Interest in guanine (G) quadruplexes [1] has intensified over the past decade because they are found in human telomeres,[2] the chromosome ends that govern gene stability, and other parts of the genome, especially in promoters.[3] They have been shown to inhibit the activity of telomerase, an enzyme which maintains the proper length of telomere DNAs and is overexpressed in many types of cancer. [4] As such, the G-quadruplex DNA has been an attractive target for cancer therapy. [5] Despite the wide interest in the topology of G-quadruplexes, their physical properties are not well-understood. [6] There are large differences between the kinetic data obtained in solution using fluorescence and on surfaces using surface plasmon resonance (SPR) approaches, [7] possibly due to destabilization by fluorophore labelling [7a-c] and surface hindrance in the indirect hybridization based SPR approach.[7d] The kinetics and thermodynamics are expected to be different for G-quadruplexes formed from uni-, bi-, and tetra-molecular DNA strands. Furthermore, G-quadruplexes formed from a single DNA strand may have different conformations, [6.7] where minor changes in the DNA sequence may result in significant differences in their thermodynamic properties. [6] By taking measurements directly on unlabelled individual single molecules, one at a time, avoiding ensemble averaging, single-molecule (SM) force spectroscopy (FS) based on atomic force microscopy (AFM) has been demonstrated as a powerful tool to gain insights to a wide range of biological problems: protein folding, [8] protein-ligand interactions, [9] and DNA base pairing. [10] Given the large number of G-quadruplex topologies possible,[1.2] SM-FS can potentially provide unique insights into their structure, function, and stability. Further, the recent development of theoretical analysis for SM approaches has enabled reliable estimates of the kinetic and thermodynamic parameters. [11] To the best of our knowledge, the AFM SM-FS has not been used to study G-quadruplex.[12] Herein, we present the first AFM based SM-FS study on a bi-molecular G-quadruplex system.We used a bi-molecular G-quadruplex formed between the AFM probe and a gold surface as the model system in this SM-FS study (see Fig. 1). The DNA sequences and their abbreviations are given in NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript surface were functionalized with a self-assembled monolayer (SAM) of the 3G or 4G DNA, [13] each having two G-rich domains, diluted by the spacer DNA at a ratio of 1:5.[13] Such dilution greatly reduces the possibility for the 3G/4G DNAs from forming intra-surface quadruplexes,[13d] while ensuring the forming of an inter-surface G-quadruplex when the probe and surface are brought into close proximity, and the observation of specific Gquadruplex SM rupture events when they are pulled apart. A representative force-distance curve for the 4G/3G DNAs is shown in Fig. 1, which highlights an adhesion event followed by a stretching and the rupture of the G-qu...

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Force spectroscopy allows to test out the free energy landscapes of molecular interactions by repeatedly applying a load to the molecular interaction and detecting the rupture events. At present the dependency of the rupture forces on the pulling speed or the shape of the detected rupture force distributions are analyzed to get information about the underlying free energy landscape. But all of these models contain approximations and basic assumptions. We present a fast and completely model free way to extract the force dependent dissociation rates and free energies directly from the force curve data. Using the Ni-NTA-His6 interaction as a model system and comparing the resulting parameters with results from other techniques, we demonstrate the correctness and practicability of this method. The presented approach, which is applicable to any force spectroscopic methods, makes it possible to test or validate directly any energy landscape models without any basic assumptions.