Bio-Assembly The tremendous advance in understanding, utilization and control of biomolecules provides a dramatically enhanced set of reagents, allowing precise control over the assembly of nano-scale components into larger construct. Next generation materials developed at the interface between traditional inorganic materials and biological polymers can form the basis of novel device technologies by utilizing the highly cooperative, self-assembling capability of biopolymers to direct the formation of 3-dimensional constructs. Integrating nano-scale materials into biological architectures offers intriguing potentials for novel electronic frameworks. Biomaterials represent a burgeoning field in which the demonstration and control of bio-compatibility between the inorganic nanomaterials and the biological scaffolding, as well as the maintenance of bioactivity of the biological framework is crucial to the development of the field. We demonstrate that bio-compatibility and bio-activity are maintained for biomaterials composed of duplex DNA appended with 1.4 nm Au particles. We used highly selective proteins that induce sequence-specific structural perturbations on the DNA. Electron microscopy imaging provides a direct assessment of the bio-activity of the DNA-Au assemblies. (Figure 6) These results lay a foundation for interfacing more complex and diverse protein-DNA-nanomaterial systems, and mechanism for the analysis of the resultant conjugate structures.

NSET - Nano-Surface Energy Transfer

Nanoparticles represent a new frontier in technology, blurring the lines and defining a sort-of conjugated system between physics, chemistry, biology and materials. A materials scientist may be interested in the ability to construct a metal or semiconductor with certain geometries for the pure challenge of the work, while at the same time, a physicist or spectroscopist may be very interested in studying that same nanomaterial to understand how size and geometry have influenced the behavior of its electrons. By taking advantage of well-characterized biological systems (eg. proteo-nucleic interactions) we are able to probe the electrons of small gold nanoparticles by observing the behavior of strategically placed nearby fluorophores at specific distances from the nanoparticle surface. Although a nanomaterial is very different from organic molecules or inorganic coordination compounds, there are certain expectations we may still hold as to the interaction a nanomaterial may have with such a system. If a metallic nanoparticle such as 1.4nm gold maintains band structure similar to the bulk metal, there would still be a population of free electrons available in the conduction band, each electron moving through the lattice with the normal scattering phenomena, (ie. electron-electron scattering or surface potential scattering). Because a 1.4nm particle is below the normal mean free-path of an electron in gold at normal temperatures, the only scattering event an electron will feel is that of the surface potential. In other words, we expect that the electrons spend all of their time at the surface of the particle. (See Figure 7.) If we place a common fluorophore such as fluorescein nearby such a particle, then we see that the fluorescence quantum efficiency of the dye begins to decrease with a 1/R^4 distance dependence (see Figure 8.) The basis of Nano-Surface Energy Transfer (NSET) comes from the damping of the fluorophore's oscillating dipole by the gold metal's free electrons as a through-space mechanism. Because the gold particle's electrons are homogenously oriented, the constraint on dipole-dipole coupling has been greatly relaxed and thus gives rise to energy transfer efficiency at much larger distances. The experiments and math describing the interactions of fluorophores above metal surfaces have already been described by many experimenters and mathematicians, where we are only taking advantage of the groundbreaking work others have laid down for us, (see JACS 2005.)

Applications of NSET - Ribozyme Kinetics

A major advantage of nanoparticles for bio-related research is the size of a nanoparticle relative to relevant macromolecules. For example, a 1.4nm particle is roughly the same size as the footprint of a dsDNA strand which means that we are not dealing with yarn taped to a bowling ball, we are dealing with a ping-pong ball on a yard hose. This size-comparison becomes important if we want to ask ourselves about maintaining the activity of the macromolecule to which the nanoparticle is appended. To answer this question about activity we turned to a well-characterized and important system: the hammerhead ribozyme. Ribozymes have become increasingly popular in biochemistry research because they have the potential of being powerful gene expression and viral therapy agents. Current ribozyme research seems bound to slower but well-standardized traditional biological techniques as a means of analysis, (PAGE gels, radioactive labeling, etc.) We have been able to show experimentally that rapid detection of ribozyme kinetics and activity is possible by monitoring energy transfer processes to small gold nanoparticles, (see Figure 9.) Nano-surface energy transfer (NSET) allows for real-time monitoring of ribozyme folding and cleavage events, while maintaining bio-compatibility and without altering ribozymal activity. Figure 10 shows a comparison of the kinetics of a hammerhead ribozyme as measured by standard PAGE Gel techniques and as measured by quenching of a fluorescein labeled substrate strand. The kinetics show here that for this reaction NSET is just as valid a technique as PAGE, although much faster, with infinite time resolution and can be performed on very small amounts of sample. You may now ask yourself, "why not just use FRET? It's got the same advantages." - Not true! Although similar to FRET, NSET offers a number of advantages over this classical technique. A major advantage of NSET is the ability to observe simultaneous quenching events of a wide variety of organic dyes covering energies from the visible to the IR. Continuous wave photoluminescence experiments have been able to validate the effectiveness of this technique which increases measurable distances out 2X further (>20nm) than traditional FRET and can allow simultaneous analysis of ribozymal activity on different localizations of the hammerhead moiety. This technique is effective for, but not limited to ribozyme kinetics and could include any study desiring to observe dynamic distance changes in a molecule or macromolecule.

Interested in learning more?
Contact Steve Yun

or read the published articles:

"Nanometal Surface Energy Transfer in Optical Rulers, Breaking the FRET Barrier" C.S. Yun, A. Javier, T. Jennings, M. Fisher, S. Hira, S. Peterson, B. Hopkins, N.O. Reich, and G.F. Strouse, J. Am. Chem. Soc.127(9), 3115-3119 (2005). [view article-PDF]

"Assembly of Nanomaterials Using Bio-Scaffolding." Yun, C.S.; Major, J.L.; Strouse, G.F. Mat. Res. Soc. Symp. Proc., 642, J2.3 (2001). [ view article - PDF ]