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Single molecule experiments have demonstrated a progressive transition from a B-to an L-form helix as DNA is gently stretched and progressively unwound. The particular sequence of a DNA segment defines both base stacking and hydrogen bonding that affect the partitioning and conformations of the two phases. Naturally or artificially modified bases alter H-bonds and base stacking and DNA with diaminopurine (DAP) replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded DAP:T base pairs. Both unmodified and DAP-substituted DNA transitioned from a B-to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing and the ease of this transition may prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length than B-DNA. However, left-handed DAP-substituted DNA was twice as stiff as unmodified L-DNA. Unmodified DNA and DAP-substituted DNA have very distinct mechanical characteristics at physiological levels of negative supercoiling and tension.

2,6-diaminopurine (DAP) is a nucleobase analog of adenine. When incorporated into double-stranded DNA (dsDNA), it forms three hydrogen bonds with thymine. Rare in nature, DAP substitution alters the physical characteristics of a DNA molecule without sacrificing sequence specificity. Here, we show that in addition to stabilizing double-strand hybridization, DAP substitution also changes the mechanical and conformational properties of dsDNA. Thermal melting experiments reveal that DAP substitution raises melting temperatures without diminishing sequence-dependent effects. Using a combination of atomic force microscopy (AFM), magnetic tweezer (MT) nanomechanical assays, and circular dichroism spectroscopy, we demonstrate that DAP substitution increases the flexural rigidity of dsDNA yet also facilitates conformational shifts, which manifest as changes in molecule length. DAP substitution increases both the static and dynamic persistence length of DNA (measured by AFM and MT, respectively). In the static case (AFM), in which tension is not applied to the molecule, the contour length of DAP-DNA appears shorter than wild-type (WT)-DNA; under tension (MT), they have similar dynamic contour lengths. At tensions above 60 pN, WT-DNA undergoes characteristic overstretching because of strand separation (tension-induced melting) and spontaneous adoption of a conformation termed S-DNA. Cyclic overstretching and relaxation of WT-DNA at near-zero loading rates typically yields hysteresis, indicative of tension-induced melting; conversely, cyclic stretching of DAP-DNA showed little or no hysteresis, consistent with the adoption of the S-form, similar to what has been reported for GC-rich sequences. However, DAP-DNA overstretching is distinct from GC-rich overstretching in that it happens at a significantly lower tension. In physiological salt conditions, evenly mixed AT/GC DNA typically overstretches around 60 pN. GC-rich sequences overstretch at similar if not slightly higher tensi

The tethered particle motion (TPM) technique involves an analysis of the Brownian motion of a bead tethered to a slide by a single DNA molecule. We describe an improved experimental protocol with which to form the tethers, an algorithm for analyzing bead motion visualized using differential interference contrast microscopy, and a physical model with which we have successfully simulated such DNA tethers. Both experiment and theory show that the statistics of the bead motion are quite different from those of a free semiflexible polymer. Our experimental data for chain extension versus tether length fit our model over a range of tether lengths from 109 to 3477 base pairs, using a value for the DNA persistence length that is consistent with those obtained under similar solution conditions by other methods. Moreover, we present the first experimental determination of the full probability distribution function of bead displacements and find excellent agreement with our theoretical prediction. Our results show that TPM is a useful tool for monitoring large conformational changes such as DNA looping

Many proteins "bind'' DNA through positively charged amino acids on their surfaces. However, to overcome significant energetic and topological obstacles, proteins that bend or package DNA might also modulate the stiffness that is generated by repulsions between phosphates within DNA. Much previous work describes how ions change the flexibility of DNA in solution, but when considering macromolecules such as chromatin in which the DNA contacts the nucleosome core each turn of the double helix, it may be more appropriate to assess the. exibility of DNA on charged surfaces. Mica coated with positively charged molecules is a convenient substrate upon which the. exibility of DNA may be directly measured with a scanning force microscope. In the experiments described below, the. exibility of DNA increased as much as fivefold depending on the concentration and type of polyamine used to coat mica. Using theory that relates charge neutralization to. exibility, we predict that phosphate repulsions were attenuated by similar to 50% in the most flexible DNA observed. This simple method is an important tool for investigating the physiochemical causes and molecular biological effects of DNA. exibility, which affects DNA biochemistry ranging from chromatin stability to viral encapsulation.

This dissertation is a report on an interdisciplinary investigation consisting of an application of random walk techniques to problems in ecology, particularly to the spread of Hantavirus epidemic among rodents that live on an open terrain. The population of mice that we consider is made up of infectious disease-carrying mice and susceptible mice that are disease-free, and each mouse has its own home range around which it executes a random walk. We describe an event of infection transmission in such a population via reaction-diffusion theory. Our simple model consists of two mice, one infected and the other susceptible, the disease being passed upon encounter as the two mice move on the terrain. The existence of home ranges of the mice is included in the model by representing each mouse to be a Smoluchowski random walker. Such a simple model is appropriate for a dilute population where only one infected-susceptible mice pair is considered to meet at a time. However the calculation helps the understanding of underlying microscopic processes of an epidemic outbreak in an arbitrary population density. The two-mice model is formulated in an arbitrary number of dimensions and explicit calculation in 1-dimension is performed first. We uncover an interesting effect of the home ranges on the characteristics of infection-transmission event. We find that there is an optimal configuration of the home ranges for which infection-transmission occurs most efficiently. Furthermore, the practical application of our model to higher dimensions requires an extension of the theory to circumvent a seemingly well-known problem in reaction-diffusion theory that the `reaction' site cannot be a 0-dimensional object for problems considered in higher dimension than 1. We develop a detailed resolution and present a practical extension with an explicit calculation demonstrated in 2-dimensions. Our work is, thus, useful in two ways. One is the further development of reaction-diffusion theory to t

Tethered particle motion (TPM) experiments can be used to detect time-resolved loop formation in a single DNA molecule by measuring changes in the length of a DNA tether. Interpretation of such experiments is greatly aided by computer simulations of DNA looping which allow one to analyze the structure of the looped DNA and estimate DNA-protein binding constants specific for the loop formation process. We here present a new Monte Carlo scheme for accurate simulation of DNA configurations subject to geometric constraints and apply this method to Lac repressor mediated DNA looping, comparing the simulation results with new experimental data obtained by the TPM technique. Our simulations, taking into account the details of attachment of DNA ends and fluctuations of the looped subsegment of the DNA, reveal the origin of the double-peaked distribution of RMS values observed by TPM experiments by showing that the average RMS value for anti-parallel loop types is smaller than that of parallel loop types. The simulations also reveal that the looping probabilities for the anti-parallel loop types are significantly higher than those of the parallel loop types, even for loops of length 600 and 900 base pairs, and that the correct proportion between the heights of the peaks in the distribution can only be attained when loops with flexible Lac repressor conformation are taken into account. Comparison of the in silico and in vitro results yields estimates for the dissociation constants characterizing the binding affinity between O1 and Oid DNA operators and the dimeric arms of the Lac repressor. © 2014 Biton et al.

The applications of the scanning tunneling microscope (STM) in air are usually restricted to good conducting materials as clean metals, doped and passivated semiconductors, or to some molecular adsorbates deposited onto graphite. In order to study poor conducting materials as biological molecules, we have built a very low current STM. This instrument can routinely be operated at 0.1 pA while having a bandwidth of 7 kHz. The advantages of using very low currents are illustrated by imaging 5-nm-thick purple membranes. These membranes can only be imaged at currents smaller than 2 pA. © 1995 American Institute of Physics.