Mechanisms of Small Molecule-DNA Interactions Probed by Single-molecule Force Spectroscopy

There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA–ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules

A General Mechanism for Competitor-induced Dissociation of Molecular Complexes

The kinetic stability of non-covalent macromolecular complexes controls many biological phenomena. Here we find that physical models of complex dissociation predict that competitor molecules will, in general, accelerate the breakdown of isolated bimolecular complexes by occluding rapid rebinding of the two binding partners. This prediction is largely independent of molecular details. We confirm the prediction with single-molecule fluorescence experiments on a well-characterized DNA strand dissociation reaction. Contrary to common assumptions, competitor-induced acceleration of dissociation can occur in biologically relevant competitor concentration ranges and does not necessarily imply ternary association of competitor with the bimolecular complex. Thus, occlusion of complex rebinding may play a significant role in a variety of biomolecular processes. The results also show that single-molecule colocalization experiments can accurately measure dissociation rates despite their limited spatiotemporal resolution

Strong DNA Deformation Required for Extremely Slow DNA Threading Intercalation by a Binuclear Ruthenium Complex

DNA intercalation by threading is expected to yield high affinity and slow dissociation, properties desirable for DNA-targeted therapeutics. To measure these properties, we utilize single molecule DNA stretching to quantify both the binding affinity and the force-dependent threading intercalation kinetics of the binuclear ruthenium complex Δ,Δ-[μ‐bidppz‐(phen)4Ru2]4+ (Δ,Δ-P). We measure the DNA elongation at a range of constant stretching forces using optical tweezers, allowing direct characterization of the intercalation kinetics as well as the amount intercalated at equilibrium. Higher forces exponentially facilitate the intercalative binding, leading to a profound decrease in the binding site size that results in one ligand intercalated at almost every DNA base stack. The zero force Δ,Δ-P intercalation Kd is 44 nM, 25-fold stronger than the analogous mono-nuclear ligand (Δ-P). The force-dependent kinetics analysis reveals a mechanism that requires DNA elongation of 0.33 nm for association, relaxation to an equilibrium elongation of 0.19 nm, and an additional elongation of 0.14 nm from the equilibrium state for dissociation. In cells, a molecule with binding properties similar to Δ,Δ-P may rapidly bind DNA destabilized by enzymes during replication or transcription, but upon enzyme dissociation it is predicted to remain intercalated for several hours, thereby interfering with essential biological processes

Making Connections: Comparative Innovations in the Sciences

Looking into the Crystallin Ball: Elucidating the Role of aB-crystallin in Inflammasone Activation in Retinal Epithelial Cells Dr. Merideth Krevosky, Dr. Jeffery Bowen Inhibitors of apoptosis are upregulated in cancer, conferring cellular survival, and are downregulated in neurodegenerative and inflammatory diseases such as age-related macular-degeneration (AMD) which are characterized by increased cell death. The small heat shock protein, αB-crystallin inhibits apoptosis by disrupting activation of an enzyme critical for cell destruction. Previous work demonstrated αB-crystallin cleavage and inactivation is coincident with destructive endophthalmitis and loss of retinal function, supporting αB-crystallin’s cytoprotective role in the retina. Research implicates inflammation in retinal destruction during AMD which involves a protein complex known as the inflammasome. Studies are underway to address whether αB-crystallin interacts with inflammasome complex proteins. Our current studies support that αB-crystallin is localized to and cleaved within cellular lysosomes during inflammasome activation, supporting that loss of αB-crystallin correlates with retinal cell destruction. Since few therapeutic interventions exist for AMD, modulation of αB-crystallin expression may promote retinal cell viability and prevent vision loss. Rapid Fooling Around in the Presence of Competitors Favors Breakups Dr. Thayaparan Paramanathan The title makes common sense with human behavior, but is this true for the breaking apart of non-covalent complexes in biological systems? The formation and breaking apart of these non-covalent complexes is a key determinant of functions in molecular biology and pharmacology. Dissociation rates of complexes are conventionally assumed to depend only on the interactions between the molecules forming the complex, and not on the presence of competitors. We use a single-molecule technique, where we label the molecules with fluorescent dyes of different colors and shine them with the appropriate laser color to watch them individually. Our results suggest that, indeed, the competitor accelerates dissociation of a non-covalently bound molecular complex by occluding the rapid rebinding of binding partners. The results show that an acceleration of ligand dissociation rate with increasing competitor concentration is a natural feature of a molecular competition that can occur in biologically relevant ranges of competitor concentration

Constructing Dual Beam Optical Tweezers for Undergraduate Biophysics Research

Optical tweezing, or trapping, is a modern physics technique which allows us to use the radiation pressure from laser beams to trap micron sized particles. Optical tweezers are commonly used in graduate level biophysics research but seldom used at the undergraduate level. Our goal is to construct a dual beam optical tweezers for future undergraduate biophysical research. Dual beam optical tweezers use two counter propagating laser beams to provide a stronger trap. In this study we discuss how the assembly of the dual beam optical tweezers is done through three main phases. The first phase was to construct a custom compressed air system to isolate the optical table from the vibrations from its surroundings so that we can measure pico-newton scale forces that are observed in biological systems. In addition, the biomaterial flow system was designed with a flow cell to trap biomolecules by combining several undergraduate semester projects. During the second phase we set up the optics to image and display the inside of the flow cell. Currently we are in the process of aligning the laser to create an effective trap and developing the software to control the data collection. This optical tweezers set up will enable us to study potential cancer drug interactions with DNA at the single molecule level and will be a powerful tool in promoting interdisciplinary research at the undergraduate level