Development of a nanoprobe for measuring the molecular dynamics in living cells

In living cells, there are many huge filamentous structures, organelles, protein complexes, and nucleic acids. The protein concentration in living cells is estimated to reach several hundred mg/mL. The complex intracellular environment rises from awful molecular crowding conditions in the cytosol. To learn the dynamic molecular reactions in living cells, it is essential to clarify their physicochemical structure and features like disproportional macromolecular crowding structures, molecular diffusion, and excluded volume effect.

In this project, we have aimed to analyze the macromolecular dynamics, simulate physical structures, and finally discuss the dynamic molecular reactions in cells. Particularly we have aimed to quantitatively measure the intrinsic mRNA dynamics and reactions inside cells by developing a nanoprobe for mRNA using a fine nanoneedle. Our research will be a platform for better achievements in nanomedicine molecular science.

The amount of total mRNA in a cell is approximately 0.5-1.0 x 106 molecules. The transcript copy number is up to 5 x 103 and mostly in the range of 50to 500 copies. For example, from the results of quantitative PCR, GAPDH mRNA in a Hela cell was about 1.0 x 103 copies and its concentration was estimated about 3.4 nM. Thus, to analyze the dynamics of mRNA in living cells, we have to develop an mRNA detection system with nM sensitivity in molecular crowding conditions. To this end, we modified the nanoneedle with molecular beacon, which is a highly sensitive nuclear acid probe. The developed nanoprobe could detect as small as 1 nM RNA in solution, but the reaction time required for 100 nM of RNA was 10 min. By using the nanoprobe for GAPDH or beta-actin, we succeeded in selective detection of the mRNA inside the cells. The estimated concentration of intrinsic GAPDH mRNA was approximately 3 nM by reacted fluorescence intensity of the nanoprobe, but by reaction rate of the nanoprobe, the appeared concentration was estimated 103-fold high rather than the above concentration. Probably this contradiction will be a one of the effects of physico-chemical features on macromolecular dynamics in a cell.

Moreover, we built a measurement system for mechanical property characterization of floating round cells, which have negligible asymmetric acto-myosin tension. This system can evaluate the mechanical properties of many cell types under the same conditions of cell shape. we also succeeded in detecting the non-Newtonian behavior of molecules surrounding the cells using FCS system.

Publications

Shimizu, Y., Kihara, T., Haghparast, S.M.A., Yuba, S., Miyake, J., Simple display system of mechanical properties of cells and their dispersion, PLoS ONE, 7 (3), e34305 (2012).
Mieda, S., Amemiya, Y., Kihara, T., Okada, T., Sato, T., Fukazawa, K., Ishihara, K., Nakamura, N., Miyake, J., Nakamura, C., Mechanical force-based probing of intracellular proteins from living cells using antibody-immobilized nanoneedles, Biosens. Bioelectron., 31(4), 323-329 (2012).
Kihara, T., Haghparast, S.M.A., Shimizu, Y., Yuba, S., Miyake, J., Physical properties of mesenchymal stem cells are coordinated by the perinuclear actin cap, Biochem. Biophys. Res. Commun., 409(1), 1-6 (2011).
Kihara, T., Yoshida, N., Nakamura, C., Nakamura, N., Miyake, J., Development of a novel method to detect intrinsic mRNA in a living cell by using a molecular beacon-immobilized nanoneedle, Biosens. Bioelectron., 26(4), 1449-1454 (2010).
Kagiwada, H., Nakamura, C., Kihara, T., Kamiishi, H., Kawano, K., Nakamura, N., Miyake, J., The mechanical properties of a cell, as determined by its actin cytoskeleton, are important for nanoneedle insertion into a living cell, Cytoskeleton,, 67(8), 496-503 (2010).