Nucleosome assembly and disassembly pathways in vitro.

Structural fluctuations of nucleosomes modulate the access to internal DNA in eukaryotic cells; clearly characterisation of this fundamental process is crucial to understanding gene regulation. Here we apply PhAST (Photochemical Analysis of Structural Transitions) to monitor at a base pair level, structural alterations induced all along the DNA upon histone binding or release. By offering the first reliable, detailed comparison of nucleosome assembly and disassembly in vitro, we reveal similarities and differences between the two processes. We identify multiple, sequential intermediate states characterised by specific PhAST signals whose localisation and amplitude reflect asymmetries of DNA/histone interactions with respect to the nucleosome pseudo dyad. These asymmetries involve not only the DNA extremities but also regions close to the pseudo dyad. Localisations of asymmetries develop in a consistent manner during both assembly and disassembly processes; they primarily reflect the DNA sequence effect on the efficiency of DNA-histone binding. More unexpectedly, the amplitude component of PhAST signals not only evolves as a function of intermediate states but does so differently between assembly and disassembly pathways. Our observation of differences between assembly and disassembly opens up new avenues to define the role of the DNA sequence in processes underlying the regulation of gene expression. Overall, we provide new insights into how the intrinsic properties of DNA are integrated into a holistic mechanism that controls chromatin structure.

Out-of-Equilibrium Measurements of Kinetic Constants on a Biosensor.

Conventional measurements of kinetic constants currently in use are performed at equilibrium and may require large volumes, especially at a low association rate constant kon. If the measurements are made out of equilibrium, the values obtained may be biased by dilution of the sample with the flow of the running buffer. In some applications, the available sample volume can be very critical and requires the development of tools to measure kinetic constants with low volumes. In this paper, by combining an experimental, numerical and modeling approach, we propose a surface plasmon resonance-based method that relies on an out-of-equilibrium measurement using the effect of dilution by flow to its advantage. This new method should have a significant impact in biochemistry and medical research.

Influence of the Sequestration Effect of CTAB on the Biofunctionalization of Gold Nanorods.

Gold nanorods (GNRs) can be functionalized with multiple biomolecules allowing efficient cell targeting and delivery into specific cells. However, various issues have to be addressed prior to any clinical applications. They involve controlled biofunctionalization to be able to deliver a known dose of drug by immobilizing a known number of active molecules to GNRs while protecting their surface from degradation. The most widely used synthesis method of GNRs is seed-mediated growth. It requires the use of cetyltrimethylammonium bromide (CTAB) that acts as a strong capping agent stabilizing the colloidal solution. The problem is that not only is CTAB cytotoxic to most cells but it also induces the sequestration of biomolecules in solution during the functionalization steps of GNRs. The presence of CTAB therefore makes it difficult to control the immobilization of biomolecules to GNRs while removing CTAB from the colloidal solution, leading to the aggregation of GNRs. The sequestration effect of ssDNA in solution by CTAB was studied in detail as a function of the CTAB concentration and the nature of the solution (water or buffer) using Forster resonance energy transfer as a detection tool. The conditions in which DNA sequestration did and did not occur could be clearly defined. Using gel electrophoresis, we could demonstrate how strongly the ssDNA sequestration effect in solution impacts the GNR surface biofunctionalization.

Photochemical analysis of structural transitions in DNA liquid crystals reveals differences in spatial structure of DNA molecules organized in liquid crystalline form.

The anisotropic shape of DNA molecules allows them to form lyotropic liquid crystals (LCs) at high concentrations. This liquid crystalline arrangement is also found in vivo (e.g., in bacteriophage capsids, bacteria or human sperm nuclei). However, the role of DNA liquid crystalline organization in living organisms still remains an open question. Here we show that in vitro, the DNA spatial structure is significantly changed in mesophases compared to non-organized DNA molecules. DNA LCs were prepared from pBluescript SK (pBSK) plasmid DNA and investigated by photochemical analysis of structural transitions (PhAST). We reveal significant differences in the probability of UV-induced pyrimidine dimer photoproduct formation at multiple loci on the DNA indicative of changes in major groove architecture.

Selection, characterization, and application of DNA aptamers for detection of Mycobacterium tuberculosis secreted protein MPT64.

Rapid detection of Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), is important for global control of this disease. Aptamers have emerged as a potential rival for antibodies in therapeutics, diagnostics and biosensing due to their inherent characteristics. The aim of the current study was to select and characterize single-stranded DNA aptamers against MPT64 protein, one of the predominant secreted proteins of Mtb pathogen. Aptamers specific to MPT64 protein were selected in vitro using systematic evolution of ligands through exponential enrichment (SELEX) method. The selection was started with a pool of ssDNA library with randomized 40-nucleotide region. A total of 10 cycles were performed and seventeen aptamers with unique sequences were identified by sequencing. Dot Blot analysis was performed to monitor the SELEX process and to conduct the preliminary tests on the affinity and specificity of aptamers. Enzyme linked oligonucleotide assay (ELONA) showed that most of the aptamers were specific to the MPT64 protein with a linear correlation of R2 = 0.94 for the most selective. Using Surface Plasmon Resonance (SPR), dissociation equilibrium constant KD of 8.92 nM was obtained. Bioinformatics analysis of the most specific aptamers revealed the existence of a conserved as well as distinct sequences and possible binding site on MPT64. The specificity was determined by testing non-target ESAT-6 and CFP-10. Negligible cross-reactivity confirmed the high specificity of the selected aptamer. The selected aptamer was further tested on clinical sputum samples using ELONA and had sensitivity and specificity of 91.3% and 90%, respectively. Microscopy, culture positivity and nucleotide amplification methods were used as reference standards. The aptamers studied could be further used for the development of medical diagnostic tools and detection assays for Mtb.

High-resolution biophysical analysis of the dynamics of nucleosome formation.

We describe a biophysical approach that enables changes in the structure of DNA to be followed during nucleosome formation in in vitro reconstitution with either the canonical "Widom" sequence or a judiciously mutated sequence. The rapid non-perturbing photochemical analysis presented here provides 'snapshots' of the DNA configuration at any given moment in time during nucleosome formation under a very broad range of reaction conditions. Changes in DNA photochemical reactivity upon protein binding are interpreted as being mainly induced by alterations in individual base pair roll angles. The results strengthen the importance of the role of an initial (H3/H4)2 histone tetramer-DNA interaction and highlight the modulation of this early event by the DNA sequence. (H3/H4)2 binding precedes and dictates subsequent H2A/H2B-DNA interactions, which are less affected by the DNA sequence, leading to the final octameric nucleosome. Overall, our results provide a novel, exciting way to investigate those biophysical properties of DNA that constitute a crucial component in nucleosome formation and stabilization.

Surface plasmon resonance imaging reveals multiple binding modes of Agrobacterium transformation mediator VirE2 to ssDNA.

VirE2 is the major secreted protein of Agrobacterium tumefaciens in its genetic transformation of plant hosts. It is co-expressed with a small acidic chaperone VirE1, which prevents VirE2 oligomerization. After secretion into the host cell, VirE2 serves functions similar to a viral capsid in protecting the single-stranded transferred DNA en route to the nucleus. Binding of VirE2 to ssDNA is strongly cooperative and depends moreover on protein-protein interactions. In order to isolate the protein-DNA interactions, imaging surface plasmon resonance (SPRi) studies were conducted using surface-immobilized DNA substrates of length comparable to the protein-binding footprint. Binding curves revealed an important influence of substrate rigidity with a notable preference for poly-T sequences and absence of binding to both poly-A and double-stranded DNA fragments. Dissociation at high salt concentration confirmed the electrostatic nature of the interaction. VirE1-VirE2 heterodimers also bound to ssDNA, though by a different mechanism that was insensitive to high salt. Neither VirE2 nor VirE1-VirE2 followed the Langmuir isotherm expected for reversible monomeric binding. The differences reflect the cooperative self-interactions of VirE2 that are suppressed by VirE1.

DNA base pair resolution measurements using resonance energy transfer efficiency in lanthanide doped nanoparticles.

Lanthanide-doped nanoparticles are of considerable interest for biodetection and bioimaging techniques thanks to their unique chemical and optical properties. As a sensitive luminescence material, they can be used as (bio) probes in Förster Resonance Energy Transfer (FRET) where trivalent lanthanide ions (La3+) act as energy donors. In this paper we present an efficient method to transfer ultrasmall (ca. 8 nm) NaYF4 nanoparticles dispersed in organic solvent to an aqueous solution via oxidation of the oleic acid ligand. Nanoparticles were then functionalized with single strand DNA oligomers (ssDNA) by inducing covalent bonds between surface carboxylic groups and a 5' amine modified-ssDNA. Hybridization with the 5' fluorophore (Cy5) modified complementary ssDNA strand demonstrated the specificity of binding and allowed the fine control over the distance between Eu3+ ions doped nanoparticle and the fluorophore by varying the number of the dsDNA base pairs. First, our results confirmed nonradiative resonance energy transfer and demonstrate the dependence of its efficiency on the distance between the donor (Eu3+) and the acceptor (Cy5) with sensitivity at a nanometre scale.

Post-synthesis reshaping of gold nanorods using a femtosecond laser.

This work describes the interaction between femtosecond laser pulses (~130 fs, 800 nm) and gold nanorods (NRs) leading to reshaping of the NRs. We focus on the investigation of structural changes of the NRs and the parameters influencing the reshaping, like surface modification using sodium sulphide, laser power and the position of the longitudinal surface plasmon resonance band (l-SPR) with respect to the laser wavelength. A thermogravimetric analysis experiment is performed to examine changes in the composition of NRs upon heating. A new type of banana-shaped NPs is described and the conditions of their appearance are discussed.

SPRi-based strategy to identify specific biomarkers in systemic lupus erythematosus, rheumatoid arthritis and autoimmune hepatitis.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is a target for antinuclear autoantibodies in systemic Lupus erythematosus (SLE), rheumatoid arthritis (RA), and autoimmune hepatitis (AIH). AIM: To monitor molecular interactions between peptides spanning the entire sequence of hnRNP A2/B1 and sera from patients and healthy controls. METHODS: Sera from 8 patients from each pathology and controls were passed across a surface plasmon resonance Imagery (SPRi) surface containing 39 overlapping peptides of 17 mers covering the human hnRNP B1. Interactions involving the immobilised peptides were followed in real time and dissociation rate constants k(off) for each interaction were calculated. RESULTS: Several significant interactions were observed: i) high stability (lower k(off) values) between P55₋70 and the AIH sera compared to controls (p= 0.003); ii) lower stability (higher k(off) values) between P118₋133 and P262₋277 and SLE sera, P145₋160 and RA sera compared to controls (p=0.006, p=0.002, p=0.007). The binding curves and k(off) values observed after the formation of complexes with anti-IgM and anti-IgG antibodies and after nuclease treatment of the serum indicate that i) IgM isotypes are prevalent and ii) nucleic acids participate in the interaction between anti-hnRNAP B1 and P55₋70 and also between controls and the peptides studied. CONCLUSIONS: These results indicate that P55₋70 of hnRNP B1 is a potential biomarker for AIH in immunological tests and suggest the role of circulating nucleic acids, (eg miRNA), present or absent according to the autoimmune disorders and involved in antigen-antibody stability.

Efficient antifouling surface for quantitative surface plasmon resonance based biosensor analysis.

Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractive to analysis due to the prevalent high degree of non-specific binding. In this manuscript we explore the dynamic process of the formation of self-assembled monolayers and optimize physical and chemical properties thus reducing considerably non-specific binding while maintaining the integrity of the immobilized biomolecules. As a result, analysis of specific binding of analytes to immobilized target molecules is significantly facilitated.

Nanoengineering the second order susceptibility in semiconductor quantum dot heterostructures.

We study second-harmonic generation from single CdTe/CdS core/shell rod-on-dot nanocrystals with different geometrical parameters, which allow to fine tune the nonlinear properties of the nanostructure. These hybrid semiconductor-semiconductor nanoparticles exhibit extremely strong and stable second-harmonic emission, although the size of CdTe core is still within the strong quantum confinement regime. The orientation sensitive polarization response is analyzed by means of a pointwise additive model of the third-order tensors associated to the nanoparticle components. These findings prove that engineering of semiconducting complex heterostructures at the single nanoparticle scale can lead to extremely bright nanometric nonlinear light sources.

Characterisation of peptide microarrays for studying antibody-antigen binding using surface plasmon resonance imagery.

BACKGROUND: Non-specific binding to biosensor surfaces is a major obstacle to quantitative analysis of selective retention of analytes at immobilized target molecules. Although a range of chemical antifouling monolayers has been developed to address this problem, many macromolecular interactions still remain refractory to analysis due to the prevalent high degree of non-specific binding. We describe how we use the dynamic process of the formation of self assembling monolayers and optimise physical and chemical properties thus reducing considerably non-specific binding and allowing analysis of specific binding of analytes to immobilized target molecules. METHODOLOGY/PRINCIPAL FINDINGS: We illustrate this approach by the production of specific protein arrays for the analysis of interactions between the 65kDa isoform of human glutamate decarboxylase (GAD65) and a human monoclonal antibody. Our data illustrate that we have effectively eliminated non-specific interactions with the surface containing the immobilised GAD65 molecules. The findings have several implications. First, this approach obviates the dubious process of background subtraction and gives access to more accurate kinetic and equilibrium values that are no longer contaminated by multiphase non-specific binding. Second, an enhanced signal to noise ratio increases not only the sensitivity but also confidence in the use of SPR to generate kinetic constants that may then be inserted into van't Hoff type analyses to provide comparative DeltaG, DeltaS and DeltaH values, making this an efficient, rapid and competitive alternative to ITC measurements used in drug and macromolecular-interaction mechanistic studies. Third, the accuracy of the measurements allows the application of more intricate interaction models than simple Langmuir monophasic binding. CONCLUSIONS: The detection and measurement of antibody binding by the type 1 diabetes autoantigen GAD65 represents an example of an antibody-antigen interaction where good structural, mechanistic and immunological data are available. Using SPRi we were able to characterise the kinetics of the interaction in greater detail than ELISA/RIA methods. Furthermore, our data indicate that SPRi is well suited to a multiplexed immunoassay using GAD65 proteins, and may be applicable to other biomarkers.

A nanostructure made of a bacterial noncoding RNA.

Natural RNAs, unlike many proteins, have never been reported to form extended nanostructures, despite their wide variety of cellular functions. This is all the more striking, as synthetic DNA and RNA forming large nanostructures have long been successfully designed. Here, we show that DsrA, a 87-nt noncoding RNA of Escherichia coli, self-assembles into a hierarchy of nanostructures through antisense interactions of three contiguous self-complementary regions. Yet, the extended nanostructures, observed using atomic force microscopy (AFM) and fluorescence microscopy, are easily disrupted into >100 nm long helical bundles of DsrA filaments, including hundreds of DsrA monomers, and are surprisingly resistant to heat and urea denaturation. Molecular modeling demonstrates that this structural switch of DsrA nanostructures into filament bundles results from the relaxation of stored torsional constraints and suggests possible implications for DsrA regulatory function.

Interaction of self-assembled monolayers of DNA with electrons: HREELS and XPS studies.

We present results from high-resolution electron energy loss spectroscopy (HREELS) and XPS studies of self-assembled monolayers of DNA. The monolayers are well-organized and display sharp vibrational peaks in the HREEL spectra. The electrons interact mainly with the backbone of the DNA. The XPS results indicate that, in most of the samples studied, the phosphates on the DNA are not charged.

Electrical characterization of self-assembled single- and double-stranded DNA monolayers using conductive AFM.

We recently reported electrical transport measurements through double-stranded (ds)DNA molecules that are embedded in a self-assembled monolayer of single-stranded (ss)DNA and attached to a metal substrate and to a gold nanoparticle (GNP) on opposite ends. The measured current flowing through the dsDNA amounts to 220 nA at 2 V. In the present report we compare electrical transport through an ssDNA monolayer and dsDNA monolayers with and without upper thiol end-groups. The measurements are done with a conductive atomic force microscope (AFM) using various techniques. We find that the ssDNA monolayer is unable to transport current. The dsDNA monolayer without thiols in the upper end can transport low current on rare occasions and the dsDNA monolayer with thiols on both ends can transport significant current but with a much lower reliability and reproducibility than the GNP-connected dsDNA. These results reconfirm the ability of dsDNA to transport electrical current under the appropriate conditions, demonstrate the efficiency of an ssDNA monolayer as an insulating layer, and emphasize the crucial role of an efficient charge injection through covalent bonding for electrical transport in single dsDNA molecules.

Direct measurement of electrical transport through single DNA molecules of complex sequence.

Seemingly contradicting results raised a debate over the ability of DNA to transport charge and the nature of the conduction mechanisms through it. We developed an experimental approach for measuring current through DNA molecules, chemically connected on both ends to a metal substrate and to a gold nanoparticle, by using a conductive atomic force microscope. Many samples could be made because of the experimental approach adopted here, which enabled us to obtain reproducible results with various samples, conditions, and measurement methods. We present multi-leveled evidence for charge transport through 26-bp-long dsDNA of a complex sequence, characterized by S-shaped current-voltage curves that show currents >220 nA at 2 V. This significant observation implies that a coherent or band transport mechanism takes over for bias potentials leading to high currents (>1 nA).