Supercoiling-dependent DNA binding: quantitative modeling and applications to bulk and single-molecule experiments.

DNA stores our genetic information and is ubiquitous in applications, where it interacts with binding partners ranging from small molecules to large macromolecular complexes. Binding is modulated by mechanical strains in the molecule and can change local DNA structure. Frequently, DNA occurs in closed topological forms where topology and supercoiling add a global constraint to the interplay of binding-induced deformations and strain-modulated binding. Here, we present a quantitative model with a straight-forward numerical implementation of how the global constraints introduced by DNA topology modulate binding. We focus on fluorescent intercalators, which unwind DNA and enable direct quantification via fluorescence detection. Our model correctly describes bulk experiments using plasmids with different starting topologies, different intercalators, and over a broad range of intercalator and DNA concentrations. We demonstrate and quantitatively model supercoiling-dependent binding in a single-molecule assay, where we directly observe the different intercalator densities going from supercoiled to nicked DNA. The single-molecule assay provides direct access to binding kinetics and DNA supercoil dynamics. Our model has broad implications for the detection and quantification of DNA, including the use of psoralen for UV-induced DNA crosslinking to quantify torsional tension in vivo, and for the modulation of DNA binding in cellular contexts.

High-yield ligation-free assembly of DNA constructs with nucleosome positioning sequence repeats for single-molecule manipulation assays.

Force and torque spectroscopy have provided unprecedented insights into the mechanical properties, conformational transitions, and dynamics of DNA and DNA-protein complexes, notably nucleosomes. Reliable single-molecule manipulation measurements require, however, specific and stable attachment chemistries to tether the molecules of interest. Here, we present a functionalization strategy for DNA that enables high-yield production of constructs for torsionally constrained and very stable attachment. The method is based on two subsequent PCRs: first ∼380 bp long DNA strands are generated that contain multiple labels, which are used as "megaprimers" in a second PCR to generate ∼kbp long double-stranded DNA constructs with multiple labels at the respective ends. To achieve high-force stability, we use dibenzocyclooctyne-based click chemistry for covalent attachment to the surface and biotin-streptavidin coupling to the bead. The resulting tethers are torsionally constrained and extremely stable under load, with an average lifetime of 70 ± 3 h at 45 pN. The high yield of the approach enables nucleosome reconstitution by salt dialysis on the functionalized DNA, and we demonstrate proof-of-concept measurements on nucleosome assembly statistics and inner turn unwrapping under force. We anticipate that our approach will facilitate a range of studies of DNA interactions and nucleoprotein complexes under forces and torques.

DNA Origami Fiducial for Accurate 3D Atomic Force Microscopy Imaging.

Atomic force microscopy (AFM) is a powerful technique for imaging molecules, macromolecular complexes, and nanoparticles with nanometer resolution. However, AFM images are distorted by the shape of the tip used. These distortions can be corrected if the tip shape can be determined by scanning a sample with features sharper than the tip and higher than the object of interest. Here we present a 3D DNA origami structure as fiducial for tip reconstruction and image correction. Our fiducial is stable under a broad range of conditions and has sharp steps at different heights that enable reliable tip reconstruction from as few as ten fiducials. The DNA origami is readily codeposited with biological and nonbiological samples, achieves higher precision for the tip apex than polycrystalline samples, and dramatically improves the accuracy of the lateral dimensions determined from the images. Our fiducial thus enables accurate and precise AFM imaging for a broad range of applications.

Twisting DNA by salt.

The structure and properties of DNA depend on the environment, in particular the ion atmosphere. Here, we investigate how DNA twist -one of the central properties of DNA- changes with concentration and identity of the surrounding ions. To resolve how cations influence the twist, we combine single-molecule magnetic tweezer experiments and extensive all-atom molecular dynamics simulations. Two interconnected trends are observed for monovalent alkali and divalent alkaline earth cations. First, DNA twist increases monotonously with increasing concentration for all ions investigated. Second, for a given salt concentration, DNA twist strongly depends on cation identity. At 100 mM concentration, DNA twist increases as Na+ < K+ < Rb+ < Ba2+ < Li+ ≈ Cs+ < Sr2+ < Mg2+ < Ca2+. Our molecular dynamics simulations reveal that preferential binding of the cations to the DNA backbone or the nucleobases has opposing effects on DNA twist and provides the microscopic explanation of the observed ion specificity. However, the simulations also reveal shortcomings of existing force field parameters for Cs+ and Sr2+. The comprehensive view gained from our combined approach provides a foundation for understanding and predicting cation-induced structural changes both in nature and in DNA nanotechnology.

Quantifying epigenetic modulation of nucleosome breathing by high-throughput AFM imaging.

Nucleosomes are the basic units of chromatin and critical for storage and expression of eukaryotic genomes. Chromatin accessibility and gene readout are heavily regulated by epigenetic marks, in which post-translational modifications of histones play a key role. However, the mode of action and the structural implications at the single-molecule level of nucleosomes is still poorly understood. Here we apply a high-throughput atomic force microscopy imaging and analysis pipeline to investigate the conformational landscape of the nucleosome variants three additional methyl groups at lysine 36 of histone H3 (H3K36me3), phosphorylation of H3 histones at serine 10 (H3S10phos), and acetylation of H4 histones at lysines 5, 8, 12, and 16 (H4K5/8/12/16ac). Our data set of more than 25,000 nucleosomes reveals nucleosomal unwrapping steps corresponding to 5-bp DNA. We find that H3K36me3 nucleosomes unwrap significantly more than wild-type nucleosomes and additionally unwrap stochastically from both sides, similar to centromere protein A (CENP-A) nucleosomes and in contrast to the highly anticooperative unwrapping of wild-type nucleosomes. Nucleosomes with H3S10phos or H4K5/8/12/16ac modifications show unwrapping populations similar to wild-type nucleosomes and also retain the same level of anticooperativity. Our findings help to put the mode of action of these modifications into context. Although H3K36me3 likely acts partially by directly affecting nucleosome structure on the single-molecule level, H3S10phos and H4K5/8/12/16ac must predominantly act through higher-order processes. Our analysis pipeline is readily applicable to other nucleosome variants and will facilitate future high-resolution studies of the conformational landscape of nucleoprotein complexes.

DNA fluctuations reveal the size and dynamics of topological domains.

DNA supercoiling is a key regulatory mechanism that orchestrates DNA readout, recombination, and genome maintenance. DNA-binding proteins often mediate these processes by bringing two distant DNA sites together, thereby inducing (transient) topological domains. In order to understand the dynamics and molecular architecture of protein-induced topological domains in DNA, quantitative and time-resolved approaches are required. Here, we present a methodology to determine the size and dynamics of topological domains in supercoiled DNA in real time and at the single-molecule level. Our approach is based on quantifying the extension fluctuations-in addition to the mean extension-of supercoiled DNA in magnetic tweezers (MT). Using a combination of high-speed MT experiments, Monte Carlo simulations, and analytical theory, we map out the dependence of DNA extension fluctuations as a function of supercoiling density and external force. We find that in the plectonemic regime, the extension variance increases linearly with increasing supercoiling density and show how this enables us to determine the formation and size of topological domains. In addition, we demonstrate how the transient (partial) dissociation of DNA-bridging proteins results in the dynamic sampling of different topological states, which allows us to deduce the torsional stiffness of the plectonemic state and the kinetics of protein-plectoneme interactions. We expect our results to further the understanding and optimization of magnetic tweezer measurements and to enable quantification of the dynamics and reaction pathways of DNA processing enzymes in the context of physiologically relevant forces and supercoiling densities.

A High-throughput Pipeline to Determine DNA and Nucleosome Conformations by AFM Imaging.

Atomic force microscopy (AFM) is a powerful tool to image macromolecular complexes with nanometer resolution and exquisite single-molecule sensitivity. While AFM imaging is well-established to investigate DNA and nucleoprotein complexes, AFM studies are often limited by small datasets and manual image analysis that is slow and prone to user bias. Recently, we have shown that a combination of large scale AFM imaging and automated image analysis of nucleosomes can overcome these previous limitations of AFM nucleoprotein studies. Using our high-throughput imaging and analysis pipeline, we have resolved nucleosome wrapping intermediates with five base pair resolution and revealed how distinct nucleosome variants and environmental conditions affect the unwrapping pathways of nucleosomal DNA. Here, we provide a detailed protocol of our workflow to analyze DNA and nucleosome conformations focusing on practical aspects and experimental parameters. We expect our protocol to drastically enhance AFM analyses of DNA and nucleosomes and to be readily adaptable to a wide variety of other protein and protein-nucleic acid complexes.

Molecular structure, DNA binding mode, photophysical properties and recommendations for use of SYBR Gold.

SYBR Gold is a commonly used and particularly bright fluorescent DNA stain, however, its chemical structure is unknown and its binding mode to DNA remains controversial. Here, we solve the structure of SYBR Gold by NMR and mass spectrometry to be [2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] and determine its extinction coefficient. We quantitate SYBR Gold binding to DNA using two complementary approaches. First, we use single-molecule magnetic tweezers (MT) to determine the effects of SYBR Gold binding on DNA length and twist. The MT assay reveals systematic lengthening and unwinding of DNA by 19.1° ± 0.7° per molecule upon binding, consistent with intercalation, similar to the related dye SYBR Green I. We complement the MT data with spectroscopic characterization of SYBR Gold. The data are well described by a global binding model for dye concentrations ≤2.5 µM, with parameters that quantitatively agree with the MT results. The fluorescence increases linearly with the number of intercalated SYBR Gold molecules up to dye concentrations of ∼2.5 µM, where quenching and inner filter effects become relevant. In summary, we provide a mechanistic understanding of DNA-SYBR Gold interactions and present practical guidelines for optimal DNA detection and quantitative DNA sensing applications using SYBR Gold.

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.

A benchmark data set for the mechanical properties of double-stranded DNA and RNA under torsional constraint.

Nucleic acids are central to the storage and transmission of genetic information and play essential roles in many cellular processes. Quantitative understanding and modeling of their functions and properties requires quantitative experimental characterization. We use magnetic tweezers (MT) to apply precisely calibrated stretching forces and linking number changes to DNA and RNA molecules tethered between a surface and superparamagnetic beads. Magnetic torque tweezers (MTT) allow to control the linking number of double-stranded DNA or RNA tethers, while directly measuring molecular torque by monitoring changes in the equilibrium rotation angle upon over- or underwinding of the helical molecules. Here, we provide a comprehensive data set of double-stranded DNA and RNA under controlled stretching as a function of the linking number. We present data for extension and torque as a function of linking number in equilibrium. We report data for the critical torque of buckling and of the torsional stiffness of DNA and RNA as a function of applied force. Finally, we provide dynamic data for the hopping behavior at the DNA buckling point.

Deciphering the Gene Regulatory Landscape Encoded in DNA Biophysical Features.

Gene regulation in higher organisms involves a sophisticated interplay between genetic and epigenetic mechanisms. Despite advances, the logic in selective usage of certain genomic regions as regulatory elements remains unclear. Here we show that the inherent biophysical properties of the DNA encode epigenetic state and the underlying regulatory potential. We find that the propeller twist (ProT) level is indicative of genomic location of the regulatory elements, their strength, the affinity landscape of transcription factors, and distribution in the nuclear 3D space. We experimentally show that ProT levels confer increased DNA flexibility and surface accessibility, and thus potentially primes usage of high ProT regions as regulatory elements. ProT levels also correlate with occurrence and phenotypic consequences of mutations. Interestingly, cell-fate switches involve a transient usage of low ProT regulatory elements. Altogether, our work provides unprecedented insights into the gene regulatory landscape encoded in the DNA biophysical features.

The free energy landscape of retroviral integration.

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the replication cycle. Yet, strand transfer is intrinsically iso-energetic and it is not clear how efficient integration can be achieved. Here we investigate the dynamics of strand transfer and demonstrate that consecutive nucleoprotein intermediates interacting with a supercoiled target are increasingly stable, resulting in a net forward rate. Multivalent target interactions at discrete auxiliary interfaces render target capture irreversible, while allowing dynamic site selection. Active site binding is transient but rapidly results in strand transfer, which in turn rearranges and stabilizes the intasome in an allosteric manner. We find the resulting strand transfer complex to be mechanically stable and extremely long-lived, suggesting that a resolving agent is required in vivo.

Polymer Nanoreactors Shield Perovskite Nanocrystals from Degradation.

Halide perovskite nanocrystals (NCs) have shown impressive advances, exhibiting optical properties that outpace conventional semiconductor NCs, such as near-unity quantum yields and ultrafast radiative decay rates. Nevertheless, the NCs suffer even more from stability problems at ambient conditions and due to moisture than their bulk counterparts. Herein, we report a strategy of employing polymer micelles as nanoreactors for the synthesis of methylammonium lead trihalide perovskite NCs. Encapsulated by this polymer shell, the NCs display strong stability against water degradation and halide ion migration. Thin films comprising these NCs exhibit a more than 15-fold increase in lifespan in comparison to unprotected NCs in ambient conditions and even survive over 75 days of complete immersion in water. Furthermore, the NCs, which exhibit quantum yields of up to 63% and tunability of the emission wavelength throughout the visible range, show no signs of halide ion exchange. Additionally, heterostructures of MAPI and MAPBr NC layers exhibit efficient Förster resonance energy transfer (FRET), revealing a strategy for optoelectronic integration.

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).

Free Energy Landscape and Dynamics of Supercoiled DNA by High-Speed Atomic Force Microscopy.

DNA supercoiling fundamentally constrains and regulates the storage and use of genetic information. While the equilibrium properties of supercoiled DNA are relatively well understood, the dynamics of supercoils are much harder to probe. Here we use atomic force microscopy (AFM) imaging to demonstrate that positively supercoiled DNA plasmids, in contrast to their negatively supercoiled counterparts, preserve their plectonemic geometry upon adsorption under conditions that allow for dynamics and equilibration on the surface. Our results are in quantitative agreement with a physical polymer model for supercoiled plasmids that takes into account the known mechanical properties and torque-induced melting of DNA. We directly probe supercoil dynamics using high-speed AFM imaging with subsecond time and ∼nanometer spatial resolution. From our recordings we quantify self-diffusion, branch point flexibility, and slithering dynamics and demonstrate that reconfiguration of molecular extensions is predominantly governed by the bending flexibility of plectoneme arms. We expect that our methodology can be an asset to probe protein-DNA interactions and topochemical reactions on physiological relevant DNA length and supercoiling scales by high-resolution AFM imaging.

Zn2+-triggered self-assembly of Gonadorelin [6-D-Phe] to produce nanostructures and fibrils.

A synthetic derivative, GnRH [6-D-Phe], stable against enzymatic degradation, self-assembles and forms nanostructures and fibrils upon a pH shift in the presence of different concentrations of Zn2+ in vitro. Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed the existence of higher order assembly of Zn2+: GnRH [6-D-Phe]. Nuclear Magnetic Resonance spectroscopy (NMR) indicated a weak interaction between Zn2+ and GnRH [6-D-Phe]. Atomic Force Microscopy (AFM) showed the existence of GnRH [6-D-Phe] oligomers and fibrils. Molecular Dynamic (MD) simulation of the 10:1 Zn2+: GnRH [6-D-Phe] explored the interaction and dimerization processes. In contrast to already existing short peptide fibrils, GnRH [6-D-Phe] nanostructures and fibrils form in a Tris-buffered pH environment in a controlled manner through a temperature reduction and a pH shift. The lyophilized Zn2+: GnRH [6-D-Phe] assembly was tested as a platform for the sustained delivery of GnRH [6-D-Phe] and incorporated into two different oil vehicle matrices. The in vitro release was slow and continuous over 14 days and not influenced by the oil matrix.

Measuring Single-Molecule Twist and Torque in Multiplexed Magnetic Tweezers.

Magnetic tweezers permit application of precisely calibrated stretching forces to nucleic acid molecules tethered between a surface and superparamagnetic beads. In addition, magnetic tweezers can control the tethers' twist. Here, we focus on recent extensions of the technique that expand the capabilities of conventional magnetic tweezers by enabling direct measurements of single-molecule torque and twist. Magnetic torque tweezers (MTT) still control the DNA or RNA tether's twist, but directly measure molecular torque by monitoring changes in the equilibrium rotation angle upon overwinding and underwinding of the tether. In freely orbiting magnetic tweezers (FOMT), one end of the tether is allowed to rotate freely, while still applying stretching forces and monitoring rotation angle. Both MTT and FOMT have provided unique insights into the mechanical properties, structural transitions, and interactions of DNA and RNA. Here, we provide step-by-step protocols to carry out FOMT and MTT measurements. In particular, we focus on multiplexed measurements, i.e., measurements that record data for multiple nucleic acid tethers at the same time, to improve statistics and to facilitate the observation of rare events.

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.

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.