Background to Project

The FET-OPEN scheme allowed us to submit a highly ambitious project designed to make use of biological molecular motors to produce a nanoactuator that would link the biological and silicon worlds. The logic behind this original proposal was the knowledge that Magnetic Tweezer Setups (Figure 1) were being applied to the study of enzymes that manipulate DNA (Karymov et al., 2002; Zlatanova and Leuba, 2002) and the occurrence of a chance meeting between Dr Firman, Dr Bensimon and Prof Dekker at a Bionanotechnology Conference in Berkley, USA. At this meeting it became apparent that both Dr Bensimon’s group and Prof Dekker’s group were interested in the study of DNA translocation by enzymes such as the Type I Restriction-Modification enzyme EcoR124I, which ‘pull’ adjacent DNA through a DNA-bound enzyme complex. It was quickly decided to arrange a collaboration and seek funding and that Prof Dekker would study the EcoR124I enzyme in collaboration with Dr Firman, while Dr Bensimon would study a variety of alternate enzymes that also translocate DNA – the Mol Switch Project was thus born.

Figure 1 A Magnetic Tweezer Setup

Therefore, the concept was to make use of unusual DNA-translocators, these enzymes are DNA-based molecular motors that, unlike other DNA-based enzymes such as polymerases, do not simply track along the DNA, but instead remain bound at their recognition site and move the DNA ends relative to this site (this makes them a simple nanoactuator – Figure 2). There potential use in nanodevices is made easier because they do not depend upon surface attachment of the motor to enable relative motion of the end of the DNA (as is required for a polymerase - Harada et al., 1999; Wang et al., 1998), but only surface attachment of DNA, which is a relatively simple process.

The DNA substrate is prepared from three segments:
(1)  A PCR product with biotin incorporated into both strands of the amplicon - this will attach to a streptavidin-coated magnetic bead.
(2)  A PCR product into which digoxygenin (DIG) has been incorporated into both strands.  This allows surface attachment of the DNA by coating the surface with anti-DIG antibody.
(3)  The above two PCR products are then ligated to a suitable length of DNA, which carries a single-site for the motor to bind to.

The external magnetic field of the Magnetic Tweezer Setup will then stretch the surface attached DNA with a bead attached trapping the bead in mid-air allowing visualisation in the inverted microscope.  Beads with more than one DNA molecule attached can be identified by rotating the magnets as the resulting twisting of the two DNA molecules, around each other, reduces the height of the bead from the surface.

The height of the bead can be accurately determined (±10nm), from the images in the microscope, in real time allowing vertical motion of the bead to be visualised (e.g. Figure right).

Thermal motion due to Brownian Motion, can be seen in the initial stage of the output.  This is then replaced by rapid movement of the bead toward the surface due to motor activity.

The motor is then seen to reset and the bead moves back to its original position. and this is followed by a short excursion toward the bead (opposite direction).  This leads to a stalling event and the bead is held in place by the motor for several tens of seconds, until it is again released and a further translocation occurs.

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