Single Molecule Studies of Biological Systems by Atomic Force Microscopy (AFM)
My research group uses single molecule force spectroscopy to understand the interactions between molecules in biological systems.
Single molecule measurements can track an individual molecule over a period of time or measure many single molecule events one at a time. A single molecule can therefore act as a probe to map the local environment around it.
Our group uses an Atomic Force Microscope (AFM) to investigate single molecule interactions. An AFM is typically used to visualize objects on a surface at nanoscale resolution in three-dimensions (see AFM Gallery). However, the AFM can also measure very small forces. A force measurement always involves tethering the molecules of interest to a cantilever and to a surface, then using a piezoelectric scanner to move the surface towards the probe to allow the molecules of interest to interact before pulling them apart to measure the rupture force.
We are particularly interested in understanding the interactions between carbonic anhydrase enzyme and its inhibitors. An overactive carbonic anhydrase produces excess fluid in the eye, leading to the increase in intraocular pressure resulting in possible damage to the optic nerve in the eye. This process, also known as glaucoma, is a serious eye disease. It can be partly treated using inhibitors that bind to the carbonic anhydrase enzyme.
To measure biomolecular forces, we attach the carbonic anhydrase enzyme to a positively charged surface and the inhibitor to an AFM probe via a long tether. The rupture between the inhibitor and the enzyme leads to a specific shape on a force curve measurement referred to as the rupture event in the cartoon below. All other interactions are considered non-specific and result in a large adhesion curve.
Hundreds of these rupture events can be measured and displayed as a distribution of forces to discriminate between single molecule interactions and multiple molecule interactions.
Using microscopic models recently developed by Dudko and Hummer models, the force distribution plots can be analyzed at different pulling velocities to estimate kinetic and thermodynamic parameters for the single molecule interaction.
Nanoscale DNA surface features can be constructed on template stripped gold surfaces by using a combination of "nanoshaving" and self assembly. An AFM tip can be selectively used to remove a layer of a short double-stranded DNA in a specific area, and the shaved area can then be refilled with a longer length single-stranded DNA. The single stranded DNA in the shaved area can be hybridized with a complementary strand and the entire process of manipulation can be monitored by AFM in situ. Nanoscale DNA surface features can be constructed on template stripped gold surfaces by using a combination of "nanoshaving" and self assembly. An AFM tip can be selectively used to remove a layer of a short double-stranded DNA in a specific area, and the shaved area can then be refilled with a longer length single-stranded DNA. The single stranded DNA in the shaved area can be hybridized with a complementary strand and the entire process of manipulation can be monitored by AFM in situ.
Our group also attaches short complementary DNA strands to an AFM probe and to a surface and measures the rupture force present in a duplex DNA strand.
We have shown that the magnitude of the rupture force is dependent on the force at which the AFM probe and surface come into contact initially. By varying the initial contact force, we have demonstrated the rupture force from a DNA duplex can vary significantly.
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