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Gapped DNA and Cyclization of Short DNA Fragments

Quan Du ^*, Maria Vologodskaia ^*, Heiko Kuhn , Maxim Frank-Kamenetskii  and Alexander Vologodskii ^*

^* Department of Chemistry, New York University, New York, New York 10003; and Center for Advanced Biotechnology and Department of Biomedical Engineering, Boston University, Boston, Massachusetts 02215

Correspondence: Address reprint requests to Alexander Vologodskii, Tel.: 212-998-3599; Fax: 212-260-7905; E-mail: alex.vologodskii{at}nyu.edu.

We use the cyclization of small DNA molecules, 200 bp in length, to study conformational properties of DNA fragments with single-stranded gaps. The approach is extremely sensitive to DNA conformational properties and, being complemented by computations, allows a very accurate determination of the fragment's conformational parameters. Sequence-specific nicking endonucleases are used to create the 4-nt-long gap. We determined the bending rigidity of the single-stranded region in the gapped DNA. We found that the gap of 4 nt in length makes all torsional orientations of DNA ends equally probable. Our results also show that the gap has isotropic bending rigidity. This makes it very attractive to use gapped DNA in the cyclization experiments to determine DNA conformational properties, since the gap eliminates oscillations of the cyclization efficiency with the DNA length. As a result, the number of measurements is greatly reduced in the approach, and the analysis of the data is greatly simplified. We have verified our approach on DNA fragments containing well-characterized intrinsic bends caused by A-tracts. The obtained experimental results and theoretical analysis demonstrate that gapped-DNA cyclization is an exceedingly sensitive and accurate approach for the determination of DNA bending.

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