Precision surface-coupled optical-trapping assay with one-basepair resolution.
The most commonly used optical-trapping assays are coupled to surfaces, yet such assays lack atomic-scale ( approximately 0.1 nm) spatial resolution due to drift between the surface and trap. We used active stabilization techniques to minimize surface motion to 0.1 nm in three dimensions and decrease multiple types of trap laser noise (pointing, intensity, mode, and polarization). As a result, we achieved nearly the thermal limit (<0.05 nm) of bead detection over a broad range of trap stiffness (k(T) = 0.05-0.5 pN/nm) and frequency (Deltaf = 0.03-100 Hz). We next demonstrated sensitivity to one-basepair (0.34-nm) steps along DNA in a surface-coupled assay at moderate force (6 pN). Moreover, basepair stability was achieved immediately after substantial (3.4 pN) changes in force. Active intensity stabilization also led to enhanced force precision ( approximately 0.01%) that resolved 0.1-pN force-induced changes in DNA hairpin unfolding dynamics. This work brings the benefit of atomic-scale resolution, currently limited to dual-beam trapping assays, along with enhanced force precision to the widely used, surface-coupled optical-trapping assay.
Carter
Ashley R
AR
JILA, National Institute of Standards and Technology, University of Colorado, Boulder, Colorado, USA.
Seol
Yeonee
Y
Perkins
Thomas T
TT
D020029:Base Pairing; D004247:DNA / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D009691:Nucleic Acid Denaturation; D052898:Optical Tweezers; D012680:Sensitivity and Specificity; D013499:Surface Properties
D004247:DNA