Nanotechnology has become the ‘buzz-word’ of science in recent years and the quest for devices that are smaller and more reliable is the main thrust of this area of research.  One particularly exciting area of research is Bio-Nanotechnology.  Nature’s machines’ are generally produced at the nano-scale and provide exciting and novel ways to solve simple problems (e.g. rotary molecular motors such as ATP synthase (Adachi et al., 2000; Fillingame et al., 2000; Kinosita, 1999) , or biological transport systems such kinesin, which mimic ‘railway systems’ in the cell (Cross, 2000; Kojima et al., 1997; Vale et al., 1996) .  The use or study of biological systems in artificially produced nano-scale devices will provide an innovative way to develop this technology and may provide ideal models of how best to produce working systems.  It is likely that the use of biological systems in nano-devices will also provide important links between existing silicon-based technology and the biological world.  These links could provide new mechanisms for developing communication between the human and the silicon-driven device, with the future possibility of direct interaction between human tissue (as a source of fuel for the nano-device) and a silicon-based detector.  The type of magnetic molecular switch described in this project could find applications as a controllable micro-delivery device in specific tissues (Reed et al., 1998; Santini et al., 1999) .

In reality, 'available and useful' Nanotechnology lies in the future and current work at the nanoscale is in fact Nanoscience.  Initiation of the ‘Nanotechnology Revolution’ will require the production of the first commercial nano-device.  As discussed earlier, US Genomics believe such a device will be a single-molecule DNA sequencing device.  However, they admit such a device may still take a further ten years of development.

The main criteria for single molecule DNA sequencing is to have a simple mechanism for identify each base in the sequence.  At its simplest such a device involves a detector, a mechanism for moving DNA past the detector, a means for labelling each base to allow detection and a system for activating and controlling the device.  Many systems have been described for single molecule DNA sequencing and recently The Journal of Biotechnology (Volume 86, Issue 3) provided a useful description of developments in this area (Rigler and Seela, 2001) .  These systems range from those based on the analysis of tagged degradation products produced from the DNA to those based on hybridisation on DNA microchips.  There are also systems that detect local changes in electrical conductivity as single-stranded DNA passes through a pore in a membrane.

The innovation in this project lies with the overall concept.  Not only do we propose to use novel types of DNA-based motor(s) to develop DNA sequencing devices, we also propose to develop an innovative nano-actuator based on the use of these motors.  Therefore, within this project we describe an unusual class of DNA-based molecular motor.  Unlike DNA polymerase these motors do not track along DNA, but instead bind to a specific recognition sequence and then translocate the rest of the DNA through this bound complex (Figure 1 & 2).  Therefore, this motor can also be viewed as an integral part of a nano-actuator, which is able to move the end of the DNA toward the enzyme/DNA complex.  Since one end of the DNA molecule can be easily immobilised by surface attachment and since many ligands and objects can be easily attached to the other end of the DNA, this will provide a useful and adaptable device for Nanotechnology.  In addition, with linear DNA some of these motors are able to re‑set when they reach the end of the DNA molecule (Firman and Szczelkun, 2000) , this will provide a simple mechanism for developing parallelism in the device – an important aspect of nano-devices.  ATP, a common bio-energy source, drives the translocation process and therefore the system does not require any DNA synthesis per se.

It is this ability to act as a nano-actuator that has led us toward this innovative program to develop a nano-device to link the biological world (through the fuelling of the DNA-based molecular motor) to the silicon world of microelectronics (by detection of the moving magnetic particle using for example a micron-size Hall probe - Bending et al., 2000) .  Such a device is likely to have a wide range of analytical uses by directly linking bio-sensing to activation of intelligent silicon devices.  The most obvious use for such a device would be in ‘Lab-on-a-Chip’ technology.  However, such devices may also eventually become implantable and could make use of external magnetic fields to control the local delivery of hormones, or drugs by micro-devices located in specific tissues.

In addition, the use of magnetic tweezers for force measurement during translocation has led to the suggestion of another novel mechanism for DNA sequence determination based upon measurement of the force required to un‑pair complementary bases.  The force required to separate the two strands of a DNA molecule varies with its sequence and can in principle be used to deduce it. Such a device is likely to be used in the rapid detection/screening of single-base mismatches in DNA, the cause of many genetic disorders.

Innovative aspects of this proposal

• Development of a nano-device linking the biological- and silicon-based worlds.

The possibility of producing a molecular switch based on a nano-actuator that uses a biological molecular motor is a major step forward in the area of Bio-Nanotechnology.

• Precise localisation and positioning of the DNA/motor complex on silicon surfaces and in silicon nano‑wells or ‑channels.

This project will develop improved techniques for positioning biomolecules on etched surfaces and within channels on surfaces.  These techniques will range from positioning using the AFM, and novel self-assembly using DNA structures through to chemical attachment on derivatised surfaces.  The outcomes will provide valuable techniques for use in other biosensors and nano-devices.

• The use of a DNA-based molecular motor as a nano-actuator.

This will provide us with a robust tool for manipulation of objects at the nano-scale, which is both cheap and easy to set up.  Nature provides many other examples of molecular motors.  Demonstration that such complex assemblies of protein can be manipulated at the single molecule level and provide valuable nano-tools will lead to the development of many other systems.  Examples include devices such as biosensors and manipulators of bio-molecules for Lab-on-a-Chip technology leading eventually to implantable devices that enable new forms of communication between biological systems and the silicon world of microcomputers.

• Development of novel biocompatible magnetic particles with high-density magnetic fields.

There is an increasing demand for small magnetic particles that are bio-compatible for use in the medical industries.  Production of such particles for attachment to DNA allowing them to be moved without the problems presented by friction and inertia will provide invaluable tools for many areas.  Reduction in particle size to the nanoscale will be crucial for achieving this.

• Development of nano-detectors capable of detecting movement of magnetic nano-particles.

Innovative use of silicon-based technologies will be required to provide a useful and robust detector for movement of the magnetic bead.

• Determination of DNA sequence through force measurements with cruciform DNA

This technique is designed to overcome the problems presented by the elastic energy stored in DNA, which normally swamps the energy differences between different basepairs in the sequence.  The use of DNA coiling in the cruciform arms is a unique solution to these problems and if successful this technique will provide a rapid, yet simple system for determining single-point mutations in DNA.

• Measurement of DNA sequence using a variety of optical methods.

The innovation in this area of the project is illustrated by the use of the expanding loop produced by translocation as the means of generating the FRET signals.  By only labelling the DNA, we will overcome problems associated with labelling the motor.  This will provide a unique system for DNA sequence analysis using motor proteins. The availability of linked, moving-magnet detector will allow greater control of the DNA, including re-setting and independent measurement of rate of translocation.

The ultimate goal of this type of DNA sequencing device would be a mechanism to directly read the bases, in the major groove, as they pass the detector.  The proposed use of nanotubes carrying a fluorophore as a means of improving scanning near-field optical microscopy (SNOM) detection of the labelled bases will be an innovative first step toward such a device.