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Nucleic Acids Research, 2002, Vol. 30, No. 22 4902-4909
© 2002 Oxford University Press

Visualization of hemiknot DNA structure with an atomic force microscope

Yuri L. Lyubchenko*,1,2, Luda S. Shlyakhtenko2, Melinda Binus2, Claire Gaillard3 and François Strauss3

1 Department of Biology and 2 Department of Microbiology, Arizona State University, Tempe, AZ 85287-2701, USA and 3 Institut Jacques Monod, 2 Place Jussieu, 75251 Paris 05, France

*To whom correspondence should be addressed at Department of Microbiology, Arizona State University, PO Box 872701, Tempe, AZ 85287-2701, USA. Tel: +1 480 965 8430; Fax: +1 480 965 0098; Email: yuri.lyubchenko{at}asu.edu

The hemiknot, a novel type of DNA structure in which a loop is stabilized by threading one end of the duplex through another, has been studied in this paper. The hemiknot was obtained by reassociation of a DNA fragment with (CA/TG)n inserts of different lengths. Slow and fast migrating products were purified by gel electrophoresis and imaged by atomic force microscopy (AFM) using the aminopropylsilatrane–mica technique for sample preparation. Slow migrating product was characterized by the formation of small blobs for the short insert (60 bp) and clear loops and other morphologies for the long insert (188 bp). These structural features were found in almost 100% of the molecules of the slow migrating sample and were not present in the control sample. Measurements showed that the location of the blobs coincided with the positions of the inserts. The sample with the 188 bp insert in the 573 bp fragment had large structural irregularities. The majority of the molecules (77%) had asymmetrically located loops. The location of the loop in the molecules correlated well with the position of the insert in the fragment. The measured sizes of the loops were in agreement with the insert size. Altogether, these data support the hypothesis for hemiknot formation suggested earlier. In addition to looped structures, other morphologies of the hemiknot were identified in AFM images. Possible models for hemiknot formation and structure are discussed.


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