To explore the analytic relevance of unbinding force measurements between complementary DNA strands with an atomic force microscope, we measured the forces to mechanically separate a single DNA duplex under physiological conditions by pulling at the opposite 5-ends as a function of the loading rate (dynamic force spectroscopy). We investigated DNA duplexes with 10, 20, and 30 base pairs with loading rates in the range of 16-4,000 pN͞s. Depending on the loading rate and sequence length, the unbinding forces of single duplexes varied from 20 to 50 pN. These unbinding forces are found to scale with the logarithm of the loading rate, which is interpreted in terms of a single energy barrier along the mechanical separation path. The parameters describing the energy landscape, i.e., the distance of the energy barrier to the minimum energy along the separation path and the logarithm of the thermal dissociation rate, are found to be proportional to the number of base pairs of the DNA duplex. These single molecule results allow a quantitative comparison with data from thermodynamic ensemble measurements and a discussion of the analytic applications of unbinding force measurements for DNA.The direct measurement of forces between individual biological ligand receptor pairs is an emerging field. With the atomic force microscope (AFM), it has been possible to measure unbinding forces between single ligand receptor pairs, which are typically in the pico-Newton range (1-5). These sensitive force measurements can be performed with a high spatial resolution (5). However, the relevance of the force measurements as an (local) analytical tool depends on the understanding of the relationship between the measured forces and thermodynamic data characterizing the complex (6).The measured unbinding forces are not a fundamental property of a ligand-receptor pair but depend on the loading rate that is applied to the complex: If the load on the complex increases sufficiently slowly, there is time for thermal fluctuations to drive the system over the energy barrier, and the unbinding force will be small (7). A scaling of the force with the logarithm of the loading rate is expected for a single energy barrier along the unbinding path (8) and was found in AFM experiments on unfolding of protein domains (9, 10) and on rupture force measurements of the P-selectin͞ligand complex (11). With a different force probe technique, it has been possible to measure the loading rate dependence of the unbinding forces of biotin͞(strept)avidin complexes (12). In this system the loading rate dependence of the force is more complicated, indicating that more than one energy barrier is present along the separation path.The aim of our paper is to present a deeper understanding of the forces that arise if one separates the two strands of a DNA duplex by pulling at both 5Ј-ends. For this purpose, we have measured the loading rate dependence of the unbinding forces between complementary DNA strands to get information about the energy profile of the separation...
Antibody single-chain Fv fragment (scFv) molecules that are specific for f luorescein have been engineered with a C-terminal cysteine for a directed immobilization on a f lat gold surface. Individual scFv molecules can be identified by atomic force microscopy. For selected molecules the antigen binding forces are then determined by using a tip modified with covalently immobilized antigen. An scFv mutant of 12% lower free energy for ligand binding exhibits a statistically significant 20% lower binding force. This strategy of covalent immobilization and measuring well separated single molecules allows the characterization of ligand binding forces in molecular repertoires at the single molecule level and will provide a deeper insight into biorecognition processes.Atomic force microscopy (AFM) has been a versatile tool for imaging the surface structure of individual molecules and of molecular assemblies of a wide variety of biological specimens (1-5). Furthermore, molecular binding forces between a small number of various ligand and receptor molecules have been measured by AFM (6-15). In these force spectroscopy studies, one interacting partner is immobilized on the AFM cantilever and the other on a surface, and the interaction is probed with force-distance curves. The rupture force, the maximum force at the moment of detachment, which is obtained upon withdrawing the cantilever from the surface, is taken as a measure of the interaction between ligand and receptor.In this paper we report on combining AFM imaging and force spectroscopy at the level of individually selected and addressed molecules. We used recombinant antibody singlechain Fv (scFv) fragments as a versatile model system to study unbinding forces of single molecules. Antibody scFv fragments, in which the variable domain of the heavy chain (V H ) is fused by means of a (Gly 4 Ser) 3 linker to the variable domain of the light chain V L , are the minimal size antibody molecules that still comprise the complete antigen binding site (16). Unlike whole antibodies, they do not contain additional domains whose unfolding under force may give rise to structural changes (17, 18) that might influence the unbinding event. scFv fragments are particularly interesting models, because they can be generated against all conceivable antigenic targets, and mutants with various binding properties can be engineered.The scFv proteins were immobilized in a directed orientation on an ultraflat gold surface, their position was detected with AFM, and then the binding force of a spatially well isolated molecule was measured by using a tip endowed with immobilized antigen. To achieve correct determinations of the interaction force between scFv fragments and the cognate antigen fluorescein, their immobilization was carefully designed such that no detachment from the surface occurred within the time of the experiment. Furthermore, by sufficiently spacing the individual molecules on the surface, interactions could be restricted to single protein molecules. Thus, individual mole...
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