|
| |
Abstract
Type I
Restriction-Modification (R-M) enzymes are biological molecular motors (Firman
& Szczelkun, 2000) that translocate DNA prior to their normal function
of DNA cleavage (for a recent review see
Murray, 2000). This DNA
translocation is ATP-dependent, with ATP acting as the ‘fuel’ for the motor
and one ATP molecule is consumed for each base-pair of translocated DNA.
Translocation occurs at 550bp sec-1 (0.2mm sec-1) and
each holoenzyme enzyme contains two motor subunits (Seidel
et al., 2004. We have shown that the EcoR124I R-M enzyme readily
dissociates into a single-motor complex (R1 complex), which is
unable to cleave DNA (Janscák
et al., 1998), but can still translocate DNA (Seidel
et al., 2004). In addition, we have produced point mutations of the
restriction subunit (HsdR), which are unable to cleave DNA, but can still
translocate (Janscak et
al., 1999). Therefore, we have available a motor that can be readily
used as a nanoactuator. This nanoactuator can produce useful work, which
has been well characterised at the single molecule level (Seidel
et al., 2005, Seidel et
al., 2004. We propose to develop this nanoactuator as a single-molecule
reporting system, which can be incorporated into a wide range of biosensing
systems, and in this poster we will show initial work funded under the EC
Project BioNano-Switch. This includes a simple system for detection of
thrombin, through release of an immobilised motor subunit, and single
molecule detection of the translocation events.
|
