Mol Switch
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The Mol Switch Project can be divided into two major efforts:

1. Demonstration of the Mol Switch Device: Use of a biological molecular motor to produce a nanoactuator, which pulls a magnetic bead toward a surface, and detection of the movement of the magnetic bead in such a way as to produce an electronic output.
2. Demonstration of single molecule DNA sequencing: Use of the molecular motor to pull fluorescently labelled DNA toward a fluorescently-tagged version of the motor, resulting in Fluorescent Resonant Energy transfer (FRET).  Time-resolved fluorescence measurements would allow positioning of the fluorophore relative to the bound motor and thus a degree of DNA sequencing.

The Mol Switch Device:

The molecular motors to be used in the project are unusual in that they translocate DNA (rather than making a one-dimensional walk along the DNA, as polymerases do).  The motors bind to DNA at a specific site and then translocate the rest of the DNA through the bound motor, following the helical thread of the DNA (see animation to right).  This produces supercoiled loops of DNA in the expanding loop and 'pulls' the free end of the toward the motor.

The key to the Mol Switch Device is that the free end of the DNA is attached to a paramagnetic bead, which can be used to stretch the DNA, but can also produce a molecular dynamo effect allowing an electronic output from the moving magnet.

Single Molecule DNA Sequencing:

Because the molecular motor pulls the DNA through itself, it is possible to fluorescently tag the motor and the DNA and then obtain a time-resolved FRET signal as the two fluorophores approach each other following DNA translocation.  This is illustrated in the figure at the right.  We have demonstrated labelling of the EcoR124I molecular motor subunit and FRET between the labelled motor suitably labelled DNA.  The potential exists for time-resolved FRET between motor and labelled DNA.

The problem with DNA sequencing using light emission or fluorescence is that it is not possible to discriminate individual basepairs, which are separated by only 0.34nm.  However, such a system could be used to detect Single Nucleotide Polymorphisms through PCR-incorporation of bases at specific locations within the DNA substrate.  It is not required that individual bases be discriminated to detect the SNP associated with a specific disease and to map associated changes or the precise nature of the SNP.

The Molecular Motors:

1. EcoR124I:
   The Type I Restriction-Modification (R M) system EcoR124I is a member of a unique group of DNA-based molecular motors. These motors belong to a large superfamily (SF-II) of helicase-like enzymes (Flaus and Owen-Hughes, 2001) including Type III R M enzymes, chromatin remodelling factors and a few chimeric enzymes. The R M enzyme EcoR124I, as with other Type I R M enzymes, is composed of a number of subunits encoded by the genes hsdR, hsdM and hsdS. The products of all three genes are required for DNA cleavage (restriction), producing the endonuclease, or REase, which has a stoichiometry of R₂M₂S₁ (for a review see Murray, 2000).

2. FtsK:
   FtsK is another bacterial protein with a DNA translocation activity that is used to transport chromosomal DNA during the late stages of cell division (Aussel et al., 2002; Capiaux et al., 2002; Lau et al., 2003; Lesterlin et al., 2004). DNA translocation by FtsK was studied in vitro using a Magnetic Tweezer setup to analyse the activity (translocation rate, processivity, step-size, etc.) of this enzyme. It also allowed us to quantify the torque produced in DNA by observing the supercoils induced into the molecule during translocation by a single FtsK complex (Saleh et al., 2005b). These studies have shown that this motor is one of the fastest amongst the DNA translocating motors with a maximum rate of translocation of 6.7 kbp/s (Saleh et al., 2004) and a processivity of ~10 kbps.

Building the Mol Switch device:

During the three years of the Mol Switch Project (2003-2005) we have successfully demonstrated that micron-sized beads can be translocated by the above molecular motors, shown that they can self assemble on surface attached DNA within a microfluidics-based flowcell and demonstrated that the moving magnetic bead can be detected using a scanning-Hall Effect Sensor.  However, within both the budget limit of the Project and the Consortium Member's capabilities it was not possible to fabricate a suitable Lab-on-a-Chip that would be a prototype Mol Switch Device (see animation to the right).

Further funding has been sought from the EC 6th Framework Programme NEST (New and Emerging science and Technologies) under the Pathfinder Call for project in the area of Synthetic Biology.