The scourge of single molecule manipulation experiments is the non-specific interactions of the surfaces with DNA and proteins.  We have developed some ad-hoc protocols that need to be improved and rationalised to make a consistently working device.  Determine the effect of large surface areas of silicon and motor activity and motor stability using sensitive assays for the presence of residual ATP after translocation.  Determination of the sensitivity of these assays.  Construction of a variety of structures in silicon (wells, slits, arrays) using etching techniques and electron-beam patterning. Test various means of silanizing silicon surfaces with PEG-silanes, oligo-silanes and other functionalised silanes to reduce these non-specific interactions.  To localise the DNA-bound beads in specific wells we will use a combination of STM/AFM/optical /magnetic tweezers and micro-fluidic pumps.  Couple these manipulation tools with an epifluorescence visualisation set-up (evanescent wave, 1 or 2 photons).  All three participants have developed independent techniques for positioning single molecules on surfaces and this Workpackage will allow exploration of the best technique(s) for this process.

Outcomes:

(a) Surface Passivation

We have developed surface passivation protocols that reproducibly allow us to specifically tether magnetic beads to adequate surfaces via one, or, two DNA molecules. We have, also, developed surface treatment and passivation protocols (based on the use of PEG-silanes) to reduce fluorescent background in experiments requiring the joint manipulation and visualization of single molecules.

The flow dynamics of micron-size magnetic beads driven by external magnetic field in microfluidic glass channels was investigated. It was demonstrated that the used channels are too large (minimum size is 10 mm) for the successful bead detection by MFM. Smaller Si channels were successfully implemented for bead immobilization and moving along the channel.

(b) Positioning of single molecules and microfluidics

The observation of an available cysteine at the N-terminus of the HsdR (motor) subunit of the EcoprrI molecular motor, and its subsequent production as a hybrid protein, has allowed the Ports group to investigate surface attachment using this cysteine, to enable attachment to gold and silicon.  We have shown that the HsdR(prrI) subunit can be attached to both surfaces and that this surface attached protein can self-assemble to produce both R₁- and R₂‑complexes with and without DNA.  This data has been confirmed, using AFM visualisation and surface plasmon resonance.  We are now investigating functionality of the surface-attached complexes.  This will allow precise location of motor complexes, within any device, using Dip Pen Nanolithography (DPN) or, other surface patterning approaches.

Single molecule positioning could also be achieved by a patterned surface treatment, of which we have some experience. Namely, one would generate patches of binding sites for one DNA extremity (i.e. areas coated with anti-Dig proteins).

The AFM was also used to investigate surface attachment of the motor, but non-specific interactions hampered this work. As part of the investigation of surface attachment we have investigated various methods for surface passivation.

ENS/CNRS have developed a microfluidic system based on PDMS technology. A mould was generated, by regular machining techniques, defining a channel (actually a rift) a few mm wide, a few cm long and approximately 100 microns deep. That mould was used to generate a channel with these characteristics, in a soft PDMS polymer that was baked at about 80°C. The PDMS block is positioned on top of a microscope coverslip to define the reaction chamber and small tubing plugged in this PDMS stamp is used to exchange buffers. With lithographic techniques to generate smaller channels in PDMS, we could even reduce the dimensions of that cell, but, at the moment, we see no urgency in such a task.  We have miniaturized the flow cell for implementing the magnetic trap technique in a micro-fluidic environment.

(c)  Microinjection and epifluorescence systems

ENS/CNRS have developed a Total Internal Reflection (TIR) microscope at three excitation wavelengths that allows us to visualize by regular fluorescence and by Fluorescent Energy Transfer (FRET) the interactions of a variety of labelled molecules (proteins, fluorescently labelled ATP) with their DNA substrate. This set-up is combined with a magnetic trap system that also allows for the manipulation of single molecule. Experiments on helicase, DNA polymerases and various DNA translocases (FtsK, EcoR124I, etc.) are planned in collaboration with the groups at Delft and Portsmouth.