(a) TuDelft and CNRS/ENS will provide reports describing progress toward the measurement of the forces generated by the molecular motors under study.  These initial measurements will be produced using pre-existing set-ups and the data can be compared to data from the AFM studies above.

(b) In addition, these reports will provide information for the design of customised magnetic tweezer set-ups that will be developed.

(c) Finally, this report will describe potential mechanisms for introducing varying concentrations of ATP (the fuel for the motor) and the initial observations on the force generated.

Outcomes:

(a) Measurement of Translocation by AFM

Visualisation of translocated DNA, using the AFM, was first shown by the TUDelft Group and was published in  a paper in NAR (van Noort et al., 2003).  However, this technology has been distributed to other Partners and is now used by NPL and Ports for rapid analysis of motor activity.

(b) Custom-Building of Magnetic Tweezers

Both TUDelft and CNRS/ENS have produced purpose-built Magnetic Tweezer Set-ups.  They have calibrated these and shown they are able to measure DNA translocation.  The rate of translocation has been determined and the forces produced, measured for a variety of motors.  This data is now published (Saleh et al., 2004, 2005; Seidel et al., 2004,2005)).  The ENS group is involved in the creation of a start-up company that will commercialize the magnetic tweezer set-up to the academic and industrial research community.

A new molecular motor has been discovered that is the fastest and one of the most processive and robust DNA translocase yet reported. It translocates DNA at a rate up to 10 kbps/sec for a few seconds and against a load larger than 50pN. It has been observed to work repeatedly and reliably under various buffer conditions. We have characterized its dynamic properties (rate, on-time, processivity, torque generated, step-size, ATP consumption,  etc.). It presents the best candidate yet for a DNA motor to be used in a Mol Switch device.

(c) Influence of [ATP] on rate of translocation

Ports have determined the characteristics of ATPase activity for the EcoR124I molecular motor, and derivatives thereof, using standard ‘bulk’ biochemical methods.  These data have been compared to single-molecule data, obtained with the Magnetic Tweezers and a full understanding of motor activity has emerged (Seidel et al., 2005).  The measurements of the translocation of EcoR124I have been extended and refined. New and valuable information concerning the mechanism of translocation could be gained from the measured data and some of this is published in Seidel et al (2005).