Nanophotonic waveguide enhanced Raman spectroscopy of biological submonolayers

Characterizing a monolayer of biological molecules has been a major challenge. We demonstrate nanophotonic wave-guide enhanced Raman spectroscopy (NWERS) of monolayers in the near-infrared region, enabling real-time measurements of the hybridization of DNA strands and the density of sub-monolayers of biotin-streptavidin complex immobilized on top of a photonics chip. NWERS is based on enhanced evanescent excitation and collection of spontaneous Raman scattering near nanophotonic waveguides, which for a one centimeter silicon nitride waveguide delivers a signal that is more than four orders of magnitude higher in comparison to a confocal Raman microscope. The reduced acquisition time and specificity of the signal allows for a quantitative and real-time characterization of surface species, hitherto not possible using Raman spectroscopy. NWERS provides a direct analytic tool for monolayer research and also opens a route to compact microscope-less lab-on-a-chip devices with integrated sources, spectrometers and detectors fabricated using a mass-producible CMOS technology platform

Structure and mechanical characterization of DNA i-motif nanowires by molecular dynamics simulation

We studied the structure and mechanical properties of DNA i-motif nanowires by means of molecular dynamics computer simulations. We built up to 230 nm long nanowires, based on a repeated TC5 sequence from crystallographic data, fully relaxed and equilibrated in water. The unusual stacked C*C+ stacked structure, formed by four ssDNA strands arranged in an intercalated tetramer, is here fully characterized both statically and dynamically. By applying stretching, compression and bending deformation with the steered molecular dynamics and umbrella sampling methods, we extract the apparent Young's and bending moduli of the nanowire, as wel as estimates for the tensile strength and persistence length. According to our results, the i-motif nanowire shares similarities with structural proteins, as far as its tensile stiffness, but is closer to nucleic acids and flexible proteins, as far as its bending rigidity is concerned. Furthermore, thanks to its very thin cross section, the apparent tensile toughness is close to that of a metal. Besides their yet to be clarified biological significance, i-motif nanowires may qualify as interesting candidates for nanotechnology templates, due to such outstanding mechanical properties.Comment: 25 pages, 1 table, 7 figures; preprint submitted to Biophysical Journa

Sequence Dependence of Transcription Factor-Mediated DNA Looping

DNA is subject to large deformations in a wide range of biological processes. Two key examples illustrate how such deformations influence the readout of the genetic information: the sequestering of eukaryotic genes by nucleosomes, and DNA looping in transcriptional regulation in both prokaryotes and eukaryotes. These kinds of regulatory problems are now becoming amenable to systematic quantitative dissection with a powerful dialogue between theory and experiment. Here we use a single-molecule experiment in conjunction with a statistical mechanical model to test quantitative predictions for the behavior of DNA looping at short length scales, and to determine how DNA sequence affects looping at these lengths. We calculate and measure how such looping depends upon four key biological parameters: the strength of the transcription factor binding sites, the concentration of the transcription factor, and the length and sequence of the DNA loop. Our studies lead to the surprising insight that sequences that are thought to be especially favorable for nucleosome formation because of high flexibility lead to no systematically detectable effect of sequence on looping, and begin to provide a picture of the distinctions between the short length scale mechanics of nucleosome formation and looping.Comment: Nucleic Acids Research (2012); Published version available at http://nar.oxfordjournals.org/cgi/content/abstract/gks473? ijkey=6m5pPVJgsmNmbof&keytype=re

Monitoring transient elastic energy storage within the rotary motors of single FoF1-ATP synthase by DCO-ALEX FRET

The enzyme FoF1-ATP synthase provides the 'chemical energy currency' adenosine triphosphate (ATP) for living cells. Catalysis is driven by mechanochemical coupling of subunit rotation within the enzyme with conformational changes in the three ATP binding sites. Proton translocation through the membrane-bound Fo part of ATP synthase powers a 10-step rotary motion of the ring of c subunits. This rotation is transmitted to the gamma and epsilon subunits of the F1 part. Because gamma and epsilon subunits rotate in 120 deg steps, we aim to unravel this symmetry mismatch by real time monitoring subunit rotation using single-molecule Forster resonance energy transfer (FRET). One fluorophore is attached specifically to the F1 motor, another one to the Fo motor of the liposome-reconstituted enzyme. Photophysical artifacts due to spectral fluctuations of the single fluorophores are minimized by a previously developed duty cycle-optimized alternating laser excitation scheme (DCO-ALEX). We report the detection of reversible elastic deformations between the rotor parts of Fo and F1 and estimate the maximum angular displacement during the load-free rotation using Monte Carlo simulationsComment: 14 pages, 7 figure

Ab initio RNA folding

RNA molecules are essential cellular machines performing a wide variety of functions for which a specific three-dimensional structure is required. Over the last several years, experimental determination of RNA structures through X-ray crystallography and NMR seems to have reached a plateau in the number of structures resolved each year, but as more and more RNA sequences are being discovered, need for structure prediction tools to complement experimental data is strong. Theoretical approaches to RNA folding have been developed since the late nineties when the first algorithms for secondary structure prediction appeared. Over the last 10 years a number of prediction methods for 3D structures have been developed, first based on bioinformatics and data-mining, and more recently based on a coarse-grained physical representation of the systems. In this review we are going to present the challenges of RNA structure prediction and the main ideas behind bioinformatic approaches and physics-based approaches. We will focus on the description of the more recent physics-based phenomenological models and on how they are built to include the specificity of the interactions of RNA bases, whose role is critical in folding. Through examples from different models, we will point out the strengths of physics-based approaches, which are able not only to predict equilibrium structures, but also to investigate dynamical and thermodynamical behavior, and the open challenges to include more key interactions ruling RNA folding.Comment: 28 pages, 18 figure

Extracting physical chemistry from mechanics: a new approach to investigate DNA interactions with drugs and proteins in single molecule experiments

In this review we focus on the idea of establishing connections between the mechanical properties of DNAligand complexes and the physical chemistry of DNA-ligand interactions. This type of connection is interesting because it opens the possibility of performing a robust characterization of such interactions by using only one experimental technique: single molecule stretching. Furthermore, it also opens new possibilities in comparing results obtained by very different approaches, in special when comparing single molecule techniques to ensemble-averaging techniques. We start the manuscript reviewing important concepts of the DNA mechanics, from the basic mechanical properties to the Worm-Like Chain model. Next we review the basic concepts of the physical chemistry of DNA-ligand interactions, revisiting the most important models used to analyze the binding data and discussing their binding isotherms. Then, we discuss the basic features of the single molecule techniques most used to stretch the DNA-ligand complexes and to obtain force x extension data, from which the mechanical properties of the complexes can be determined. We also discuss the characteristics of the main types of interactions that can occur between DNA and ligands, from covalent binding to simple electrostatic driven interactions. Finally, we present a historical survey on the attempts to connect mechanics to physical chemistry for DNA-ligand systems, emphasizing a recently developed fitting approach useful to connect the persistence length of the DNA-ligand complexes to the physicochemical properties of the interaction. Such approach in principle can be used for any type of ligand, from drugs to proteins, even if multiple binding modes are present

Traction force microscopy on soft elastic substrates: a guide to recent computational advances

The measurement of cellular traction forces on soft elastic substrates has become a standard tool for many labs working on mechanobiology. Here we review the basic principles and different variants of this approach. In general, the extraction of the substrate displacement field from image data and the reconstruction procedure for the forces are closely linked to each other and limited by the presence of experimental noise. We discuss different strategies to reconstruct cellular forces as they follow from the foundations of elasticity theory, including two- versus three-dimensional, inverse versus direct and linear versus non-linear approaches. We also discuss how biophysical models can improve force reconstruction and comment on practical issues like substrate preparation, image processing and the availability of software for traction force microscopy.Comment: Revtex, 29 pages, 3 PDF figures, 2 tables. BBA - Molecular Cell Research, online since 27 May 2015, special issue on mechanobiolog

Focus on chirality of HIV-​1 non-​nucleoside reverse transcriptase inhibitors

Chiral HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) are of great interest since one enantiomer is often more potent than the corresponding counterpart against the HIV-1 wild type (WT) and the HIV-1 drug resistant mutant strains. This review exemplifies the various studies made to investigate the effect of chirality on the antiretroviral activity of top HIV-1 NNRTI compounds, such as nevirapine (NVP), efavirenz (EFV), alkynyl- and alkenylquinazolinone DuPont compounds (DPC), diarylpyrimidine (DAPY), dihydroalkyloxybenzyloxopyrimidine (DABO), phenethylthiazolylthiourea (PETT), indolylarylsulfone (IAS), arylphosphoindole (API) and trifluoromethylated indole (TFMI) The chiral separation, the enantiosynthesis, along with the biological properties of these HIV-1 NNRTIs, are discussed