Modeling the effect of intercalators on the high-force stretching behavior of DNA

DNA is structurally and mechanically altered by the binding of intercalator molecules. Intercalation strongly affects the force-extension behavior of DNA, in particular the overstretching transition. We present a statistical model that captures all relevant findings of recent force-extension experiments. Two predictions from our model are presented. The first suggests the existence of a novel hyper-stretching regime in the presence of intercalators and the second, a linear dependence of the overstretching force on intercalator concentration, is verified by re-analyzing available experimental data. Our model pins down the physical principles that govern intercalated DNA mechanics, providing a predictive understanding of its limitations and possibilities.Comment: 5 pages, 4 figure

Physics of viral dynamics

Viral capsids are often regarded as inert structural units, but in actuality they display fascinating dynamics during different stages of their life cycle. With the advent of single-particle approaches and high-resolution techniques, it is now possible to scrutinize viral dynamics during and after their assembly and during the subsequent development pathway into infectious viruses. In this Review, the focus is on the dynamical properties of viruses, the different physical virology techniques that are being used to study them, and the physical concepts that have been developed to describe viral dynamics

Fluctuating Nonlinear Spring Model of Mechanical Deformation of Biological Particles

We present a new theory for modeling forced indentation spectral lineshapes of biological particles, which considers non-linear Hertzian deformation due to an indenter-particle physical contact and bending deformations of curved beams modeling the particle structure. The bending of beams beyond the critical point triggers the particle dynamic transition to the collapsed state, an extreme event leading to the catastrophic force drop as observed in the force (F)-deformation (X) spectra. The theory interprets fine features of the spectra: the slope of the FX curves and the position of force-peak signal, in terms of mechanical characteristics --- the Young's moduli for Hertzian and bending deformations E_H and E_b, and the probability distribution of the maximum strength with the strength of the strongest beam F_b^* and the beams' failure rate m. The theory is applied to successfully characterize the $FX$ curves for spherical virus particles --- CCMV, TrV, and AdV

Mechanical Characterization of Liposomes and Extracellular Vesicles, a Protocol

Both natural as well as artificial vesicles are of tremendous interest in biology and nanomedicine. Small vesicles (<200 nm) perform essential functions in cell biology and artificial vesicles (liposomes) are used as drug delivery vehicles. Atomic Force Microscopy (AFM) is a powerful technique to study the structural properties of these vesicles. AFM is a well-established technique for imaging at nanometer resolution and for mechanical measurements under physiological conditions. Here, we describe the procedure of AFM imaging and force spectroscopy on small vesicles. We discuss how to image vesicles with minimal structural disturbance, and how to analyze the data for accurate size and shape measurements. In addition, we describe the procedure for performing nanoindentations on vesicles and the subsequent data analysis including mechanical models used for data interpretation

Real-time observation of DNA looping dynamics of Type IIE restriction enzymes NaeI and NarI

Many restriction enzymes require binding of two copies of a recognition sequence for DNA cleavage, thereby introducing a loop in the DNA. We investigated looping dynamics of Type IIE restriction enzymes NaeI and NarI by tracking the Brownian motion of single tethered DNA molecules. DNA containing two endonuclease recognition sites spaced a few 100 bp apart connect small polystyrene beads to a glass surface. The position of a bead is tracked through video microscopy. Protein-mediated looping and unlooping is then observed as a sudden specific change in Brownian motion of the bead. With this method we are able to directly follow DNA looping kinetics of single protein–DNA complexes to obtain loop stability and loop formation times. We show that, in the absence of divalent cations, NaeI induces DNA loops of specific size. In contrast, under these conditions NarI mainly creates non-specific loops, resulting in effective DNA compaction for higher enzyme concentrations. Addition of Ca(2+) increases the NaeI-DNA loop lifetime by two orders of magnitude and stimulates specific binding by NarI. Finally, for both enzymes we observe exponentially distributed loop formation times, indicating that looping is dominated by (re)binding the second recognition site

Revealing the Competition between Peeled-Ssdna, Melting Bubbles and S-DNA during DNA Overstretching using Fluorescence Microscopy

Understanding the structural changes occurring in double-stranded (ds)DNA during mechanical strain is essential to build a quantitative picture of how proteins interact and modify DNA. However, the elastic response of dsDNA to tension is only well-understood for forces < 65 pN. Above this force, torsionally unconstrained dsDNA gains ∼70% of its contour length, a process known as overstretching. The structure of overstretched DNA has proved elusive, resulting in a rich and controversial debate in recent years. At the centre of the debate is the question of whether overstretching yields a base-paired elongated structure, known as S-DNA, or instead forms single-stranded (ss)DNA via base-pair cleavage. Here, we show clearly, using a combination of fluorescence microscopy and optical tweezers, that both S-DNA and base-pair melted structures can exist, often concurrently, during overstretching. The balance between the two models is affected strongly by temperature and ionic strength. Moreover, we reveal, for the first time, that base-pair melting can proceed via two entirely different processes: progressive strand unpeeling from a free end in the backbone, or by the formation of ‘bubbles' of ssDNA, nucleating initially in AT-rich regions. We demonstrate that the mechanism of base-pair melting is governed by DNA topology: strand unpeeling is favored when there are free ends in the DNA backbone. Our studies settle a long running debate, and unite the contradictory dogmas of DNA overstretching. These findings have important implications for both medical and biological sciences. Force-induced melting transitions (yielding either peeled-ssDNA or melting bubbles) may play active roles in DNA replication and damage repair. Further, the ability to switch easily from DNA containing melting bubbles to S-DNA may be particularly advantageous in the cell, for instance during the formation of RNA within transcription bubbles. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved

Revealing the Competition between Peeled-Ssdna, Melting Bubbles and S-DNA during DNA Overstretching using Fluorescence Microscopy

Understanding the structural changes occurring in double-stranded (ds)DNA during mechanical strain is essential to build a quantitative picture of how proteins interact and modify DNA. However, the elastic response of dsDNA to tension is only well-understood for forces < 65 pN. Above this force, torsionally unconstrained dsDNA gains ∼70% of its contour length, a process known as overstretching. The structure of overstretched DNA has proved elusive, resulting in a rich and controversial debate in recent years. At the centre of the debate is the question of whether overstretching yields a base-paired elongated structure, known as S-DNA, or instead forms single-stranded (ss)DNA via base-pair cleavage. Here, we show clearly, using a combination of fluorescence microscopy and optical tweezers, that both S-DNA and base-pair melted structures can exist, often concurrently, during overstretching. The balance between the two models is affected strongly by temperature and ionic strength. Moreover, we reveal, for the first time, that base-pair melting can proceed via two entirely different processes: progressive strand unpeeling from a free end in the backbone, or by the formation of ‘bubbles' of ssDNA, nucleating initially in AT-rich regions. We demonstrate that the mechanism of base-pair melting is governed by DNA topology: strand unpeeling is favored when there are free ends in the DNA backbone. Our studies settle a long running debate, and unite the contradictory dogmas of DNA overstretching. These findings have important implications for both medical and biological sciences. Force-induced melting transitions (yielding either peeled-ssDNA or melting bubbles) may play active roles in DNA replication and damage repair. Further, the ability to switch easily from DNA containing melting bubbles to S-DNA may be particularly advantageous in the cell, for instance during the formation of RNA within transcription bubbles. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved