Spatial recognition and mapping of proteins using DNA aptamers

Analytical Chemistry, 2012

Aptamers that bind small molecules can serve as basic biosensing platforms. Evaluation of the binding constant between an aptamer and a small molecule helps to determine the effectiveness of the aptamer-based sensors. Binding constants are often measured by a series of experiments with varying ligand or aptamer concentrations. Such experiments are time-consuming, material nonprudent, and prone to low reproducibility. Here, we use laser tweezers to determine the dissociation constant for aptamer−ligand interactions at the single-molecule level from only one ligand concentration. Using an adenosine 5′-triphosphate disodium salt (ATP) binding aptamer as an example, we have observed that the mechanical stabilities of aptamers bound with ATP are higher than those without a ligand. Comparison of the change in free energy of unfolding (ΔG unfold ) between these two aptamers yields a ΔG of 33 ± 4 kJ/mol for the binding. By applying a Hess-like cycle at room temperature, we obtained a dissociation constant (K d ) of 2.0 ± 0.2 μM, a value consistent with the K d obtained from our equilibrated capillary electrophoresis (CE) (2.4 ± 0.4 μM) and close to that determined by affinity chromatography in the literature (6 ± 3 μM). We anticipate that our laser tweezers and CE methodologies may be used to more conveniently evaluate the binding between receptors and ligands and also serve as analytical tools for force-based biosensing.

Journal of Structural Biology, 1997

During the last years, atomic force microscopy (AFM) has developed from a microscopy tool for solid state surface science towards a method employed in many scientific disciplines such as biology to investigate individual molecules on a nanometer scale. This article describes the current status of the imaging possibilities of AFM on RNA, IgG and gold-labelled cell adhesion molecules, as well as of measurements of intermolecular binding forces between biomolecules in order to investigate their molecular structure, function and elasticity. _____________________________________ 165 Volume 119

Micron, 2014

Probing the interaction forces that drive biomolecular recognition on cell surfaces is essential for understanding diverse biological processes. Force spectroscopy has been a widely used dynamic analytical technique, allowing measurement of such interactions at the molecular and cellular level. The capabilities of working under near physiological environments, combined with excellent force and lateral resolution make atomic force microscopy (AFM)-based force spectroscopy a powerful approach to measure biomolecular interaction forces not only on non-biological substrates, but also on soft, dynamic cell surfaces. Over the last few years, AFM-based force spectroscopy has provided biophysical insight into how biomolecules on cell surfaces interact with each other and induce relevant biological processes. In this review, we focus on describing the technique of force spectroscopy using the AFM, specifically in the context of probing cell surfaces. We summarize recent progress in understanding the recognition and interactions between macromolecules that may be found at cell surfaces from a force spectroscopy perspective. We further discuss the challenges and future prospects of the application of this versatile technique.

Journal of Molecular Recognition, 2007

The capability of atomic force microscopes (AFM) to generate atomic or nanoscale resolution images of surfaces has deeply transformed the study of materials. However, high resolution imaging of biological systems has proved more difficult than obtaining atomic resolution images of crystalline surfaces. In many cases, the forces exerted by the tip on the molecules (1-10 nN) either displace them laterally or break the noncovalent bonds that hold the biomolecules together. Here, we apply a force microscope concept based on the simultaneous excitation of the first two flexural modes of the cantilever. The coupling of the modes generated by the tip-molecule forces enables imaging under the application of forces ($35 pN) which are smaller than those needed to break noncovalent bonds. With this instrument we have resolved the intramolecular structure of antibodies in monomer and pentameric forms. Furthermore, the instrument has a force sensitivity of 0.2 pN which enables the identification of compositional changes along the protein fragments.

Single-molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2-adrenergic receptor (β2-AR), a G protein-coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2-AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright © 2014 John Wiley & Sons, Ltd.

ACS Nano, 2017

Single-molecule force spectroscopy (SMFS) is a powerful technique to characterize the energy landscape of individual proteins, the mechanical properties of nucleic acids, and the strength of receptor-ligand interactions. Atomic-force-microscopy (AFM)-based SMFS benefits from ongoing progress in improving the precision and stability of cantilevers and the AFM itself. Underappreciated is that the accuracy of such AFM studies remains hindered by inadvertently stretching molecules at an angle while measuring only the vertical component of the force and extension, degrading both measurements. This inaccuracy is particularly problematic in AFM studies using double-stranded DNA and RNA due to their large persistence length (p ≈ 50 nm), often limiting such studies to other SMFS platforms (e.g., custom-built optical and magnetic tweezers). Here, we developed an automated algorithm that aligns the AFM tip above the DNA's attachment point to a coverslip. Importantly, this algorithm was performed at low force (10-20 pN) and relatively fast (15-25 s), preserving the connection between the tip and the target molecule. Our data revealed large uncorrected lateral offsets for 100-nm and 650-nm DNA molecules [24 ± 18 nm (mean ± std. dev.) and 180 ± 110 nm, respectively]. Correcting this offset yielded a 3-fold improvement in accuracy and precision when characterizing DNA's overstretching transition. We also demonstrated high throughput by acquiring 88 geometrically corrected force-extension curves of a single, individual 100-nm DNA molecule in ~40 min and versatility by aligning polyprotein-and PEG-based protein-ligand assays. Importantly, our software-based algorithm was implemented on a commercial AFM, so it can be broadly adopted. More generally, this work illustrates how to enhance AFM-based SMFS by developing more sophisticated data-acquisition protocols.

Applied Surface Science, 2019

Previous literature on dynamic force spectroscopy of the thrombin-aptamer complex has shown significantly different force magnitudes even at similar AFM tip velocities, implying the need for a more elaborate evaluation of the force interaction. In this study, we employ a combination of experimental and computational methods to reveal comprehensively how the unbinding force can possibly depend on the loading rate, coverage density, binding rate, linker stiffness and binding buffer composition. In addition, we utilize enhanced analytical techniques that include autocorrelation function, probability density convolution, and Poisson distribution to determine the dissociation force quanta of multiple bond dissociation from force spectroscopy experiments. We then predict the unbinding force measured in the experiments by using atomistic simulation data with significantly broader loading rate range following a continuum model. The dissociation forces of thrombin-aptamer complex are found to be lower than forces associated with the breakdown of G-quadruplex on aptamer, while comparable to forces associated with DNA melting, which indicates the complex dissociation could be more likely attributed to the bond breakage between thrombin and aptamer, rather than the collapse of G-quadruplex on aptamer.

Angewandte Chemie International Edition, 2008

The forces governing cell-cell adhesion are vitally important to many biological processes, including cell differentiation, tissue growth, tumorigenesis, and proper functioning of the vertebrate immune response. The strengths of these interactions are typically characterized through the attachment of single living cells to probes that are capable of force measurement, such as suction micropipettes. More recently, optical tweezers have been applied to capture single cells and to measure these forces with high accuracy, but this technique is limited to applying forces in the piconewton range. Atomic force microscopy (AFM) provides an attractive alternative to these methods, because it is capable of quantifying forces in the piconewton to nanonewton range, and this technique has indeed been used to measure the mechanical properties of live single cells and to study adhesion forces at the single-cell level. Several fundamental adhesion measurements have been achieved by coating AFM cantilevers with fibronectin or lectins that bind to carbohydrate moieties on the cell surface, but especially in the latter case the cell-binding molecules themselves have been reported to have a degree of cytotoxicity that can influence the cellular properties being evaluated. Thus, while these studies highlight the utility of AFM for the measurement of cell receptor-ligand interactions, an expanded set of cantilever attachment methods will be needed for the study of cell-cell interactions over widely varying time scales.

Trends in Cell Biology, 1999

Ultramicroscopy, 2003

The distribution of the receptor-associated protein (RAP) binding protein and the adhesion forces between RAP and its binding protein on living fibroblast cells were examined using an atomic force microscope (AFM). The distribution of RAP binding protein was obtained on 256 (16 Â 16) locations in 2 Â 2 mm sections over the surface of living cells. The adhesion forces between RAP and the binding protein were measured with an AFM tip functionalized with RAP. In the presence of RAP in the scanning solution, the number of force curves with large adhesion force decreased. These results indicate that the adhesive forces observed here represent specific binding between RAP and the binding protein. This method will be a useful application of AFM to examine receptors on cell surfaces in high resolution. r