Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling.

Shallow genome-wide cell-free DNA (cfDNA) sequencing holds great promise for non-invasive cancer monitoring by providing reliable copy number alteration (CNA) and fragmentomic profiles. Single nucleotide variations (SNVs) are, however, much harder to identify with low sequencing depth due to sequencing errors. Here we present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which leverages RCA and consensus calling based on genome-wide long-read nanopore sequencing to enable simultaneous multimodal tumor fraction estimation through SNVs, CNAs, and fragmentomics.

Exploring protein-mediated compaction of DNA by coarse-grained simulations and unsupervised learning.

Protein-DNA interactions and protein-mediated DNA compaction play key roles in a range of biological processes. The length scales typically involved in DNA bending, bridging, looping, and compaction (≥1 kbp) are challenging to address experimentally or by all-atom molecular dynamics simulations, making coarse-grained simulations a natural approach. Here, we present a simple and generic coarse-grained model for DNA-protein and protein-protein interactions and investigate the role of the latter in the protein-induced compaction of DNA.

Epigenetic Histone Modifications H3K36me3 and H4K5/8/12/16ac Induce Open Polynucleosome Conformations via Different Mechanisms.

Nucleosomes are the basic compaction unit of chromatin and nucleosome structure and their higher-order assemblies regulate genome accessibility. Many post-translational modifications alter nucleosome dynamics, nucleosome-nucleosome interactions, and ultimately chromatin structure and gene expression. Here, we investigate the role of two post-translational modifications associated with actively transcribed regions, H3K36me3 and H4K5/8/12/16ac, in the contexts of tri-nucleosome arrays that provide a tractable model system for quantitative single-molecule analysis, while enabling us to probe nucleosome-nucleosome interactions.

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.

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.

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.

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.

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.

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.

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.

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.

Voice Reactivity as a Response to Acute Hypobaric Hypoxia at High Altitude.

Although the understanding of hypobaric hypoxia is increasing, it remains a hazard in aviation medicine. This study examined the feasibility of detecting voice markers sensitive to acute hypobaric hypoxia in an early presymptomatic (PRE-SYMP) stage. Eight subjects qualified with hypobaric training completed a series of standardized speech tests in a hypobaric chamber at 20,000 ft and 25,000 ft (6096 and 7620 m) of altitude.

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.

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.

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.

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.

Zn-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 Zn in vitro. Attenuated Total Reflection Fourier Transform Infrared spectroscopy (ATR-FTIR) revealed the existence of higher order assembly of Zn: GnRH [6-D-Phe]. Nuclear Magnetic Resonance spectroscopy (NMR) indicated a weak interaction between Zn and GnRH [6-D-Phe].

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.

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.

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.

Twist-Bend Coupling and the Torsional Response of Double-Stranded DNA.

Recent magnetic tweezers experiments have reported systematic deviations of the twist response of double-stranded DNA from the predictions of the twistable wormlike chain model. Here we show, by means of analytical results and computer simulations, that these discrepancies can be resolved if a coupling between twist and bend is introduced. We obtain an estimate of 40±10  nm for the twist-bend coupling constant.