Session X40: Focus Session: Single Molecule Biological Physics - Nucleic Acids

From force-fields to photons: MD simulations of dye-labeled nucleic acids and Monte Carlo modeling of FRET

Fluorescence resonance energy transfer (FRET) is a powerful technique for understanding the structural fluctuations and transformations of RNA, DNA and proteins. Molecular dynamics (MD) simulations provide a window into the nature of these fluctuations on a different, faster, time scale. We use Monte Carlo methods to model and compare FRET data from dye-labeled RNA with what might be predicted from the MD simulation. With a few notable exceptions, the contribution of fluorophore and linker dynamics to these FRET measurements has not been investigated. We include the dynamics of the ground state dyes and linkers in our study of a 16mer double-stranded RNA. Water is included explicitly in the simulation. Cyanine dyes are attached at either the 3' or 5' ends with a 3 carbon linker, and differences in labeling schemes are discussed.\\[4pt] Work done in collaboration with Peker Milas, Benjamin D. Gamari, and Louis Parrot.   

Quantifying screening ion excesses surrounding stretched, charged polymers

We present the results of a combined theoretical/experimental study in which we have applied thermodynamic identities to infer -- from single molecule force-extension curves taken at different salt concentrations -- how the number of screening ions associated with a charged polymer changes as a function of its end to end extension. This number, which can change only through non-linear screening mechanisms, turns out to depend non-trivially on both the concentration of salt and the inherent rigidity of the polymer. In the case of a flexible polymer, such as ssDNA, our data indicates that the excess can change substantially between the fully extended and globule states. The effect is reduced for semi-flexible polymers, such as dsDNA, at physiologically-relevant salt concentrations, but it can again become substantial at lower salt concentrations. Based on these findings, we argue that small ion entropic effects should often contribute substantially to free energy differences between the competing conformational states of charged polymers -- both \textit{in vivo} and in certain polymer-based materials systems.   

Euler buckling and nonlinear kinking of double-stranded DNA

Bare double-stranded DNA is a stiff biopolymer with a persistence length of roughly 53 nm under physiological conditions. Cells and viruses employ extensive protein machinery to overcome this stiffness and bend, twist, and loop DNA to accomplish tasks such as packaging, recombination, gene regulation, and repair. The mechanical properties of DNA are of fundamental importance to the mechanism and thermodynamics of these processes, but physiologically relevant curvature has been difficult to access experimentally. We designed and synthesized a DNA hairpin construct in which base-pairing interactions generated a compressive force on a short segment of duplex DNA, inducing Euler buckling followed by bending to thermally inaccessible radii of curvature. The efficiency of F\"{o}rster resonance energy transfer (FRET) between two fluorophores covalently linked to the hairpin indicated the degree of buckling. Bulk and single-molecule measurements yielded distinctly different force-compression curves for intact DNA and for strands with single nicks, base pair mismatches, and damage sites. These results suggest that changes in local mechanical properties may play a significant role in the recognition of these features by DNA-binding proteins.   

Measuring fluctuations in shear stretched DNAs using site specific labeling

We report a new technique for measuring the internal dynamics of surface tethered DNAs in shear flow. Previous studies have used end labeling or intercolating dyes which label the entire length of the DNA. Neither prior method can resolve the internal longitudinal fluctuations of the DNA. Our technique accomplishes this by site specific labeling of five sites in lambda phage DNA using EcoRI labeled with fluorescent quantum dots. We used our technique to determine the two point cross correlation functions of the longitudinal and transverse fluctuations of the DNA under shear flow. Our technique allows us to test current models of the non-equilibrium fluctuations of DNA in shear flow in a way previously inaccessible.   

Electric-field Assisted Deposition of the DNA on Polymer Surface

Recently, the interaction of DNA with surfaces has been widely studied for its range of applications, including mapping, sequencing and analyzing DNAs. In this study, the Lambda DNA molecules were aligned in 6:50(0.1M NaOH:0.02M MES) buffer solution with different electric fields and deposited onto polymethylmetacrylate (PMMA) surfaces by dipping and retracting PMMA coated silicon wafers into the solution. Electric field was set up with platinum wire and gold plated Si wafer. The DNA strands were dyed with YoYo-1 and observed using a fluorescence microscope. The efficiency of deposition was optimized with respect to DNA concentration, DNA length and electric field. The results indicate that the density and possibly the lengths of the DNA deposited on surface can be controlled by this method. Enhancement of adsorption density of greater than twenty-fold were found using electric field strengths of 10v/cm. This study is supported by NSF-DMR-MRSEC program.   

Using Microcontact Printing as a Novel Method for Patterned Dyeing of Surface-adsorbed DNA

We use microcontact printing (MCP)$^{1}$ to stain individual DNA molecules adsorbed and combed onto a polymer-coated silicon surface. Polydimethylsiloxane (PDMS) stamps with micron-sized features have been used to selectively stain lambda DNA molecules with SyBr Gold dye. DNA was deposited out of dilute solution onto polymethylmethacrylate (PMMA) layers, 70nm thick, spun-coated on Si wafers, producing linearly stretched and aligned molecules. The stamps were soaked in dye solutions for one minute, followed by wiping of excess solution with a swap. The stamp was pressed onto the surface, varying the pressure and time of application (typically 5-10 minutes) to control the staining. The DNA molecules were imaged with a fluorescence microscope equipped with a cooled CCD camera. Single molecules of DNA were successfully dyed and imaged with stamps having a grating pattern either parallel to or perpendicular to the DNA orientation. Supported by NSF-DMR MRSEC program.   

Stretching DNA Molecules on a Polymer Surface

DNA's stretched form is one of great importance to the study of its structural characteristics and sequence. In our experiment, we studied the effects of stretching on lambda DNA, deposited onto Polydimethyl siloxane (PDMS, silicone) using the evaporating drop method. The DNA was dyed with SyBr Gold dye, or YOYO dye, which does not drastically affect the stretching properties of the DNA molecules while being deposited. Different DNA concentrations were used to optimize the density of the DNA on the surface. Once deposited, the DNA was imaged using a confocal microscope, for further measurements and to image stretching, in situ. To stretch the DNA molecules after deposition onto PDMS, the PDMS sample was placed onto a modified linear stage, pinched at the ends. The DNA length was measured throughout stretching. The result shows we successfully stretched DNA strands by 68{\%} without breakage of the strands and without the strands coming off of the PDMS surface. This study is supported by NSF-DMR-MRSEC program.   

Polarization and Angle Dependence of Fluorescence from Aligned DNA

DNA molecules can be deposited and aligned on various surfaces and imaged by confocal microscopy when labeled with fluorescent dyes. SyBr Gold dye, is known to possess a high angle and polarization dependence. We measured the emission intensity for various incident angles as a function of incident polarization angle. Samples were created by means of dipping PMMA-coated silicon wafers into dyed DNA solutions with DC electric field setup or drop evaporation. The blue laser as the imaging light source was mounted on an optical rail with a polarizer with rotatable half wave plate to change the incident polarization relative to the DNA molecular orientation. When applied to samples dyed using SyBr Gold, a clear change in the intensity of imaged DNA strands was observed though a range of input polarization angle. We have shown that it is possible to optimize the conditions in which aligned DNA is imaged using confocal microscopy by varying the polarization and angle of incidence of laser light on the sample. This study is supported by NSF-DMR-MRSEC program.   

Calorimetric and Low-Frequency Dielectric Studies of Mesoscopic Ordering in Solutions of Engineered DNA Hairpin Fragments

Calorimetry (both AC and MDSC) from $20$ to $100$~$^o$C, as well as low-frequency ($0.1$ to $100$~kHz) isothermal dielectric measurements have been performed on solutions of DNA fragments as a function of concentration. Custom hairpin DNA fragments were obtained with $13$-base unit length and samples made in solution at various concentration. Results show a reproducible heat capacity $C_p$ signature on heating and cooling scans. This thermal behavior of a diluted oligonucleotide chain is very different from that seen for mesoscopic ordering of liquid crystals. The AC Cp peak vanishes and new features are revealed as the temperature scan rate is lowered to $0.017$~K~min$^{-1}$. The observed real, $\epsilon'$, and imaginary, $\epsilon''$, permittivity of the suspended DNA show features indicating low-frequency dynamics that in turn suggests large-scale ordering or agglomeration of the DNA hairpin loops.   

Modeling of DNA zipper reaction rates

DNA zippers are a thermodynamically driven system consisting of three DNA oligonucleotides. Two of the strands are designed to create a small helix the third is designed to invade and separated the helix. A zipper system consisting of a normal strand (N), a weak strand (W), and an opening strand (O). N is made up of normal DNA bases, while W is engineered with inosine bases substituted for guanine. Inosine forms one less hydrogen bond with cytosine than guanine. By varying the number and order of inosine, W is engineered to provide less than natural bonding affinities to N in forming the [N:W] helix. When O is introduced (a natural complement of N), it competitively displaces W from [N:W] and forms [N:O]. DNA zippers have been used to create new DNA devices such as springs and tweezers and to create functionalized DNA origami structures. Currently, The basic principles and interactions of DNA zippers are not well understood. Here we will report the results on an investigation of several different DNA zipper constructs designed to aid in the creation of a mathematical prediction of the reaction rate for DNA zippers.   

Moving Beyond Watson-Crick Models of Coarse Grained DNA

DNA structure possesses several levels of complexity, ranging from the sequence of bases (primary structure) to base pairing (secondary structure) to its three-dimensional shape (tertiary structure) and can produce a wide variety of conformations in addition to canonical double stranded DNA. By including non-Watson-Crick interactions in a coarse-grained model, we developed a system that not only can capture the traditional B-form double helix, but also can adopt a wide variety of other DNA conformations. In our experimentally parameterized, coarse-grained DNA model we are able to reproduce the microscopic features of double-stranded DNA without the need for explicit constraints and capture experimental melting curves for a number of short DNA hairpins. We demonstrate the utility of the model by simulating more complex tertiary structures such as the folding of the thrombin aptamer, which includes G-quartets, and strand invasion during triplex formation. Our results highlight the importance of non-canonical interactions in DNA coarse- grained models.   

Direct comparison of the theory of molecular solvation with molecular dynamics and experiment

The reference interaction site model (RISM) provides complete equilibrium sampling of bulk solvent and solvent around a solute of arbitrary shape and size at a fraction of the computational cost of explicit solvent molecular dynamics (MD). Though based on first principles, approximations must be made to achieve numerical solutions. In this study, we first compare RISM to MD and experimental results for bulk solutions of aqueous monovalent ions using the Joung-Cheatham parameters with SPC/E and TIP3P water subject to several approximate closures. Then, using the same parameters and approximations, we evaluate the distribution of water and ions around a 24 base pair strand of DNA, once again, comparing to MD results and experimental observables. In both cases RISM gives the correct qualitative behavior and, often, the correct quantitative behavior. However, this strongly depends on the closure relation used, with higher order, HNC-like closures usually giving better results.   

Information-based measure of chirality for biomolecules

Homochirality is a common property of biomolecules such as DNA, RNA and proteins. In particular, {\scshape d}-ribose and {\scshape d}-deoxyribose enantiomers are found within living cells, while their mirror images, the {\scshape l}-enantiomers, are not known to occur naturally even though the configurations are highly stable. On the other hand, proteins are formed by {\scshape l}-amino acids, not by their mirror images. Why? In this work, we propose the use of Fisher Information (FI) $I$ as a measure of chirality or dissimilarity between enantiomers. We performed Hartree-Fock (HF) and Density Functional Theory (DFT) calculations to obtain the electronic wave function $\Psi (x,y,z)$ and corresponding density function $\rho(x,y,z)$ for each of the natural and synthetic forms of oligoribonucleotides and alanine amino acid. The four wave functions $\Psi (x,y,z)$ are used to compute the FI evaluated from two different view points: a coherent viewpoint, which includes the phase part of each $\Psi (x,y,z)$, and an incoherent or classical viewpoint, which ignores the phase. Our goal is to describe the extent to which the information content in chiral molecules ({\scshape d}- and {\scshape l}-) plays a role in selecting one or the other isomer in nature.