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. (See molecular representation below.)

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 Travis Jennings

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 ]