Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins

doi:10.1073/pnas.0511048103

Nanomechanical measurements of the sequence-dependent folding landscapes of single nucleic acid hairpins

*National Institute for Nanotechnology, National Research Council of Canada, Edmonton, AB, Canada T6G 2V4; and
Departments of ^†Biological Sciences,
^¶Biochemistry, and
^‖Applied Physics, Stanford University, Stanford, CA 94305

Edited by Kiyoshi Mizuuchi, National Institutes of Health, Bethesda, MD, and approved February 27, 2006 (received for review December 21, 2005)

Abstract

Nucleic acid hairpins provide a powerful model system for probing the formation of secondary structure. We report a systematic study of the kinetics and thermodynamics of the folding transition for individual DNA hairpins of varying stem length, loop length, and stem GC content. Folding was induced mechanically in a high-resolution optical trap using a unique force clamp arrangement with fast response times. We measured 20 different hairpin sequences with quasi-random stem sequences that were 6–30 bp long, polythymidine loops that were 3–30 nt long, and stem GC content that ranged from 0% to 100%. For all hairpins studied, folding and unfolding were characterized by a single transition. From the force dependence of these rates, we determined the position and height of the energy barrier, finding that the transition state for duplex formation involves the formation of 1–2 bp next to the loop. By measuring unfolding energies spanning one order of magnitude, transition rates covering six orders of magnitude, and hairpin opening distances with subnanometer precision, our results define the essential features of the energy landscape for folding. We find quantitative agreement over the entire range of measurements with a hybrid landscape model that combines thermodynamic nearest-neighbor free energies and nanomechanical DNA stretching energies.

Footnotes

^§To whom correspondence should be sent. E-mail: michael.woodside{at}nrc-cnrc.gc.ca
Author contributions: M.T.W., D.H., and S.M.B. designed research; M.T.W. and W.M.B.-P. performed research; M.T.W., W.M.B.-P., K.L., and K.T. contributed new reagents/analytic tools; M.T.W. analyzed data; M.T.W., D.H., and S.M.B. wrote the paper; and M.T.W. built the apparatus.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

Abbreviations:

FEC,

force–extension curve;

WLC,

worm-like chain.