loading rate correlation produced kinetic parameters that describe the PNA–DNA interaction, which were the thermal force scale (fβ) to be 17.5 pN, the distance to the energy barrier for dissociation (Δx) to be 0.23 nm, and the dissociation rate constant at zero force (koff) to be 2.5 s−1, respectively. The results highlighted the stronger binding affinity between PNA and DNA than between DNA and DNA. And by changing the DNA sequence to d(AAGAAA) with a single-base mismatch, no specific rupture events were observed, confirming the high sensitivity of PNA–DNA hybridization to base mismatch. This single-molecule force study probes into the energetic and kinetic aspects of PNA–DNA interactions that are not accessible by conventional bulk methods, leading to deeper understanding of PNA–DNA interactions.

Kinesin Regulation Dynamics through Cargo Delivery, a Single Molecule Investigation

Kinesins are microtubule-based motors that deliver cargo to their destinations in a highly regulated manner. Although numerous regulators of cargo delivery have been identified in recent years, the regulation mechanism for kinesin through the cargo delivery and recycling process is not known. By performing single molecule fluorescence imaging measurements in Chlamydomonas flagella, which are 200 nm in diameter and 10 microns in length with 9 sets of doublet microtubules, we tracked intraflagellar transport (IFT) trains, BBSome cargoes, and kinesin-2 motors through the entire cargo delivery process and determined their reorganization and recycling dynamics. Upon arrival at the microtubule plus end at the flagellar tip, (1) IFT trains and BBSome cargoes remain intact and dissociate away from the kinesins and microtubules together, diffuse, and reorganize along the flagellar membrane over the course of 2.3 seconds before commencing retrograde travel. (2) Kinesin motors remain bound to and diffuse along microtubules for 1.3 seconds before dissociating into the flagellar lumen for recycling.

How Does the Replication Machinery Deal with Roadblocks: A Single-Molecule Investigation

DNA replication is a fundamental process in life. Decades of study have elucidated the most important mechanistic principles underlying the duplication of DNA, but most often these processes are studied and represented as if they occur on naked DNA. We set out to employ single-molecule techniques to study the effect of intracellular crowding on the process of replication. We develop methods that allow us to mechanically stretch DNA molecules and use fluorescence microscopy to visualize replication at the single-molecule level. In particular, we reconstitute in vitro the E. coli replication reaction and observe how common DNA-binding proteins such as the histone-like proteins H-NS and HU affect the replication kinetics.

Probing of PNA-DNA Hybrid Duplex Stability with AFM Force Spectroscopy

Peptide nucleic acids (PNA) are synthetic polymers the neutral peptide backbone of which provides elevated stability to PNA-PNA and hybrid PNA-DNA duplex. It was demonstrated recently (J.Org.Chem.2011, 76, 5614-5627) that additional modification of the backbone such as incorporation of diethylene glycol (miniPEG) further increases the duplex thermal stability, so the hybrid duplex with 10 miniPEG units (γ-PNA) has the melting temperature 23°C higher compared to the regular PNA-DNA duplex. Here we applied AFM force spectroscopy to probe the strength of the γ-PNA-DNA duplex (5′GAGTAG GTAG-3’) containing ten miniPEG-modified units. Single-stranded PNA and DNA oligonucleotides containing terminal thiol groups were immobilized on amino-functionalized AFM tip and mica substrate, respectively via bifunctional PEG tethers and the interaction between the DNA and γ-PNA polymers was analyzed by multiple approach retraction cycles over various locations on the mica substrate. Such single molecule probing experiments produced force curves with well-defined rupture events corresponding to the dissociation of the duplex formed during the approach step. The experiments were performed at various pulling rates (300-3000 nm/sec) enabled us to characterize the hybrid duplex stability using dynamic force spectroscopy (DFS) approach. The DFS measurements yielded rupture forces varying in the range 60-70 pN. The data analyzed in the framework of the Bell-Evans approach yielded a dissociation constant, koff ∼ 10−9 sec−1 and rupture distance x-1∼ 1.7 nm. Similar published DFS measurements of DNA duplex are characterized by koff with ∼ 108 times less values, which is in line with elevated stabilities of the γ-PNA-DNA duplexes compared with the DNA duplexes.The work was supported by NIH grants 5P01 GM091743-03 and 5R01 GM096039-04 to YLL. γ-PNA was synthesized at PNA innovation.

Processivity of Kinesin-2 Results from Rear-Head Gating and Not Front-Head Gating

The kinesin-2 family motor KIF3A/B coordinates with dynein to bidirectionally transport intraflagellar particles, melanosomes and neuronal vesicles. Compared to kinesin-1, kinesin-2 is less processive and its processivity is more sensitive to load, suggesting that the gating mechanisms that their control processivity may differ. To understand the motor roles that front-head gating and rear-head gating, we carried out stopped flow kinetics experiments using mant nucleotides, steady state assays, and single-molecule investigations to characterize the entire kinetic cycle a functional mouse KIF3A homodimer that exhibits similar motility to full-length KIF3A/B. Upon first encounter with the microtubule lattice, the motor exchanges mADP with an on-rate of 18 μM-1 s-1 and an off-rate of 27 s-1. When AMPPNP was used to entrap the motor in a two-head bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. Similar lack of front-head gating was found with mATP and when the neck linker domain was shortened from 17 to 14 residues to enhance intramolecular strain. In contrast, microtubule pelleting and single-molecule microscopy assays found that in ADP the motor dissociates with an off-rate of 2.1 s-1 and a KD of 0.5 μM, similar to its behavior in ATP. Hence, kinesin-2 processivity results from rear-head gating and not front-head gating. Based on the kinetics measurements, finally we propose a complete model of the kinesin-2 hydrolysis cycle that accounts for all of the kinetics and motility data. This study provides the direct evidence that rear-head gating does and not front-head gating does not play a role in kinesin-2 processivity and suggests that kinesin-2 mechanochemistry is specifically adapted for bidirectional transport.

Modification of a single-molecule AFM probe with highly defined surface functionality.

Single-molecule force spectroscopy with an atomic force microscope has been widely used to study inter- and intramolecular interactions. To obtain data consistent with single molecular events, a well-defined method is critical to limit the number of molecules at the apex of an AFM probe to one or to a few. In this paper, we demonstrate an easy method for single-molecule probe modification by using the Cu-catalyzed alkyne–azide cycloaddition reaction. Excess terminal alkynes were covalently attached to the probe, and a bi-functional molecule containing an azide at one end and a carboxylic acid at the other was dissolved in the reaction solution. By simply contacting the probe and the Cu substrate, controlled carboxylation on the probe apex could be achieved, since the ‘click’ reaction requires the co-exist of alkyne, azide and Cu(I). The finite contact area would result in a highly defined surface functionality of the probe down to single molecule level with high reproducibility.

Single-molecule investigation of G-quadruplex folds of the human telomere sequence in a protein nanocavity.

Human telomeric DNA consists of tandem repeats of the sequence 5'-TTAGGG-3' that can fold into various G-quadruplexes, including the hybrid, basket, and propeller folds. In this report, we demonstrate use of the α-hemolysin ion channel to analyze these subtle topological changes at a nanometer scale by providing structure-dependent electrical signatures through DNA-protein interactions. Whereas the dimensions of hybrid and basket folds allowed them to enter the protein vestibule, the propeller fold exceeds the size of the latch region, producing only brief collisions. After attaching a 25-mer poly-2'-deoxyadenosine extension to these structures, unraveling kinetics also were evaluated. Both the locations where the unfolding processes occur and the molecular shapes of the G-quadruplexes play important roles in determining their unfolding profiles. These results provide insights into the application of α-hemolysin as a molecular sieve to differentiate nanostructures as well as the potential technical hurdles DNA secondary structures may present to nanopore technology.

Extraction of rotational correlation times from noisy single molecule fluorescence trajectories.

Monitoring single molecule probe rotations is an increasingly common approach to studying dynamics of complex systems, including supercooled liquids. Even with advances in fluorophore design and detector sensitivity, such measurements typically exhibit low signal to noise and signal to background ratios. Here, we simulated and analyzed orthogonally decomposed fluorescence signals of single molecules undergoing rotational diffusion in a manner that mimics experimentally collected data of probes in small molecule supercooled liquids. The effects of noise, background, and trajectory length were explicitly considered, as were the effects of data processing approaches that may limit the impact of noise and background on assessment of environmental dynamics. In many cases, data treatment that attempts to remove noise and background were found to be deleterious. However, for short trajectories below a critical signal to background threshold, a thresholding approach that successfully removed data points associated with noise and spared those associated with signal allowed for assessment of environmental dynamics that was as accurate and precise as would be achieved in the absence of noise.

Molecular processes studied at a single-molecule level using DNA origami nanostructures and atomic force microscopy.

DNA origami nanostructures allow for the arrangement of different functionalities such as proteins, specific DNA structures, nanoparticles, and various chemical modifications with unprecedented precision. The arranged functional entities can be visualized by atomic force microscopy (AFM) which enables the study of molecular processes at a single-molecular level. Examples comprise the investigation of chemical reactions, electron-induced bond breaking, enzymatic binding and cleavage events, and conformational transitions in DNA. In this paper, we provide an overview of the advances achieved in the field of single-molecule investigations by applying atomic force microscopy to functionalized DNA origami substrates.

Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles.

This protocol describes how to monitor individual naturally supercoiled circular DNA plasmids bound via peptide nucleic acid (PNA) handles between a bead and a surface. The protocol was developed for single-molecule investigation of the dynamics of supercoiled DNA, and it allows the investigation of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti-digoxigenin-coated glass cover slide, and one conjugated with biotin for attachment to a submicron-sized streptavidin-coated polystyrene bead. Plasmids are constructed, purified and incubated with the PNA handles. The dynamics of the construct is analyzed by tracking the tethered bead using video microscopy: less supercoiling results in more movement, and more supercoiling results in less movement. In contrast to other single-molecule methodologies, the current methodology allows for studying DNA in its naturally supercoiled state with constant linking number and constant writhe. The protocol has potential for use in studying the influence of supercoils on the dynamics of DNA and its associated proteins, e.g., topoisomerase. The procedure takes ~4 weeks.

Watching individual molecules flex within lipid membranes using SERS

Interrogating individual molecules within bio-membranes is key to deepening our understanding of biological processes essential for life. Using Raman spectroscopy to map molecular vibrations is ideal to non-destructively ‘fingerprint’ biomolecules for dynamic information on their molecular structure, composition and conformation. Such tag-free tracking of molecules within lipid bio-membranes can directly connect structure and function. In this paper, stable co-assembly with gold nano-components in a ‘nanoparticle-on-mirror’ geometry strongly enhances the local optical field and reduces the volume probed to a few nm3, enabling repeated measurements for many tens of minutes on the same molecules. The intense gap plasmons are assembled around model bio-membranes providing molecular identification of the diffusing lipids. Our experiments clearly evidence measurement of individual lipids flexing through telltale rapid correlated vibrational shifts and intensity fluctuations in the Raman spectrum. These track molecules that undergo bending and conformational changes within the probe volume, through their interactions with the environment. This technique allows for in situ high-speed single-molecule investigations of the molecules embedded within lipid bio-membranes. It thus offers a new way to investigate the hidden dynamics of cell membranes important to a myriad of life processes.

Single molecule investigation of Ag+ interactions with single cytosine-, methylcytosine- and hydroxymethylcytosine-cytosine mismatches in a nanopore.

Both cytosine-Ag-cytosine interactions and cytosine modifications in a DNA duplex have attracted great interest for research. Cytosine (C) modifications such as methylcytosine (mC) and hydroxymethylcytosine (hmC) are associated with tumorigenesis. However, a method for directly discriminating C, mC and hmC bases without labeling, modification and amplification is still missing. Additionally, the nature of coordination of Ag+ with cytosine-cytosine (C-C) mismatches is not clearly understood. Utilizing the alpha-hemolysin nanopore, we show that in the presence of Ag+, duplex stability is most increased for the cytosine-cytosine (C-C) pair, followed by the cytosine-methylcytosine (C-mC) pair, and the cytosine-hydroxymethylcytosine (C-hmC) pair, which has no observable Ag+ induced stabilization. Molecular dynamics simulations reveal that the hydrogen-bond-mediated paring of a C-C mismatch results in a binding site for Ag+. Cytosine modifications (such as mC and hmC) disrupted the hydrogen bond, resulting in disruption of the Ag+ binding site. Our experimental method provides a novel platform to study the metal ion-DNA interactions and could also serve as a direct detection method for nucleobase modifications.