Thayaparan Paramanathan

Biophysical characterization of DNA binding from single molecule force measurements.

Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.

Force spectroscopy reveals the DNA structural dynamics that govern the slow binding of Actinomycin D

Actinomycin D (ActD) is a small molecule with strong antibiotic and anticancer activity. However, its biologically relevant DNA-binding mechanism has never been resolved, with some studies suggesting that the primary binding mode is intercalation, and others suggesting that single-stranded DNA binding is most important. To resolve this controversy, we develop a method to quantify ActD’s equilibrium and kinetic DNA-binding properties as a function of stretching force applied to a single DNA molecule. We find that destabilization of double stranded DNA (dsDNA) by force exponentially facilitates the extremely slow ActD-dsDNA on and off rates, with a much stronger effect on association, resulting in overall enhancement of equilibrium ActD binding. While we find the preferred ActD–DNA-binding mode to be to two DNA strands, major duplex deformations appear to be a pre-requisite for ActD binding. These results provide quantitative support for a model in which the biologically active mode of ActD binding is to pre-melted dsDNA, as found in transcription bubbles. DNA in transcriptionally hyperactive cancer cells will therefore likely efficiently and rapidly bind low ActD concentrations (∼10 nM), essentially locking ActD within dsDNA due to its slow dissociation, blocking RNA synthesis and leading to cell death.

Mechanically Manipulating the DNA Threading Intercalation Rate

The dumbbell shaped binuclear ruthenium complex DeltaDelta-P requires transiently melted DNA in order to thread through the DNA bases and intercalate DNA. Because such fluctuations are rare at room temperature, the binding rates are extremely low in bulk experiments. Here, single DNA molecule stretching is used to lower the barrier to DNA melting, resulting in direct mechanical manipulation of the barrier to DNA binding by the ligand. The rate of DNA threading depends exponentially on force, consistent with theoretical predictions. From the observed force dependence of the binding rate, we demonstrate that only one base pair must be transiently melted for DNA threading to occur.

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.

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.

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 implyternary 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 spatio temporal resolution.