Force unfolding kinetics of RNA using optical tweezers. I. Effects of experimental variables on measured results.
Experimental variables of optical tweezers instrumentation that affect RNA folding/unfolding kinetics were investigated. A model RNA hairpin, P5ab, was attached to two micron-sized beads through hybrid RNA/DNA handles; one bead was trapped by dual-beam lasers and the other was held by a micropipette. Several experimental variables were changed while measuring the unfolding/refolding kinetics, including handle lengths, trap stiffness, and modes of force applied to the molecule. In constant-force mode where the tension applied to the RNA was maintained through feedback control, the measured rate coefficients varied within 40% when the handle lengths were changed by 10-fold (1.1-10.2 Kbp); they increased by two- to threefold when the trap stiffness was lowered to one-third (from 0.1 to 0.035 pN/nm). In the passive mode, without feedback control and where the force applied to the RNA varied in response to the end-to-end distance change of the tether, the RNA hopped between a high-force folded-state and a low-force unfolded-state. In this mode, the rates increased up to twofold with longer handles or softer traps. Overall, the measured rates remained with the same order-of-magnitude over the wide range of conditions studied. In the companion article on pages 3010-3021, we analyze how the measured kinetics parameters differ from the intrinsic molecular rates of the RNA, and thus how to obtain the molecular rates.
Wen
Jin-Der
JD
Department of Chemistry, University of California, Berkeley, California, USA.
Manosas
Maria
M
Li
Pan T X
PT
Smith
Steven B
SB
Bustamante
Carlos
C
Ritort
Felix
F
Tinoco
Ignacio
I
D016477:Artifacts; D003198:Computer Simulation; D004548:Elasticity; D007700:Kinetics; D008846:Micromanipulation / Q000379:methods*; D008956:Models, Chemical; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D052898:Optical Tweezers; D012313:RNA / Q000737:chemistry* / Q000648:ultrastructure*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013314:Stress, Mechanical
D012313:RNA