How AFM characterizes DNA-Protein Interactions
Atomic Force Microscopy (AFM) has been applied to many field since its recent development, including electro-physiological substance mapping, fabricated materials characterization, and bioorganic structure characterization. Recent advancements in bioorganic characterization has enabled researchers to observe molecular processes on a nanoscale level (1 nm resolution) with little to no modification to the target structure.
With the AFM microscope, the localization and energy associated with protein binding to DNA can be observed and quantified to better understand the mechanistics behind one of the most crucial biological processes. Observations can be made on air or in liquid (using a fluid cell to contain the cantilever and the sample both submerged in solvent--or more applicably, buffer) after DNA has first been adsorbed onto the substrate. One technique utilizes functionalization of the substrate to selectively bind part or all of the sample, which would provide good restriction of sample movement to collect accurate data.
Adsorption utilizes an 'atomically flat substrate' which is usually mica, meaning that there is little inherit height difference in the surface that will bind DNA. Imaging in air utilizes a buffer containing divalent cations (such as Ni2+ and Mg 2+) to form a stable complex between the ion and the negatively-charged mica and DNA, after which the buffer can be evaporated. Adsorption techniques for liquid measurements would depend on the parameters of the experiment, including electrophysiology of the components, buffer conditions, and time-scale of the measurements.
Liquid measurements provide better analysis of true physiological conditions by allowing measurements to be acquired while the samples are in buffer. Moreover the buffer can be changed "during the course of the experiment," inferring that the fluid cell allows for immediate manipulation of the buffer solution which can be controlled against time. Sample preparation for DNA-protein interations in liquid requires mica samples pretreated with nickel to adsorb the DNA, the adsorptions strength of which can be modified by varying the buffer concentration of monovalent and divalent cations.
The uses of AFM appear are emerging in new fields and increasing in those already utilizing AFM and other Scanning Probe Microscopy techniques. AFM can be used to image processes in real-time, such as the progression of RNA polymerase along the DNA molecule over time. Scanning Probe Acceleration Microscopy calculates the acceleration of the tip as it approaches and detaches from the sample, quantifying the "second derivative of the deflection signal to recover the tip acceleration trajectory" to reveal the forces acting between tip and substrate. Manipulating, altering, and combining techniques would provide even more information about the dynamics of biomolecular interaction on a physical and electrical scale.
(1) Landousy, F. & Le Cam, E. (2006) "Probing DNA-Protein Interactions with Atomic Force Microscopy." Veeco. 6 pgs. www.veeco.com
(2) Justin Legleiter, Matthew Park, Brian Cusick, and Tomasz Kowalewski. (2006) "Scanning probe acceleration microscopy (SPAM) in fluids: Mapping mechanical properties of surfaces at the nanoscale" Published online before print 21 March 2006. PNAS vol. 103.13, 4813-4818
With the AFM microscope, the localization and energy associated with protein binding to DNA can be observed and quantified to better understand the mechanistics behind one of the most crucial biological processes. Observations can be made on air or in liquid (using a fluid cell to contain the cantilever and the sample both submerged in solvent--or more applicably, buffer) after DNA has first been adsorbed onto the substrate. One technique utilizes functionalization of the substrate to selectively bind part or all of the sample, which would provide good restriction of sample movement to collect accurate data.
Adsorption utilizes an 'atomically flat substrate' which is usually mica, meaning that there is little inherit height difference in the surface that will bind DNA. Imaging in air utilizes a buffer containing divalent cations (such as Ni2+ and Mg 2+) to form a stable complex between the ion and the negatively-charged mica and DNA, after which the buffer can be evaporated. Adsorption techniques for liquid measurements would depend on the parameters of the experiment, including electrophysiology of the components, buffer conditions, and time-scale of the measurements.
Liquid measurements provide better analysis of true physiological conditions by allowing measurements to be acquired while the samples are in buffer. Moreover the buffer can be changed "during the course of the experiment," inferring that the fluid cell allows for immediate manipulation of the buffer solution which can be controlled against time. Sample preparation for DNA-protein interations in liquid requires mica samples pretreated with nickel to adsorb the DNA, the adsorptions strength of which can be modified by varying the buffer concentration of monovalent and divalent cations.
The uses of AFM appear are emerging in new fields and increasing in those already utilizing AFM and other Scanning Probe Microscopy techniques. AFM can be used to image processes in real-time, such as the progression of RNA polymerase along the DNA molecule over time. Scanning Probe Acceleration Microscopy calculates the acceleration of the tip as it approaches and detaches from the sample, quantifying the "second derivative of the deflection signal to recover the tip acceleration trajectory" to reveal the forces acting between tip and substrate. Manipulating, altering, and combining techniques would provide even more information about the dynamics of biomolecular interaction on a physical and electrical scale.
(1) Landousy, F. & Le Cam, E. (2006) "Probing DNA-Protein Interactions with Atomic Force Microscopy." Veeco. 6 pgs. www.veeco.com
(2) Justin Legleiter, Matthew Park, Brian Cusick, and Tomasz Kowalewski. (2006) "Scanning probe acceleration microscopy (SPAM) in fluids: Mapping mechanical properties of surfaces at the nanoscale" Published online before print 21 March 2006. PNAS vol. 103.13, 4813-4818