Microscopic demonstration of static- and dynamic-binding of Eco RI enzyme to extended DNA at a single-molecule level

The restriction enzyme Eco RI is a nanomachine of nature that is a homodimeric protein and has the molecular weight of 60 kDa in the dimer. The physiological function of this machine is degradation of foreign DNA invading cells, and this degradation requires binding to a specific sequence of six base pairs on DNA, GAATTC. Eco RI binds preferably to the GAATTC sequence with the highest affinity to form stable complexes, while binding to other base-pair sequences of DNA with much lower affinity. The difference in the two distinct affinities between sequences, called specificity, enables Eco RI to distinguish the specific GAATTC sequence from flanking nonspecific sequences. The specificity of Eco RI has been applied to in situ optical genome mapping, in which ordered positions of the sequence of interest are visualized along huge chromosomal DNA. Oana et al. has employed photolabeled Eco RI as a landmark for GAATTC sequences to determine their relative positions on asingle DNA molecule in a polyacrylamide solution. Optical mapping with Eco Rl should be facilitated if larger numbers of DNA molecules are stretched, arrayed, and fixed simultaneously, because higher concentrations of DNA enhances binding of the enzyme to DNA and extension of DNA manifests the distance of Eco RI molecules bound onto the DNA from a DNA end. One of the aims of the present study is to show this advantage of an optical mapping with extended DNA.