Single-molecule dynamics of the Eco RI enzyme using stretched DNA: its application to in situ sliding assay and optical DNA mapping

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 sensi ng ability to microscopic DNA mapping. Visible measurement of positions of the GAATTC sequence was accomplished with fluorescently labeled Ecc, RI and DNA that was stretched by dielectrophoresis and suspended above a glass su rface. Statistics showed that Eco RI molecules were trapped at particular p ositions on stretched DNA where GAATTC sequences are located. Dwell time at trapped positions agreed with the lifetime of the specific DNA-Eco RI comp lex previously measured. In a flowing stream, Eca RI molecules moved along stretched DNA and were trapped at putative GAATTC sequences, providing evid ence of sliding as a mechanism for relocation of Eco RI on DNA. This single -molecule-based method can he a lab on a chip for mapping genomic DNA and a nalyzing motility of DNA-binding nanomachines.