High-throughput AFM analysis reveals unwrapping pathways of H3 and CENP-A nucleosomes.

Nucleosomes, the fundamental units of chromatin, regulate readout and expression of eukaryotic genomes. Single-molecule experiments have revealed force-induced nucleosome accessibility, but a high-resolution unwrapping landscape in the absence of external forces is currently lacking. Here, we introduce a high-throughput pipeline for the analysis of nucleosome conformations based on atomic force microscopy and automated, multi-parameter image analysis. Our data set of ∼10 000 nucleosomes reveals multiple unwrapping states corresponding to steps of 5 bp DNA. For canonical H3 nucleosomes, we observe that dissociation from one side impedes unwrapping from the other side, but in contrast to force-induced unwrapping, we find only a weak sequence-dependent asymmetry. Notably, centromeric CENP-A nucleosomes do not unwrap anti-cooperatively, in stark contrast to H3 nucleosomes. Finally, our results reconcile previous conflicting findings about the differences in height between H3 and CENP-A nucleosomes. We expect our approach to enable critical insights into epigenetic regulation of nucleosome structure and stability and to facilitate future high-throughput AFM studies that involve heterogeneous nucleoprotein complexes.

Ru(TAP)32+ uses multivalent binding to accelerate and constrain photo-adduct formation on DNA.

Ru(ii)-complexes with polyazaaromatic ligands can undergo direct electron transfer with guanine nucleobases on blue light excitation that results in DNA lesions with phototherapeutic potential. Here we use single molecule approaches to demonstrate DNA binding mode heterogeneity and evaluate how multivalent binding governs the photochemistry of [Ru(TAP)3]2+ (TAP = 1,4,5,8-tetraazaphenanthrene).

Correlative Atomic Force and Single-Molecule Fluorescence Microscopy of Nucleoprotein Complexes.

Correlative imaging by fluorescence and atomic force microscopy provides a versatile tool to extract orthogonal information on structurally heterogeneous biomolecular assemblies. In this chapter, we describe an integrated setup for correlative fluorescence and force microscopy. We present factors influencing data quality, as well as step-by-step protocols for sample preparation, data acquisition, and data processing that yield nanoscale topographic resolution, high image registration accuracy, and single-fluorophore sensitivity. We demonstrate the capabilities of the approach through simultaneous characterization of mesoscale geometry and composition in a multipart nucleoprotein complex.

Silver nanowires for highly reproducible cantilever based AFM-TERS microscopy: towards a universal TERS probe.

Tip-enhanced Raman scattering (TERS) microscopy is a unique analytical tool to provide complementary chemical and topographic information of surfaces with nanometric resolution. However, difficulties in reliably producing the necessary metallized scanning probe tips has limited its widespread utilisation, particularly in the case of cantilever-based atomic force microscopy. Attempts to alleviate tip related issues using colloidal or bottom-up engineered tips have so far not reported consistent probes for both Raman and topographic imaging. Here we demonstrate the reproducible fabrication of cantilever-based high-performance TERS probes for both topographic and Raman measurements, based on an approach that utilises noble metal nanowires as the active TERS probe. The tips show 10 times higher TERS contrasts than the most typically used electrochemically-etched tips, and show a reproducibility for TERS greater than 90%, far greater than found with standard methods. We show that TERS can be performed in tapping as well as contact AFM mode, with optical resolutions around or below 15 nm, and with a maximum resolution achieved in tapping-mode of 6 nm. Our work illustrates that superior TERS probes can be produced in a fast and cost-effective manner using simple wet-chemistry methods, leading to reliable and reproducible high-resolution and high-sensitivity TERS, and thus renders the technique applicable for a broad community.

Orthogonal Probing of Single-Molecule Heterogeneity by Correlative Fluorescence and Force Microscopy.

Correlative imaging by fluorescence and force microscopy is an emerging technology to acquire orthogonal information at the nanoscale. Whereas atomic force microscopy excels at resolving the envelope structure of nanoscale specimens, fluorescence microscopy can detect specific molecular labels, which enables the unambiguous recognition of molecules in a complex assembly. Whereas correlative imaging at the micrometer scale has been established, it remains challenging to push the technology to the single-molecule level. Here, we used an integrated setup to systematically evaluate the factors that influence the quality of correlative fluorescence and force microscopy. Optimized data processing to ensure accurate drift correction and high localization precision results in image registration accuracies of ∼25 nm on organic fluorophores, which represents a 2-fold improvement over the state of the art in correlative fluorescence and force microscopy. Furthermore, we could extend the Atto532 fluorophore bleaching time ∼2-fold, by chemical modification of the supporting mica surface. In turn, this enables probing the composition of macromolecular complexes by stepwise photobleaching with high confidence. We demonstrate the performance of our method by resolving the stoichiometry of molecular subpopulations in a heterogeneous EcoRV-DNA nucleoprotein ensemble.

Methyltransferase-directed covalent coupling of fluorophores to DNA.

We report an assay for determining the number of fluorophores conjugated to single plasmid DNA molecules and apply this to compare the efficiency of fluorophore coupling strategies for covalent DNA labelling. We compare a copper-catalyzed azide-alkyne cycloaddition reaction, amine to N-hydroxysuccinimidyl ester coupling reaction and strain-promoted azide-alkyne cycloaddition reaction for fluorescent DNA labelling. We found increased labelling efficiency going from the amine to N-hydroxysuccinimidyl ester coupling reaction to the copper-catalyzed azide-alkyne cycloaddition and found the highest degree of DNA labelling with the strain-promoted azide-alkyne cycloaddition reaction. We also examined the effect of labelling on the DNA structure using atomic force microscopy. We observe no distortions or damage to the DNA that was labeled using the amine to N-hydroxysuccinimidyl ester and strain-promoted azide-alkyne cycloaddition coupling reactions. This was in contrast to the copper-catalyzed azide-alkyne cycloaddition reaction, which, despite the use of copper-coordinating ligands in the labelling mixture, leads to some structural DNA damage (single-stranded DNA breaks).

Covalent modification of graphene and graphite using diazonium chemistry: tunable grafting and nanomanipulation.

We shine light on the covalent modification of graphite and graphene substrates using diazonium chemistry under ambient conditions. We report on the nature of the chemical modification of these graphitic substrates, the relation between molecular structure and film morphology, and the impact of the covalent modification on the properties of the substrates, as revealed by local microscopy and spectroscopy techniques and electrochemistry. By careful selection of the reagents and optimizing reaction conditions, a high density of covalently grafted molecules is obtained, a result that is demonstrated in an unprecedented way by scanning tunneling microscopy (STM) under ambient conditions. With nanomanipulation, i.e., nanoshaving using STM, surface structuring and functionalization at the nanoscale is achieved. This manipulation leads to the removal of the covalently anchored molecules, regenerating pristine sp(2) hybridized graphene or graphite patches, as proven by space-resolved Raman microscopy and molecular self-assembly studies.

Bottom-up synthesis of liquid-phase-processable graphene nanoribbons with near-infrared absorption.

Structurally defined, long (>100 nm), and low-band-gap (∼1.2 eV) graphene nanoribbons (GNRs) were synthesized through a bottom-up approach, enabling GNRs with a broad absorption spanning into the near-infrared (NIR) region. The chemical identity of GNRs was validated by IR, Raman, solid-state NMR, and UV-vis-NIR absorption spectroscopy. Atomic force microscopy revealed well-ordered self-assembled monolayers of uniform GNRs on a graphite surface upon deposition from the liquid phase. The broad absorption of the low-band-gap GNRs enables their detailed characterization by Raman and time-resolved terahertz photoconductivity spectroscopy with excitation at multiple wavelengths, including the NIR region, which provides further insights into the fundamental physical properties of such graphene nanostructures.