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

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

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

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

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

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

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

Functional characterization of infiltrating T lymphocytes in human hepatic allografts

We have employed recently developed techniques in T-cell culturing to study the nature and function of infiltrating hepatic allograft T cells. Using the rationale that intragraft T cells are activated during cell mediated damage to the allograft, we were able to show that these cells would propagate and remain functionally active in the presence of the T-cell growth factor, IL-2. In several instances, phenotyiic analysis of cells grown in this manner was very similar to that found within the graft. Both proliferative and cytotoxic responses could be detected from the cultured cell lines. The majority of the proliferative responses were donor-directed and immunogenetic analysis could define donor-directed HLA reactivity, to either class I or class II antigens, or both. Monoclonal anti-HLA antibodies inhibition profiles verified the apparent HLA reactivity. In a smaller percentage of cases, only IL-2 responsiveness could be detected, and no HLA reactivity could be determined. Cytotoxicity could be detected against both class I and class II antigens, however, those cells which demonstrated a greater magnitude of donor-directed cytotoxicity appeared to be directed against class I antigens. A significant correlation between donor-directed proliferation of biopsy cultured lymphocytes and cellular rejection was found. This model appears to be useful in delineating functions of the intragraft T-cell population during rejection. © 1986

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