Single-Molecule Dynamics of the Eco RI Enzyme using Stretched DNA: its Application to In Situ Sliding Assay and Optical DNA Mapping

Abstract

The Eco RI enzyme is a nanomachine responsible for gene integrity in cells, and has a sensor that discriminates its specific binding sequence on DNA (GAATTC) from flanking nonspecific sequences. We applied this sequence sensing ability to microscopic DNA mapping. Visible measurement of positions of the GAATTC sequence was accomplished with fluorescently labeled Eco RI and DNA that was stretched by dielectrophoresis and suspended above a glass surface. Statistics showed that Eco RI molecules were trapped at particular positions on stretched DNA where GAATTC sequences are located. Dwell time at trapped positions agreed with the lifetime of the specific DNA- Eco RI complex previously measured. In a flowing stream, Eco RI molecules moved along stretched DNA and were trapped at putative GAATTC sequences, providing evidence of sliding as a mechanism for relocation of Eco RI on DNA. This single-molecule-based method can be a lab on a chip for mapping genomic DNA and analyzing motility of DNA-binding nanomachines.