title: Combining temperature and force to study folding of an RNA hairpin.
abstract: RNA folding in cells typically occurs at mesophilic temperatures. However, in vitro, RNA can be unfolded either by increasing temperature to values that are much higher than physiological, or by mechanically pulling structures apart at ambient temperature. To directly study RNA folding at physiological temperatures and to unify thermodynamics measured by melting and pulling, we developed temperature-controlled optical tweezers (thermal tweezers) that can be used to mechanically unfold single RNA molecules at mesophilic temperatures. Folding of a 20-base-pair tetraloop hairpin was studied under different ionic conditions and at temperatures ranging from 22 °C to 42 °C. At each temperature, single hairpin molecules were held at constant force, and their two-state folding equilibria were monitored. The change in free energy derived from these measurements was used to construct a phase diagram of RNA structure using force and temperature as variables. Furthermore, we derived ΔG(0pN,T), the folding free energy at zero force and temperature T, by subtracting the stretching energy of unfolded RNA from the reversible mechanical work done to unfold the hairpin. ΔG(0pN,T) and its salt dependence agree reasonably well with the predictions by the nearest neighbor model. Under each ionic condition, ΔG(0pN,T) depended linearly on temperature, yielding ΔH(exp) and ΔS(exp) that also matched the predictions. The combination of force and temperature to study RNA folding is a step toward unifying thermodynamics measured by thermal melting and mechanical unfolding, and opens a new path for directly monitoring temperature induced RNA structural changes, as it occurs often in biology.
authors.affiliation: Nanoscale Engineering Graduate Program, SUNY College of Nanoscale Science and Engineering, University at Albany, SUNY, 1400 Washington Ave, Albany, NY 12222, USA.
authors.forename: William
authors.initials: W
authors.lastname: Stephenson
authors.forename: Sean
authors.initials: S
authors.lastname: Keller
authors.forename: Rachel
authors.initials: R
authors.lastname: Santiago
authors.forename: James E
authors.initials: JE
authors.lastname: Albrecht
authors.forename: Papa Nii
authors.initials: PN
authors.lastname: Asare-Okai
authors.forename: Scott A
authors.initials: SA
authors.lastname: Tenenbaum
authors.forename: Michael
authors.initials: M
authors.lastname: Zuker
authors.forename: Pan T X
authors.initials: PT
authors.lastname: Li
mesh_terms: D052898:Optical Tweezers; D012313:RNA / Q000737:chemistry*; D059370:RNA Folding; D013696:Temperature
chemical_list: D012313:RNA