Eung-Sam Kim†, Jung Sook Kim‡, Yoonhee Lee‡, Kwan Yong Choi†§, and Joon Won Park†‡*
†School of Interdisciplinary Bioscience and Bioengineering, ‡Department of Chemistry, Division of Integrative Biosciences and Biotechnology, and §Department of Life Science, Division of Molecular and Life Sciences, Pohang University of Science and Technology, San 31 Hyoja-dong, Pohang, 790-784, South Korea
*Address correspondence to Joon Won Park
Nick-sealing of a single DNA duplex was studied with the use of atomic force microscopy (AFM). To form a nick between a 47mer DNA and a 24mer DNA, the complementary 71mer template DNA immobilized on an AFM tip was hybridized with the 47mer DNA and brought into contact with the 24mer DNA on a substrate surface. The AFM tip and substrate surface were modified with dendron molecules to ensure the formation of a single DNA duplex. When a single nick in the DNA duplex was sealed by DNA ligase during a pause, an increase in the unbinding force was observed after the pause. The change from 24.0 ± 4.4 piconewtons (pN) to 62.8 ± 14.6 pN matched well with the resulting DNA length (71 bp). Additionally, a 30 s pause showed a 3-fold higher nick-sealing probability (60%) than a 10 s pause, while the probability did not increase with a 120 s pause. In the presence of free 47mer DNAs in solution, the single nick-sealing event could be repeated at other positions.
Keywords: DNA ligation; single-molecule manipulation; atomic force microscopy; unbinding force measurement; dendron-modified surface