Direct Visualization of DNA Recognition by Restriction Endonuclease EcoRI

Background/Purpose: Restriction endonuclease is found in bacteria and can precisely cut off the DNA sequence specificity invaded from outside. Using the restriction endonuclease to cleave the double-stranded DNA at specific recognition nucleotide sequences is regarded as important biotechnology. However, traditional approaches cannot directly observe the process of specific binding of DNA by the restriction endonuclease. Methods: Single-molecule biotechnologies, including an optical tweezers system and single-molecule fluorescence detection system, were proposed to directly observe how the restriction endonuclease EcoRI found the recognition-binding site. Results: Overall, the study showed that EcoRI conjugated quantum dots (QDs) possessed the ability to recognize its specific binding sites. In addition, the distributions of the QD-EcoRI trajectory in the transverse direction were nearly the same; however, the difference occurred in the longitudinal direction along the DNA axis during the early and late stages of EcoRI recognition. Conclusion: These results suggest one-dimensional sliding of EcoRI existed along the DNA contour during EcoRI recognition. In addition, when EcoRI slided along the DNA contour, it would require more than 400 seconds for EcoRI to correctly find its DNA target site.