Following the DNA Ligation of a Single Duplex Using Atomic Force Microscopy

Abstract

Nick-sealing of a single DNA duplex was studied with the use of atomic force microscopy (AFM). To form a nick between a 47 mer DNA and a 24 mer DNA, the complementary 71 mer template DNA immobilized on an AFM tip was hybridized with the 47 mer DNA and brought into contact with the 24 mer 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 47 mer DNAs in solution, the single nick-sealing event could be repeated at other positions.

Covalent Positioning of Single DNA Molecules for Nanopatterning

“…According to the AFM manufacturer, the lateral resolution is less than 2 nm while the vertical (i.e., z-directional) directional is less than 0.5 nm. The details of the AFM set-up, position control strategy for the AFM tip, and classification of force-displacement curves were described in our previous study [ 24 ]. Briefly, the AFM tip with a c71mer DNA was made to approach the 24mer DNA spot on the silicon substrate ( Figure 1 A).…”

“…Furthermore, this modification significantly increased the probability of observing and mapping the desired single interactions between the AFM tip and the surface. We previously reported the in situ monitoring of single DNA nick-sealing by the enzymatic action of DNA ligase present in the reaction environment [ 24 ]. The work suggested the possibility of single-molecule patterning based on DNA ligation, which resulted in a covalent bond of patterned DNA molecules on the surface.…”

Principles and Applications of Force Spectroscopy Using Atomic Force Microscopy

“…The manipulation of single molecules using AFM force mapping has been extended to lithography. An AFM tip tethering a DNA molecule has been used as a single‐molecule template for the ligation of DNA . By locating the tip on the probe DNA spot, DNA would be covalently immobilized in the desired pattern.…”

Recent Advances in Nanobiotechnology and High-Throughput Molecular Techniques for Systems Biomedicine

“…For example, the control of lateral spacing of capture molecules with nanoscale dendron molecules can lead to more sensitive detection of target biomolecules or enzymatic reactions on the dendron-modified glass surface (Kim et al, 2011a;2012d). This dendron modification can make it possible to monitor a single nick-sealing event in situ by DNA ligase using atomic force microscopy (AFM) (Kim et al, 2012c). The label-free AFM approach can reveal the specific protein-protein interaction (Kim et al, 2011b) and visualize the dynamic translocation of a single myosin molecule along an actin fiber (Kodera et al, 2010).…”