Wilfried Grange

Rapid and label-free nanomechanical detection of biomarker transcripts in human RNA

The availability of entire genome sequences has triggered the development of microarrays for clinical diagnostics that measure the expression levels of specific genes. Methods that involve labelling can achieve picomolar detection sensitivity, but they are costly, labour-intensive and time-consuming. Moreover, target amplification or biochemical labelling can influence the original signal. We have improved the biosensitivity of label-free cantilever-array sensors by orders of magnitude to detect mRNA biomarker candidates in total cellular RNA. Differential gene expression of the gene 1-8U, a potential marker for cancer progression or viral infections, has been observed in a complex background. The measurements provide results within minutes at the picomolar level without target amplification, and are sensitive to base mismatches. This qualifies the technology as a rapid method to validate biomarkers that reveal disease risk, disease progression or therapy response. We foreseee cantilever arrays being used as a tool to evaluate treatment response efficacy for personalized medical diagnostics.

Quantitative time-resolved measurement of membrane protein- ligand interactions using microcantilever array sensors

Membrane proteins are central to many biological processes, and the interactions between transmembrane protein receptors and their ligands are of fundamental importance in medical research. However, measuring and characterizing these interactions is challenging. Here we report that sensors based on arrays of resonating microcantilevers can measure such interactions under physiological conditions. A protein receptor--the FhuA receptor of Escherichia coli--is crystallized in liposomes, and the proteoliposomes then immobilized on the chemically activated gold-coated surface of the sensor by ink-jet spotting in a humid environment, thus keeping the receptors functional. Quantitative mass-binding measurements of the bacterial virus T5 at subpicomolar concentrations are performed. These experiments demonstrate the potential of resonating microcantilevers for the specific, label-free and time-resolved detection of membrane protein-ligand interactions in a micro-array format.

Temperature Dependence of Unbinding Forces between Complementary DNA Strands

Force probe techniques such as atomic force microscopy can directly measure the force required to rupture single biological ligand receptor bonds. Such forces are related to the energy landscape of these weak, noncovalent biological interactions. We report unbinding force measurements between complementary strands of DNA as a function of temperature. Our measurements emphasize the entropic contributions to the energy landscape of the bond.

Initiation of transcription-coupled repair characterized at single-molecule resolution

Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.

Interaction of cationic surfactants with DNA: a single-molecule study

The interaction of cationic surfactants with single dsDNA molecules has been studied using force-measuring optical tweezers. For hydrophobic chains of length 12 and greater, pulling experiments show characteristic features (e.g. hysteresis between the pulling and relaxation curves, force-plateau along the force curves), typical of a condensed phase (compaction of a long DNA into a micron-sized particle). Depending on the length of the hydrophobic chain of the surfactant, we observe different mechanical behaviours of the complex (DNA-surfactants), which provide evidence for different binding modes. Taken together, our measurements suggest that short-chain surfactants, which do not induce any condensation, could lie down on the DNA surface and directly interact with the DNA grooves through hydrophobic–hydrophobic interactions. In contrast, long-chain surfactants could have their aliphatic tails pointing away from the DNA surface, which could promote inter-molecular interactions between hydrophobic chains and subsequently favour DNA condensation.

Optical tweezers system measuring the change in light momentum flux

This article describes the design of a dual-beam optical tweezers (OT) instrument which, in contrast to conventional single-beam OT, directly measures the change in light momentum flux when a trapped object experiences a force. Consequently, no local calibration is needed to measure the force acting on a trapped particle. The instrument has a high trapping efficiency and forces up to 200 pN can be measured. In addition, the above-mentioned system operates in conjunction with a three-dimensional steerable single-beam OT.

DNA Mechanics Affected by Small DNA Interacting Ligands

We have investigated the mechanics of individual DNA strands exposed to DNA binding ligands. The interaction of these agents with individual dsDNA strands measured by optical tweezers clearly indicates the ligand-DNA binding mode. As expected, if the compound is intercalating then an increase of contour length is detected. Groove binders affect the overstretching capabilities of the formerly “naked” dsDNA strand. We interacted SYBR®Green I with naked dsDNA. The binding mode of this compound, which is used for nucleic acid gel staining, is not known. The mechanics of the interaction of SYBR® is revealed by optical tweezers experiments. The force extension curves on single dsDNA fragments show a groove-binding mode, which does not affect the contour length of the molecule but significantly alters the overstretching behaviour of the dsDNA.

Molecular Recognition and Adhesion of Individual DNA Strands Studied by Dynamic Force Microscopy

The development of versatile scanning probe methods such as atomic force microscopy (AFM) makes it today possible to study bio-adhesion on a single molecule level. In this paper, we present AFM-force-spectroscopy experiments on complementary DNA strands. From such experiments, intrinsic thermodynamical properties (energy landscape) of these weak non covalent bonds can be determined.

Fast quantitative single-molecule detection at ultralow concentrations.

The applicability of single-molecule fluorescence assays in liquids is limited by diffusion to concentrations in the low picomolar range. Here, we demonstrate quantitative single-molecule detection at attomolar concentrations within 1 min by excitation and detection of fluorescence through a single-mode optical fiber in presence of turbulent flow. The combination of high detectability and short measurement times promises applications in ultrasensitive assays, sensors, and point-of-care medical diagnostics.

Analyzing Gene Expression Using Combined Nanomechanical Cantilever Sensors

In diseases such as cancer or during viral infections gene expression is greatly altered. These changes in gene activity can be analysed at different levels of cellular activity, like transcription activation, transcription and translation. Currently, no simple method is available to detect all these biochemical signals simultaneously and rapidly. Micromechanical cantilever sensor array technology is applied, because it has the advantage that sample preconditioning like labelling and amplification is not required. Furthermore, DNA, RNA, protein or combinations thereof could be detected in parallel on a single cantilever array. With such a device, diagnosis and therefore treatment of diseases can be improved. Here we present successive detection of DNA hybridization and antigen using the same micromechanical cantilever sensor array.