Toward an Effective Control of DNA's Submolecular Conformation on a Surface

DNA adopts different conformations based on its environment. We reveal conditions that either preserve the DNA's physiological B-conformation, even upon its placement in UHV, or lead to a partial B-form to A-form reorganization upon DNA's deposition on a surface. We use high resolution AFM to image DNA with a well-defined number of base pairs deposited on mica. To enable the DNA's adhesion, we either add divalent cations to the DNA solution or functionalize the surface with a silane layer. The contour length of DNA on the silane is always in perfect agreement with the B-form conformation, whereas cation-deposited DNA is always, in some cases up to 20% shorter. We varied the equilibration time, the DNA length, and sequence and compared nicked to non-nicked molecules, thus identifying several factors controlling the DNA's length. We performed TERS measurements confirming spectroscopically that cation-deposited DNA undergoes a partial B-form to A-form conformational transition upon drying and pinpointed positions along the DNA where this transition was more probable, namely the ends of the molecules. Controlling the conformation of DNA is essential for its nanotechnology applications such as nanotemplating. Our findings could also shed a whole new light on DNA polymer physics, the mechanisms of DNA binding to surfaces, or the abundant contradictory data on DNA's electrical behavior.

Amplitude and phase reconstruction for Low-Energy Electron Holography of individual proteins

Hannah Julia Ochner

Single-molecule imaging methods are of importance in structural biology, and specifically in the imaging of proteins, since they can elucidate conformational variability and structural changes that might be lost in imaging methods relying on averaging processes. Low-energy electron holography (LEEH) is a promising technique for imaging individual proteins with negligible radiation damage, which can be combined with a sample preparation process by native electrospray ion beam deposition (native ES-IBD) ensuring the creation of chemically pure samples suitable for holographic imaging.The central step in the analysis of data measured by LEEH is the numerical reconstruction of the object from the experimentally acquired holograms.Full information about the imaged object consists of both amplitude and phase information imprinted on the scattered wave during the interaction of the electron beam with the molecule and encoded in the hologram created by the interference of the scattered wave and the transmitted incident wave. A propagation-based algorithm with the goal of reconstructing the wave field in the plane of the object is presented and applied to holograms of individual proteins prepared by ES-IBD and measured with a low-energy electron holography microscope. The information retrieved from the reconstruction of the amplitude distribution in the object plane is discussed by analysing holograms of antibodies, demonstrating that the inherent conformational variability of these molecules can be mapped by LEEH. The influence of the sample preparation process on the surface conformations is tracked by tuning the landing energy of the proteins during deposition.To complement the amplitude data, an iterative phase retrieval algorithm is implemented to reconstruct the phase distribution in the object plane along with the amplitude distribution. The algorithm's performance and robustness is thoroughly evaluated and simulations regarding multiple scattering effects and element-dependent variations in scattering strength are carried out to provide a reference for the interpretation of phase data retrieved from experimentally acquired holograms. The iterative phase retrieval algorithm is then applied to protein data, indicating that both molecular density and charges can be related to features in the phase reconstructions, while the presence of metals does not correlate with specific phase signals at the current resolution obtainable from the experimental data.Since proteins are inherently three-dimensional, approaches towards three-dimensional reconstruction schemes are discussed, which will be the focus of future work.

EPFL2022

Fabrication of solid-state Nanopores for DNA analysis

Maïté Volatier

One of revolutionary technologies to sequence single molecules, such as DNA, is using nanopore devices. A single-channel with nanometer-scale aperture allows an extremely rapid electric analysis of such single molecules. The objective of this work is to fabricate nanopores into robust, solid-state membranes to achieve better performances. The technological challenge is in the small required dimension of the nanopore, around 50-100 nm in diameter and depth. To reach this objective, we used two different techniques: the ion track technique and a microfabrication method associated with Focused Ion Beam (FIB). A literature review has been done on the ion track technique. Threshold values of the ion track formation have been found for different insulator materials (polymers, SiO2, SiN). We did experiments with the linear accelerator UNILAC (Universal Linear Accelerator) at the GSI (Darmstadt, Germany) where we irradiated our samples with heavy gold ions (11.4 MeV/nucleon). On very thin membranes, we expect that a direct perforation would occur, and we chose SiN membranes and SU-8 thin films as samples. However, the experiment of ion bombardment was not possible to be done early enough, thus TEM analysis have not been done yet. If the direct perforation by ion bombardment is not possible, a chemical etching step would be necessary. About the microfabrication method associated with Focused Ion Beam (FIB) perforation, we did experiments at the Center of Micro Nano Technology (CMI) in EPFL. First trial with FIB was done on SiN membranes. In order to obtain required aperture size, reduction of membrane thickness before perforation by FIB was attempted, but this process was difficult to be achieved because of charging. To overcome this dimension limitation we chose a silicon membrane. With silicon membranes, oxidation would be performed after perforation by FIB, since channel opening will be reduced by SiO2 growth. Moreover, this oxide layer allows a functionalisation of the inner surface of the nanopore by silane chemistry. That should improve the resolution of DNA analysis. Though the aperture size reduction by oxidation has not been done yet, refining and perforation by FIB will be done according to this idea. We should reach ca. 100 nm-aperture size. Even if validation measurements are not easy, our specifications should be reached with the oxidation. More experiments and analysis have to be performed for further improvement.

2004

Effects of base-pair sequence, nicks and gaps on DNA minicircle shapes

Arnaud Amzallag

DNA is a long polymer with the form of a double helix of about two nanometers diameter (2.10-9 meters). It is composed of nucleotides whose sequence carries the information of heredity. The four possible nucleotides have slightly different geometries, and their sequence along the DNA molecule are thought to influence the shape and the stiffness of the double helix on a scale of few tens to a few hundred base pairs, and thereby its biological activity. DNA minicircles (or miniplasmids) are closed loops with lengths of the order of a few tens or hundreds of base pairs in which the double helix bends around to close on its own tail with some number of twists. Its minimal energy shape and its energy of formation depend on the sequence of base pairs, and can be efficiently computed by polymer and rod models, if shape and stiffness parameters of DNA are provided as inputs. This structure is therefore an interesting experimental motif to test sequence-dependent mechanical properties of the DNA molecule. The purpose of this thesis is to explore the experimental methods to determine the shape and free energy of formation of DNA minicircles. The shapes have been be determined from cryo-electron micrographs. Efficiency of formation has been investigated by a novel method called annealing-cyclization. Cryo-electron microscopy allows observation of very small molecules (here 17 or 11 nm diameter) in vitrified water, in order to keep the 3D shape of the molecules as close as possible to the shape they had in solution. In Chapter 1, I used stereo cryo-electron micrographs to determine and compare the three-dimensional shape of 95 individual DNA minicircles of 158 base pairs, which were identical in sequence except within a 18 bp block which contained either a TATA box sequence or a CAP site. I defined the notion of shape-distance which I used to estimate the error of reconstruction, and I detected clusters of shapes using an appropriate sorting algorithm. However the cluster did not seem to be associated with the variable sequence (TATA or CAP). I then analyzed (in Chapter 2) two-dimensional shapes of shorter DNA minicircles (94 bp) determined by negative staining electron microscopy, designed with either two nicks (breaks in one of the strands) or two gaps (missing nucleotides) at diametrically opposite sites of the minicircles. I observed that the gapped minicircles have an elongated shape with respect to the nicked minicircles, and I used this result together with the results of atomic level molecular dynamics simulations to conclude that the gap flexibility, perhaps together with base unpairing at the gap site, is responsible for this elongated minicircle shape. Finally, I proposed a ligase-free assay to measure the minicircle formation efficiency, which can give a high yield of nicked minicircles. The method avoids the use of ligase and the associated concerns about the effect of ligase concentration on the measurements. I determined an equation from a chemical model of the reaction that fits the experimental data, and I defined the Ja factor which gives a measure of the cyclization yield independent of DNA initial concentration. This method seems to confirm that minicircles with two gaps cyclize more efficiently that minicircles with two nicks, probably because of the gap flexibility. As a perspective, the conclusions of this thesis could be used for the design of minicircle constructs whose shape would be sensitive enough to sequence mutation in order to be detected by electron microscopy. Such shapes could be then compared, thanks to the shape-distance tool defined herein, to shapes computed with known or putative DNA models.

EPFL2008