Nanocapillaries combined with optical tweezers as a single molecule technique for studying DNA-protein complexes

Interactions of proteins with DNA are essential for carrying out DNA's biological functions and performing a cellular cycle. Such processes as DNA replication, expression and repair are performed by an organised action of various proteins. To better understand the function of protein machinery many methods have been developed over the years. They can be divided into two categories: single molecule and bulk techniques. In comparison to bulk experiments, where the effect of an ensemble of proteins is measured, single molecule techniques analyse each molecule one by one. This fact allows to detect rare events and avoid averaging over the population. Moreover, some single molecule techniques can be used for mechanical manipulation of biomolecules, i.e. twisting, stretching, etc. The objective of this thesis was to make a single molecule technique combining nanocapillaries and optical tweezers for the characterisation of DNA-protein complexes in physiological conditions. There were three main steps in this thesis: 1) building and characterisation of the setup 2) using it for detection and characterisation of DNA-protein complexes and 3) localisation and discrimination of DNA-protein complexes. On the first step of the project we combined two single molecule techniques: optical tweezers and glass nanocapillaries. We characterised the electrophoretic force acting on DNA in this setup by using nanocapillaries with openings of different sizes, at different applied voltages and with DNA molecules of different lengths. We observed that the position-dependent electrophoretic force acting on the DNA depends on all above-mentioned parameters. We modelled the system and found out that this effect is due to a non-uniform distribution of the potential inside the nanocapillary, which originates from its elongated shape. After having built and characterised the setup, we detected proteins bound to DNA during their controlled translocation through the opening. The proteins were visualised by a sudden decrease in the force acting on the bare DNA followed up by its slow restoration when the capillary was moved further away. We made a stochastic model to explain this force profile. From the fits of the model to experimental results we extracted the effective charges of DNA-protein complexes inside the nanocapillary. In the case of all three proteins (RecA, EcoRI and RNAP) the effective charge was of opposite sign than the one in solution. We attributed this fact to the dominant impact of the drag force in comparison to the electrostatic force inside the nanocapillary. On the last step of the project we showed the ability to localise and discriminate DNA-protein complexes in our setup using dCas9 and RNAP proteins. During controlled translocation of the DNA-protein complexes we measured multiple parameters, including protein's location on the DNA, work required to translocate the complex, and conductance change. We demonstrated that the measured location of the proteins is shifted from the designed binding site. We made a model that explained this phenomenon and that can account for the shift in our experiments. In addition, protein-specific work and conductance parameters allowed us to discriminate between RNAP and dCas9 proteins

Identification of single nucleotides in MoS2 nanopores

Ultrathin membranes have drawn much attention due to their unprecedented spatial resolution for DNA nanopore sequencing. However, the high translocation velocity (3000-50000 nt/ms) of DNA molecules moving across such membranes limits their usability. To this end, we have introduced a viscosity gradient system based on room-temperature ionic liquids (RTILs) to control the dynamics of DNA translocation through a nanometer-size pore fabricated in an atomically thin MoS2 membrane. This allows us for the first time to statistically identify all four types of nucleotides with solid state nanopores. Nucleotides are identified according to the current signatures recorded during their transient residence in the narrow orifice of the atomically thin MoS2 nanopore. In this novel architecture that exploits high viscosity of RTIL, we demonstrate single-nucleotide translocation velocity that is an optimal speed (1-50 nt/ms) for DNA sequencing, while keeping the signal to noise ratio (SNR) higher than 10. Our findings pave the way for future low-cost and rapid DNA sequencing using solid-state nanopores.Comment: Manuscript 24 pages, 4 Figures Supporting Information 24 pages, 12 Figures, 2 Tables Manuscript in review Nature Nanotechnology since May 27th 201

Relevance of the Drag Force during Controlled Translocation of a DNA–Protein Complex through a Glass Nanocapillary

Combination of glass nanocapillaries with optical tweezers allowed us to detect DNA-protein complexes in physiological conditions. In this system, a protein bound to DNA is characterized by a simultaneous change of the force and ionic current signals from the level observed for the bare DNA. Controlled displacement of the protein away from the nanocapillary opening revealed decay in the values of the force and ionic current. Negatively charged proteins EcoRI, RecA, and RNA polymerase formed complexes with DNA that experienced electrophoretic force lower than the bare DNA inside nanocapillaries. Force profiles obtained for DNA-RecA in our system were different than those in the system with nanopores in membranes and optical tweezers. We suggest that such behavior is due to the dominant impact of the drag force comparing to the electrostatic force acting on a DNA-protein complex inside nanocapillaries. We explained our results using a stochastic model taking into account the conical shape of glass nanocapillaries

Single Molecule Localization and Discrimination of DNA–Protein Complexes by Controlled Translocation Through Nanocapillaries

Through the use of optical tweezers we performed controlled translocations of DNA-protein complexes through nanocapillaries. We used RNA polymerase (RNAP) with two binding sites on a 7.2 kbp DNA fragment and a dCas9 protein tailored to have five binding sites on lambda-DNA (48.5 kbp). Measured localization of binding sites showed a shift from the expected positions on the DNA that we explained using both analytical fitting and a stochastic model. From the measured force versus stage curves we extracted the non equilibrium work done during the translocation of a DNA-protein complex and used it to obtain an estimate of the effective charge of the complex. In combination with conductivity measurements, we provided a proof of concept for discrimination between different DNA protein complexes simultaneous to the localization of their binding sites

Measurement of the Position-Dependent Electrophoretic Force on DNA in a Glass Nanocapillary

The electrophoretic force on a single DNA molecule inside a glass nanocapillary depends on the opening size and varies with the distance along the symmetrical axis of the nanocapillary. Using optical tweezers and DNA-coated beads, we measured the stalling forces and mapped the position-dependent force profiles acting on DNA inside nanocapillaries of different sizes. We showed that the stalling force is higher in nanocapillaries of smaller diameters. The position-dependent force profiles strongly depend on the size of the nanocapillary opening, and for openings smaller than 20 nm, the profiles resemble the behavior observed in solid-state nanopores. To characterize the position-dependent force profiles in nanocapillaries of different sizes, we used a model that combines information from both analytical approximations and numerical calculations

ComEA Is Essential for the Transfer of External DNA into the Periplasm in Naturally Transformable Vibrio cholerae Cells

The DNA uptake of naturally competent bacteria has been attributed to the action of DNA uptake machineries resembling type IV pilus complexes. However, the protein(s) for pulling the DNA across the outer membrane of Gram-negative bacteria remain speculative. Here we show that the competence protein ComEA binds incoming DNA in the periplasm of naturally competent Vibrio cholerae cells thereby promoting DNA uptake, possibly through ratcheting and entropic forces associated with ComEA binding. Using comparative modeling and molecular simulations, we projected the 3D structure and DNAbinding site of ComEA. These in silico predictions, combined with in vivo and in vitro validations of wild-type and sitedirected modified variants of ComEA, suggested that ComEA is not solely a DNA receptor protein but plays a direct role in the DNA uptake process. Furthermore, we uncovered that ComEA homologs of other bacteria (both Gram-positive and Gram-negative) efficiently compensated for the absence of ComEA in V. cholerae, suggesting that the contribution of ComEA in the DNA uptake process might be conserved among naturally competent bacteria

Conversion of glucose to lactic acid derivatives with mesoporous Sn-MCM-41 and microporous titanosilicates

BACKGROUND: The production of value-added products from biomass has acquired increasing importance due to the high worldwide demand for chemicals and energy, uncertain petroleum availability and the necessity of finding environmentally friendly processes. This paper reports work on the synthesis of several catalysts for the conversion of glucose to methyl lactate. RESULTS: A MCM-41 type mesoporous material containing tin (Si/Sn = 55) was developed with a uniform ordered mesoporous structure, high specific surface area and high pore volume. Sn-MCM-41 was tested in three consecutive catalytic cycles to evaluate its reusability giving methyl lactate yields of 43%, 41% and 39%, in each cycle. The slightly reduction in activity could be explained by the reduction in the accessibility of active centers due to the adsorption of reaction products and structural changes. Microporous titanosilicates and MFI-type zeolite ZSM-5 showed a lower catalytic performance, but exfoliated materials gave higher yields of methyl lactate and pyruvaldehyde dimethyl acetal than their respective layered precursors. CONCLUSIONS: Sn-MCM-41 material showed good results in the conversion of glucose to methyl lactate over three catalytic cycles and exfoliated materials facilitated the access of glucose to the catalytic sites and fast desorption of products.The authors gratefully thank the Spanish Ministry of Economy and Competitiveness (MINECO) for financial support through project MAT2010-15870, as well as the Regional Government of Aragón (DGA), the Obra Social La Caixa (GA-LC-019/2011) and the European Social Fund (ESF). C. Casado also thanks MINECO for the ‘Ramón y Cajal’ program (RYC-2011-08550)