Nanoprobes for Live-Cell Gene Detection
The ability to visualize in real time the expression dynamics and localization of specific RNAs in vivo offers tremendous opportunities for biological and disease studies including cancer detection. However, quantitative methods such as real-time polymerase chain reaction (PCR) and DNA microarrays rely on the use of cell lysates thus not able to obtain important spatial and temporal information. Fluorescence proteins and other reporter systems cannot image endogenous RNA in living cells. Fluorescence in situ hybridization (FISH) assays require washing to achieve specificity, therefore can only be used with fixed cells. Here we review the recent development of nanostructured probes for living cell RNA detection and discuss the biological and engineering issues and challenges of quantifying gene expression in vivo. In particular, we describe methods that use oligonucleotide probes, combined with novel delivery strategies, to image the relative level, localization, and dynamics of RNA in live cells. Examples of detecting endogenous messenger RNAs, as well as imaging their subcellular localization are given to illustrate the biological applications, and issues in probe design, delivery, and target accessibility are discussed. The nanostructured probes promise to open new and exciting opportunities in sensitive gene detection for a wide range of biological and medical applications.
KeywordsHairpin probe Oligonucleotide probe RNA detection Live cell Molecular beacon Fluorescence resonance energy transfer
This work was supported by the National Heart Lung and Blood Institute of the NIH as a Program of Excellence in Nanotechnology (HL80711), by the National Cancer Institute of the NIH as a Center of Cancer Nanotechnology Excellence (CA119338), and by the NIH Roadmap Initiative in Nanomedicine through a Nanomedicine Development Center award (PN2EY018244).
- 9.Behrens, S., Fuchs, B.M., Mueller, F. and Amann, R. (2003) Is the in situ accessibility of the 16S rRNA of Escherichia coli for Cy3-labeled oligonucleotide probes predicted by a three-dimensional structure model of the 30S ribosomal subunit? Appl Environ Microbiol, 69, 4935–4941.CrossRefGoogle Scholar
- 14.Dirks, R.W., Molenaar, C. and Tanke, H.J. (2001) Methods for visualizing RNA processing and transport pathways in living cells. Histochem Cell Biol, 115, 3–11.Google Scholar
- 38.Haim, L., Zipor, G., Aronov, S. & Gerst, J. E. (2007) A genomic integration method to visualize localization of endogenous mRNAs in living yeast. Nat Methods, 4, 409–412.Google Scholar
- 39.Ozawa, T., Natori, Y., Sato, M. & Umezawa, Y. (2007) Imaging dynamics of endogenous mitochondrial RNA in single living cells. Nat Methods, 4, 413–419.Google Scholar
- 40.Valencia-Burton, M., McCullough, R. M., Cantor, C. R. & Broude, N. E. (2007) RNA visualization in live bacterial cells using fluorescent protein complementation. Nat Methods, 4, 421–427.Google Scholar
- 42.Rhee, W.J., Santangelo, P.J., Jo, H., Bao, G. (2007) Target accessibility and signal specificity in live-cell detection of BMP-4 mRNA using molecular beacons. Nucleic Acids Res, submitted.Google Scholar
- 43.Lakowicz, J.R. (1999) Principles of Fluorescence Spectroscopy. 2nd ed. Springer-Verlag, New York.Google Scholar
- 45.Price, N.C. and Stevens, L. (1999) Fundamentals of Enzymology: The Cell and Molecular Biology of Catalytic Proteins. 3rd ed. Oxford University Press, New York.Google Scholar
- 53.Snyder, E.L., Dowdy, S.F. (2001) Protein/peptide transduction domains: potential to deliver large DNA molecules into cells. Curr Opin Mol Ther, 3, 147–152.Google Scholar
- 59.Troy, C.M., Derossi, D., Prochiantz, A., Greene, L.A. and Shelanski, M.L. (1996) Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J Neurosci, 16, 253–261.Google Scholar
- 60.Minamoto, T., Mai, M., Ronai, Z. (2000) K-ras mutation: early detection in molecular diagnosis and risk assessment of colorectal, pancreas, and lung cancers–a review. Cancer Detect Prev, 24, 1–12.Google Scholar
- 61.Altieri, D.C., and Marchisio, P.C. (1999) Survivin apoptosis: an interloper between cell death and cell proliferation in cancer. Lab Invest, 79, 1327–1333.Google Scholar