Skip to main content

Advertisement

SpringerLink
Log in

Nanostructured Probes for RNA Detection in Living Cells

  • Nanobioengineering
  • Published:
Annals of Biomedical Engineering Aims and scope Submit manuscript

The ability to visualize in real-time the expression level and localization of specific RNAs in living cells can offer tremendous opportunities for biological and disease studies. Here we review the recent development of nanostructured oligonucleotide 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 dual FRET (fluorescence resonance energy transfer) or single molecular beacons in combination with peptide-based or membrane-permeabilization-based delivery, to image the relative level, localization, and dynamics of RNA in live cells. Examples of detecting endogenous mRNAs, as well as imaging their subcellular localization and colocalization are given to illustrate the biological applications, and issues in molecular beacon design, probe 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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.

Similar content being viewed by others

REFERENCES

  1. Adams, M. D., M. Dubnick, A. R. Kerlavage, R. Moreno, J. M. Kelley, T. R. Utterback, J. W. Nagle, C. Fields, and J. C. Venter. Sequence identification of 2375 human brain genes. Nature 355:632–634, 1992.

    Article  PubMed  CAS  Google Scholar 

  2. Allinquant, B., P. Hantraye, P. Mailleux, K. Moya, C. Bouillot, and A. Prochiantz. Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro. J. Cell Biol. 128:919–927, 1995.

    Article  PubMed  CAS  Google Scholar 

  3. Alwine, J. C., D. J. Kemp, B. A. Parker, J. Reiser, J. Renart, G. R. Stark, and G. M. Wahl. Detection of specific RNAs or specific fragments of DNA by fractionation in gels and transfer to diazobenzyloxymethyl paper. Methods Enzymol. 68:220–242, 1979.

    Article  PubMed  CAS  Google Scholar 

  4. Barry, M. A., and A. Eastman. Identification of deoxyribonuclease II as an endonuclease involved in apoptosis. Arch. Biochem. Biophys. 300:440–450, 1993.

    Article  PubMed  CAS  Google Scholar 

  5. Bassell, G. J., C. M. Powers, K. L. Taneja, and R. H. Singer. Single mRNAs visualized by ultrastructural in situ hybridization are principally localized at actin filament intersections in fibroblasts. J. Cell Biol. 126:863–876, 1994.

    Article  PubMed  CAS  Google Scholar 

  6. Becker-Hapak, M., S. S. McAllister, and S. F. Dowdy. TAT-mediated protein transduction into mammalian cells. Methods 24:247–256, 2001.

    Article  PubMed  CAS  Google Scholar 

  7. Behrens, S., B. M. Fuchs, F. Mueller, and R. Amann. 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, 2003.

    Article  PubMed  CAS  Google Scholar 

  8. Bernacchi, S., and Y. Mely. Exciton interaction in molecular beacons: A sensitive sensor for short range modifications of the nucleic acid structure. Nucleic Acids Res. 29:E62–2, 2001.

    Article  PubMed  CAS  Google Scholar 

  9. Bonnet, G., S. Tyagi, A. Libchaber, and F. R. Kramer. Thermodynamic basis of the enhanced specificity of structured DNA probes. Proc. Natl. Acad. Sci. U.S.A. 96:6171–6176, 1999.

    Article  PubMed  CAS  Google Scholar 

  10. Bratu, D. P., B. J. Cha, M. M. Mhlanga, F. R. Kramer, and S. Tyagi. Visualizing the distribution and transport of mRNAs in living cells. Proc. Natl. Acad. Sci. U.S.A. 100:13308–13313, 2003.

    Article  PubMed  CAS  Google Scholar 

  11. Brodsky, A. S., and P. A. Silver. Identifying proteins that affect mRNA localization in living cells. Methods 26:151–155, 2002.

    Article  PubMed  CAS  Google Scholar 

  12. Brooks, H., B. Lebleu, and E. Vives. Tat peptide-mediated cellular delivery: Back to basics. Adv. Drug Deliv. Rev. 57:559–577, 2005.

    Article  PubMed  CAS  Google Scholar 

  13. Buongiorno-Nardelli, M., and F. Amaldi. Autoradiographic detection of molecular hybrids between RNA and DNA in tissue sections. Nature 225:946–948, 1970.

    Article  PubMed  CAS  Google Scholar 

  14. Crick, F. H. On protein synthesis. Symp. Soc. Exp. Biol. 12:138–163, 1958.

    PubMed  CAS  Google Scholar 

  15. Dirks, R. W., C. Molenaar, and H. J. Tanke. Methods for visualizing RNA processing and transport pathways in living cells. Histochem. Cell Biol. 115:3–11, 2001.

    PubMed  CAS  Google Scholar 

  16. Dokka, S., and Y. Rojanasakul. Novel non-endocytic delivery of antisense oligonucleotides. Adv. Drug Deliv. Rev. 44:35–49, 2000.

    Article  PubMed  CAS  Google Scholar 

  17. Forrest, K. M., and E. R. Gavis. Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila. Curr. Biol. 13:1159–1168, 2003.

    Article  PubMed  CAS  Google Scholar 

  18. Giles, R. V., C. J. Ruddell, D. G. Spiller, J. A. Green, and D. M. Tidd. Single base discrimination for ribonuclease H-dependent antisense effects within intact human leukaemia cells. Nucleic Acids Res. 23:954–961, 1995.

    Article  PubMed  CAS  Google Scholar 

  19. Giles, R. V., D. G. Spiller, J. Grzybowski, R. E. Clark, P. Nicklin, and D. M. Tidd. Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells. Nucleic Acids Res. 26:1567–1575, 1998.

    Article  PubMed  CAS  Google Scholar 

  20. Glotzer, J. B., R. Saffrich, M. Glotzer, and A. Ephrussi. Cytoplasmic flows localize injected oskar RNA in Drosophila oocytes. Curr. Biol. 7:326–337, 1997.

    Article  PubMed  CAS  Google Scholar 

  21. Hamaguchi, N., A. Ellington, and M. Stanton. Aptamer beacons for the direct detection of proteins. Anal. Biochem. 294:126–131, 2001.

    Article  PubMed  CAS  Google Scholar 

  22. Huang, Q., and T. Pederson. A human U2 RNA mutant stalled in 3′ end processing is impaired in nuclear import. Nucleic Acids Res. 27:1025–1031, 1999.

    Article  PubMed  CAS  Google Scholar 

  23. Jacobson, M. R., and T. Pederson. Localization of signal recognition particle RNA in the nucleolus of mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 95:7981–7986, 1998.

    Article  PubMed  CAS  Google Scholar 

  24. Kambhampati, D., P. E. Nielsen, and W. Knoll. Investigating the kinetics of DNA–DNA and PNA–DNA interactions using surface plasmon resonance-enhanced fluorescence spectroscopy. Biosens. Bioelectron. 16:1109–1118, 2001.

    Article  PubMed  CAS  Google Scholar 

  25. Kostrikis, L. G., S. Tyagi, M. M. Mhlanga, D. D. Ho, and F. R. Kramer. Spectral genotyping of human alleles. Science 279:1228–1229, 1998.

    Article  PubMed  CAS  Google Scholar 

  26. Kuhn, H., V. V. Demidov, J. M. Coull, M. J. Fiandaca, B. D. Gildea, and M. D. Frank-Kamenetskii. Hybridization of DNA and PNA molecular beacons to single-stranded and double-stranded DNA targets. J. Am. Chem. Soc. 124:1097–1103, 2002.

    Article  PubMed  CAS  Google Scholar 

  27. Lakowicz, J. R., Principles of Fluorescence Spectroscopy, 2nd ed. New York: Plenum Publishing, 1999.

  28. Leonetti, J. P., N. Mechti, G. Degols, C. Gagnor, and B. Lebleu. Intracellular distribution of microinjected antisense oligonucleotides. Proc. Natl. Acad. Sci. U.S.A. 88:2702–2706, 1991.

    Article  PubMed  CAS  Google Scholar 

  29. Li, J. J., R. Geyer, and W. Tan. Using molecular beacons as a sensitive fluorescence assay for enzymatic cleavage of single-stranded DNA. Nucleic Acids Res. 28:E52, 2000.

    Article  PubMed  CAS  Google Scholar 

  30. Liang, P., and A. B. Pardee. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 257:967–971, 1992.

    Article  PubMed  CAS  Google Scholar 

  31. Liu, X., and W. Tan. A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons. Anal. Chem. 71:5054–5059, 1999.

    Article  PubMed  CAS  Google Scholar 

  32. Marras, S. A., F. R. Kramer, and S. Tyagi. Efficiencies of fluorescence resonance energy transfer and contact-mediated quenching in oligonucleotide probes. Nucleic Acids Res. 30:e122, 2002.

    Article  PubMed  Google Scholar 

  33. Medley, C. D., T. J. Drake, J. M. Tomasini, R. J. Rogers, and W. Tan. Simultaneous monitoring of the expression of multiple genes inside of single breast carcinoma cells. Anal. Chem. 77:4713–4718, 2005.

    Article  PubMed  CAS  Google Scholar 

  34. Mhlanga, M. M., V. D. Fung, F. R. Kramer, and S. Tyagi. tRNA-linked molecular beacons for imaging mRNAs in the cytoplasm of living cells. Nucleic Acids Res. 33:1902–1912, 2005.

    Article  PubMed  CAS  Google Scholar 

  35. Molenaar, C., A. Abdulle, A. Gena, H. J. Tanke, and R. W. Dirks. Poly(A)+ RNAs roam the cell nucleus and pass through speckle domains in transcriptionally active and inactive cells. J. Cell Biol. 165:191–202, 2004.

    Article  PubMed  CAS  Google Scholar 

  36. Molenaar, C., S. A. Marras, J. C. Slats, J. C. Truffert, M. Lemaitre, A. K. Raap, R. W. Dirks, and H. J. Tanke. Linear 2′ O-Methyl RNA probes for the visualization of RNA in living cells. Nucleic Acids Res. 29:E89–9, 2001.

    Article  PubMed  CAS  Google Scholar 

  37. Nitin, N., P. J. Santangelo, G. Kim, S. Nie, and G. Bao. Peptide-linked molecular beacons for efficient delivery and rapid mRNA detection in living cells. Nucleic Acids Res. 32:e58, 2004.

    Article  PubMed  CAS  Google Scholar 

  38. Peng, X. H., Z. H. Cao, J. T. Xia, G. W. Carlson, M. M. Lewis, W. C. Wood, and L. Yang. Real-time detection of gene expression in cancer cells using molecular beacon imaging: New strategies for cancer research. Cancer Res. 65:1909–1917, 2005.

    Article  PubMed  CAS  Google Scholar 

  39. Piatek, A. S., T. S. Pol, A. C. Telenti, L. P. Miller, F. R. Kramer, and D. Alland. Molecular beacon sequence analysis for detecting drug resistance in Mycobacterium tuberculosis. Nat. Biotechnol. 16:359–363, 1998.

    Article  PubMed  CAS  Google Scholar 

  40. Price, N. C., and L. Stevens. Fundamentals of Enzymology: The Cell and Molecular Biology of Catalytic Proteins, 3rd ed. New York: Oxford University Press, 1999.

  41. Saiki, R. K., S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354, 1985.

    Article  PubMed  CAS  Google Scholar 

  42. Santangelo, P. J., N. Nitin, and G. Bao. Direct visualization of mRNA colocalization with mitochondria in living cells using molecular beacons. J. Biomed. Opt. 10: 44025, 2005.

    Google Scholar 

  43. Santangelo, P. J., B. Nix, A. Tsourkas, and G. Bao. Dual FRET molecular beacons for mRNA detection in living cells. Nucleic Acids Res. 32:e57, 2004.

    Article  PubMed  CAS  Google Scholar 

  44. Schena, M., D. Shalon, R. W. Davis, and P. O. Brown. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467–470, 1995.

    Article  PubMed  CAS  Google Scholar 

  45. Shav-Tal, Y., X. Darzacq, S. M. Shenoy, D. Fusco, S. M. Janicki, D. L. Spector, and R. H. Singer. Dynamics of single mRNPs in nuclei of living cells. Science 304:1797–1800, 2004.

    Article  PubMed  CAS  Google Scholar 

  46. Snyder, E. L., and S. F. Dowdy. Protein/peptide transduction domains: Potential to deliver large DNA molecules into cells. Curr. Opin. Mol. Ther. 3:147–152, 2001.

    PubMed  CAS  Google Scholar 

  47. Sokol, D. L., X. Zhang, P. Lu, and A. M. Gewirtz. Real time detection of DNA.RNA hybridization in living cells. Proc. Natl. Acad. Sci. U.S.A. 95:11538–11543, 1998.

    Article  PubMed  CAS  Google Scholar 

  48. Spiller, D. G., R. V. Giles, J. Grzybowski, D. M. Tidd, and R. E. Clark. Improving the intracellular delivery and molecular efficacy of antisense oligonucleotides in chronic myeloid leukemia cells: A comparison of streptolysin-O permeabilization, electroporation, and lipophilic conjugation. Blood 91:4738–4746, 1998.

    PubMed  CAS  Google Scholar 

  49. States, D. J., W. Gish, and S. F. Altschul. Improved sensitivity of nucleic acid database searches using application-specific scoring matrices. Methods 3:66–70, 1991.

    Article  CAS  Google Scholar 

  50. Steemers, F. J., J. A. Ferguson, and D. R. Walt. Screening unlabeled DNA targets with randomly ordered fiber-optic gene arrays. Nat. Biotechnol. 18:91–94, 2000.

    Article  PubMed  CAS  Google Scholar 

  51. Troy, C. M., D. Derossi, A. Prochiantz, L. A. Greene, and M. L. Shelanski. Downregulation of Cu/Zn superoxide dismutase leads to cell death via the nitric oxide-peroxynitrite pathway. J. Neurosci. 16:253–261, 1996.

    PubMed  CAS  Google Scholar 

  52. Tsourkas, A., M. A. Behlke, and G. Bao. Structure–function relationships of shared-stem and conventional molecular beacons. Nucleic Acids Res. 30:4208–4215, 2002.

    Article  PubMed  CAS  Google Scholar 

  53. Tsourkas, A., M. A. Behlke, S. D. Rose, and G. Bao. Hybridization kinetics and thermodynamics of molecular beacons. Nucleic Acids Res. 31:1319–1330, 2003.

    Article  PubMed  CAS  Google Scholar 

  54. Tsourkas, A., M. A. Behlke, Y. Xu, and G. Bao. Spectroscopic features of dual fluorescence/luminescence resonance energy-transfer molecular beacons. Anal. Chem. 75:3697–3703, 2003.

    Article  PubMed  CAS  Google Scholar 

  55. Tsuji, A., H. Koshimoto, Y. Sato, M. Hirano, Y. Sei-Iida, S. Kondo, and K. Ishibashi. Direct observation of specific messenger RNA in a single living cell under a fluorescence microscope. Biophys. J. 78:3260–3274, 2000.

    Article  PubMed  CAS  Google Scholar 

  56. Tyagi, S., and O. Alsmadi. Imaging native beta-actin mRNA in motile fibroblasts. Biophys. J. 87:4153–4162, 2004.

    Article  PubMed  CAS  Google Scholar 

  57. Tyagi, S., D. P. Bratu, and F. R. Kramer. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16:49–53, 1998.

    Article  PubMed  CAS  Google Scholar 

  58. Tyagi, S., and F. R. Kramer. Molecular beacons: Probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303–308, 1996.

    Article  PubMed  CAS  Google Scholar 

  59. Tyagi, S., S. A. Marras, and F. R. Kramer. Wavelength-shifting molecular beacons. Nat. Biotechnol. 18:1191–1196, 2000.

    Article  PubMed  CAS  Google Scholar 

  60. Velculescu, V. E., L. Zhang, B. Vogelstein, and K. W. Kinzler. Serial analysis of gene expression. Science 270:484–487, 1995.

    Article  PubMed  CAS  Google Scholar 

  61. Vet, J. A., A. R. Majithia, S. A. Marras, S. Tyagi, S. Dube, B. J. Poiesz, and F. R. Kramer. Multiplex detection of four pathogenic retroviruses using molecular beacons. Proc. Natl. Acad. Sci. U.S.A. 96:6394–6399, 1999.

    Article  PubMed  CAS  Google Scholar 

  62. Wadia, J. S., and S. F. Dowdy. Protein transduction technology. Curr. Opin. Biotechnol. 13:52–56, 2002.

    Article  PubMed  CAS  Google Scholar 

  63. Wadia, J. S., and S. F. Dowdy. Transmembrane delivery of protein and peptide drugs by TAT-mediated transduction in the treatment of cancer. Adv. Drug Deliv. Rev. 57:579–596, 2005.

    Article  PubMed  CAS  Google Scholar 

  64. Walev, I., S. C. Bhakdi, F. Hofmann, N. Djonder, A. Valeva, K. Aktories, and S. Bhakdi. Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O. Proc. Natl. Acad. Sci. U.S.A. 98:3185–3190, 2001.

    Article  PubMed  CAS  Google Scholar 

  65. Yamamoto, R., T. Baba, and P. K. Kumar. Molecular beacon aptamer fluoresces in the presence of Tat protein of HIV-1. Genes Cells 5:389–396, 2000.

    Article  PubMed  CAS  Google Scholar 

Download references

ACKNOWLEGMENT

This work was supported in part by National Institute of Health Grant UO1 HL80711-01 and by the Office of Science, Department of Energy Grant DE-FG02-04ER63785 to GB.

Author information

Authors and Affiliations

  1. Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA, USA

    Philip Santangelo, Nitin Nitin & Gang Bao

  2. Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Dr, Suite 2115, Atlanta, GA, 30332, USA

    Gang Bao

Authors
  1. Philip Santangelo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nitin Nitin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gang Bao
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gang Bao.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Santangelo, P., Nitin, N. & Bao, G. Nanostructured Probes for RNA Detection in Living Cells. Ann Biomed Eng 34, 39–50 (2006). https://doi.org/10.1007/s10439-005-9003-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10439-005-9003-6

Keywords

Search

Navigation