Eric A. Josephs

A single-molecule view of conformational switching of DNA tethered to a gold electrode.

Surfaces that can actively regulate binding affinities or catalytic properties in response to external stimuli are a powerful means to probe and control the dynamic interactions between the cell and its microenvironment. Active surfaces also enable novel functionalities in biosensors and biomolecular separation technologies. Although electrical stimuli are often appealing due to their speed and localization, the operation of these electrically activated surfaces has mostly been characterized with techniques averaging over many molecules. Without a molecular-scale understanding of how biomolecules respond to electric fields, achieving the ultimate detection sensitivity or localized biological perturbation with the ultimate resolution would be difficult. Using electrochemical atomic force microscopy, we are able to follow the conformational changes of individual, short DNA molecules tethered to a gold electrode in response to an applied potential. Our study reveals conformations and dynamics that are difficult to infer from ensemble measurements: defects in the self-assembled monolayer (SAM) significantly perturb conformations and adsorption/desorption kinetics of surface-tethered DNA; on the other hand, the SAM may be actively molded by the DNA at different potentials. These results underscore the importance of characterizing the systems at the relevant length scale in the development of electrically switchable biofunctional surfaces.

Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9s ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9s off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex.

Nanoscale Positioning of Individual DNA Molecules by an Atomic Force Microscope

Here we report a method to assemble nanoscale DNA structures with single-molecule precision. This assembly is accomplished by performing nanografting in the presence of short, thiolated DNA strands that have been diluted by a positively charged alkanethiol. The expected number of DNA molecules per patch can be modulated by the application of an electric potential to the surface during patterning. Our ability to position individual DNA within a controlled nanoscale environment and observe these molecules in situ will allow us to understand and potentially decouple the heterogeneity caused by the local environment from the intrinsic properties in single-molecule biophysical measurements. Additionally, our approach can potentially be extended to the molecule-by-molecule assembly of larger artificial test structures of nucleic acids or proteins.

A switchable surface enables visualization of single DNA hybridization events with atomic force microscopy.

Here we describe a novel surface that enables direct visualization of the hybridization of single DNA molecules with an unprecedented resolution using atomic force microscopy. The surface consists of single-stranded DNA probes that are covalently anchored to a self-assembled monolayer. The surface satisfies the contradictory requirements for high-resolution imaging and hybridization by switching the DNA-surface interaction between a strong state and a weak state. Our approach opens up unique opportunities in elucidating hybridization at the molecular scale.

Electrochemical Etching of Gold within Nanoshaved Self-Assembled Monolayers

Wet etching of metal substrates with patterned self-assembled monolayers (SAMs) is an inexpensive and convenient method to produce metal nanostructures. For this method to be relevant to the fabrication of high precision plasmonic structures, the kinetics of nanoscale etching process, particularly in the lateral direction, must be elucidated and controlled. We herein describe an in situ atomic force microscopy (AFM) study to characterize the etching process within patterned SAMs with nanometer resolution and in real time. The in situ study was enabled by several unique elements, including single crystalline substrates to minimize the variability of facet-dependent etch rate, high-resolution nanoshaved SAM patterns, electrochemical-potential-controlled etching, and AFM kymographs to improve temporal resolution. Our approach has successfully quantified the extent of both lateral etching and vertical etching at different potentials. Our study reveals the presence of an induction period prior to the onset of significant lateral etching, which would be difficult to observe with the limited time resolution and sample-to-sample variation of ex situ studies. By increasing the vertical etch rate during this induction period with higher potentials, gold was etched up to 40 nm in the vertical direction with minimal lateral etching. High-resolution etching was also demonstrated on single crystal gold microplates, which are high quality gold thin films suitable for plasmonics studies.

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we--for the first time--record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.

A 'Semi-Protected Oligonucleotide Recombination' Assay for DNA Mismatch Repair in vivo Suggests Different Modes of Repair for Lagging Strand Mismatches.

Abstract In Escherichia coli, a DNA mismatch repair (MMR) pathway corrects errors that occur during DNA replication by coordinating the excision and re-synthesis of a long tract of the newly-replicated DNA between an epigenetic signal (a hemi-methylated d(GATC) site or a single-stranded nick) and the replication error after the error is identified by protein MutS. Recent observations suggest that this ‘long-patch repair’ between these sites is coordinated in the same direction of replication by the replisome. Here, we have developed a new assay that uniquely allows us to introduce targeted ‘mismatches’ directly into the replication fork via oligonucleotide recombination, examine the directionality of MMR, and quantify the nucleotide-dependence, sequence context-dependence, and strand-dependence of their repair in vivo—something otherwise nearly impossible to achieve. We find that repair of genomic lagging strand mismatches occurs bi-directionally in E. coli and that, while all MutS-recognized mismatches had been thought to be repaired in a consistent manner, the directional bias of repair and the effects of mutations in MutS are dependent on the molecular species of the mismatch. Because oligonucleotide recombination is routinely performed in both prokaryotic and eukaryotic cells, we expect this assay will be broadly applicable for investigating mechanisms of MMR in vivo.