High-throughput approach for detection of DNA bending and flexibility based on cyclization

doi:10.1073/pnas.0530189100

High-throughput approach for detection of DNA bending and flexibility based on cyclization

Yongli Zhang* and
Donald M. Crothers*,†,‡

Departments of ^*Molecular Biophysics and Biochemistry and ^†Chemistry, Yale University, New Haven, CT 06511

Contributed by Donald M. Crothers

Abstract

We have developed a high-throughput approach to the labor-intensive problems of DNA cyclization, which we use to characterize DNA curvature and mechanical properties. The method includes a combinatorial approach to make the DNA constructs needed and automated real-time measurement of the kinetics using fluorescence. We validated the approach and investigated the flexibility of two kinds of nicked DNA and AT dinucleotide repeats. We found that, although the nicks hardly alter the bending flexibility, they significantly increase the torsional flexibility, and that the AT repeat has 28% (±12%) lower bending rigidity than a generic DNA sequence.

Footnotes

↵ ‡ To whom correspondence should be addressed. E-mail: donald.crothers{at}yale.edu.
Abbreviations:

NPS,

nucleosome positioning sequence;

TAMRA,

tetramethylrhodamine