Optical measurement of mechanical forces inside short DNA loops.
Knowledge of the mechanical properties of double-stranded DNA (dsDNA) is essential to understand the role of dsDNA looping in gene regulation and the mechanochemistry of molecular machines that operate on dsDNA. Here, we use a newly developed tool, force sensors with optical readout, to measure the forces inside short, strained loops composed of both dsDNA and single-stranded DNA. By varying the length of the loops and their proportion of dsDNA, it was possible to vary their internal forces from 1 pN to >20 pN. Surprisingly, internal loop forces changed erratically as the amount of dsDNA was increased for a given loop length, with the effect most notable in the smallest loop (57 nucleotides). Monte Carlo simulations based on the helical wormlike chain model accurately predict internal forces when more than half of the loop is dsDNA but fail otherwise. Mismatches engineered into the double-stranded regions increased flexibility, suggesting that Watson-Crick basepaired dsDNA can withstand high compressive forces without recourse to multibase melts. Fluorescence correlation spectroscopy further excluded transient melting (microsecond to millisecond duration) as a mechanism for relief of compressive forces in the tested dsDNAs. DNA loops with integrated force sensors may allow the comprehensive mapping of the elasticity of short dsDNAs as a function of both sequence and salt.
Biophysics Graduate Group, Department of Physics, and Department of Chemistry, University of California, Berkeley, California 94720, USA.
Hari
H
Shroff
David
D
Sivak
Jake J
JJ
Siegel
A L
AL
McEvoy
Merek
M
Siu
Andrew
A
Spakowitz
Phillip L
PL
Geissler
Jan
J
Liphardt
D001665:Binding Sites; D001703:Biophysics / Q000379:methods; D004247:DNA / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005786:Gene Expression Regulation; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D009010:Monte Carlo Method; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry; D055095:Optics and Photonics; D013050:Spectrometry, Fluorescence / Q000379:methods; D013997:Time Factors
D009841:Oligonucleotides; D004247:DNA