Force-Dependent Folding and Unfolding Kinetics in DNA Hairpins Reveals Transition-State Displacements along a Single Pathway Anna Alemany † and Felix Ritort* ,†,‡ † Small Biosystems Lab, Condensed Matter Physics Department, Universitat de Barcelona, Diagonal 647, 080028 Barcelona, Spain ‡ Ciber-BBN, Networking Research Center of Bioengineering, Biomaterials and Nanomedicine, Instituto Carlos III, 28029 Madrid, Spain * S Supporting Information ABSTRACT: Biomolecules diffusively explore their energy landscape overcoming energy barriers via thermally activated processes to reach the biologically relevant conformation. Mechanically induced unfolding and folding reactions offer an excellent playground to feature these processes at the single-molecule level by monitoring changes in the molecular extension. Here we investigate two-state DNA hairpins designed to have the transition states at different locations. We use optical tweezers to characterize the force-dependent behavior of the kinetic barrier from nonequilibrium pulling experiments by using the continuous effective barrier approach (CEBA). We introduce the mechanical fragility and the molecular transition-state susceptibility, both useful quantities to characterize the response of the transition state to an applied force. Our results demonstrate the validity of the Leffler−Hammond postulate where the transition state approaches the folded state as force increases, implying monotonically decreasing fragility with force and a non- negative transition state susceptibility at all forces. T here is a strikingly complex relationship between the conformation of a biomolecule, such as nucleic acids or proteins, and its biological function. 1 For instance, RNA riboswitches are regulatory molecules that can induce or repress the transcription of a gene depending on their conformation; and proteins with a structural function, such as keratin or collagen, usually are found in a fibrous form, whereas transport proteins such as hemoglobin are globular. In many cases the presence of misfolded states can lead to fatal diseases. Understanding the mechanisms of folding and unfolding and the general kinetic behavior of biomolecules is hence of great importance not only to unravel the link between molecular function and conformation but also to comprehend the origin of many diseases and possibly to design new therapies. In the past few years there has been a big effort to experimentally characterize the folding molecular free-energy landscape (mFEL) of biomolecules. Dynamic force spectros- copy (DFS) experiments are well-suited to study the folding/ unfolding transitions of one molecule at a time with high spatial and temporal resolution as a function of the molecular extension. By applying forces to the ends of a molecule one can observe the mechanical unfolding and folding transition and unravel the presence of intermediate and misfolded states. Additionally, from the experimentally measured force-depend- ent unfolding and folding kinetic rates it is possible to characterize the position of the transition states and the height of the corresponding kinetic barriers. However, such results can often lead to misinterpretation: In DFS experiments only the molecular extension is measured and other inaccessible reaction coordinates might be needed to properly characterize the molecular state. 2,4,5 Ideally, the molecular kinetic rates exponentially depend on the applied force according to the Bell−Evans model. 6−9 In a log-normal representation of the unfolding or folding kinetic rates versus force such an ideal model would produce straight lines (Figure 1a, solid red). Instead, a gentle curvature is expected due to the characteristic nonlinear elastic response of the released (collapsed) polymer triggered by the action of force 10,11 (Figure 1a, dashed green). In general, an extra additional curvature is observed in the experimental data at large (low) enough forces that cannot be accounted solely based on such elastic contribution (Figure 1a, dotted blue). What is the origin of such additional curvature? The most common explanation found in the literature is the existence of multiple parallel unfolding and folding pathways. 12,13 However, in a more simple scenario, a single transition state in a 1D mFEL approaches the native state upon increasing the force, as predicted by the Leffler−Hammond postulate. 14,15 This also would lead to extra-curvature effects. To prove this we have studied the mechanical folding/unfolding of DNA hairpins with different molecular fragilities (Experimental Methods). The molecular fragility μ is defined as the degree of deformability of a molecule required to spontaneously unfold due to thermal fluctuations when stretched under an applied force f. It is regulated by the force-dependent position of the transition state Received: November 16, 2016 Accepted: February 2, 2017 Published: February 2, 2017 Letter pubs.acs.org/JPCL © XXXX American Chemical Society 895 DOI: 10.1021/acs.jpclett.6b02687 J. Phys. Chem. Lett. 2017, 8, 895−900