Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Article
  • Published:

Target site selection for an RNA-cleaving catalytic DNA

Nature Biotechnology volume 17, pages 480–486 (1999)Cite this article

Abstract

A small catalytic DNA, known as the 10–23 DNA enzyme or deoxyribozyme, has been shown to efficiently hydrolyze RNA at purine-pyrimidine (R-Y) junctions in vitro. Although these potentially cleavable junctions are ubiquitous, they are often protected from deoxyribozyme activity by RNA secondary structure. We have developed a multiplex cleavage assay for screening the entire length of a target RNA molecule for deoxyribozyme cleavage sites that are efficient, both in terms of kinetics and accessibility. This strategy allowed us to simultaneously compare the RNA cleaving activity of 80 deoxyribozymes for a model target gene (HPV16 E6), and an additional 60 deoxyribozymes against the rat c-myc target. The human papilloma virus (HPV) target was used primarily to characterize the multiplex system and determine its validity. The c-myc target, coupled with a smooth muscle cell proliferation assay, allowed us to assess the relationship between in vitro cleavage efficiency and c-myc gene suppression in cell culture. The multiplex reaction approach streamlines the process of revealing effective deoxyribozymes in a functional assay and provides accessibility data that may also be applicable to site selection for other hybridization-based agents.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A schematic representation of the 10–23 general purpose RNA-cleaving DNA enzyme and the HPV16 E6/E7 target sequence.
Figure 2: Phosphorimage of a DNA sequencing gel containing the primer extension products of multiplex cleavage reactions in the E6/E7 transcript.
Figure 3: Graph comparing the normalized intensity profile derived from densitometry of (A) a partial RNase T1 digestion of the substrate, (B) the 50 nM multiplex deoxyribozyme reaction, and (C) the predicted total ΔG° for each deoxyribozyme-substrate heteroduplex.
Figure 4: Analysis of E6 deoxyribozyme-mediated target RNA cleavage and cell-free translation.
Figure 5: Multiplex selection of c-myc–cleaving deoxyribozymes.
Figure 6: Deoxyribozyme-mediated suppression of SMC proliferation.

Similar content being viewed by others

References

  1. Santoro, S.W. & Joyce, G.F. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 94, 4262 –4266 (1997).

    Article  CAS  Google Scholar 

  2. Milner, N., Mir, K.U. & Southern, E.M. Selecting effective antisense reagents on combinatorial oligonucleotide arrays. Nat. Biotechnol. 15, 537–541 (1997).

    Article  CAS  Google Scholar 

  3. Lima, W.F., Brown-Driver, V., Fox, M., Hanecak, R. & Bruice, T.W. Combinatorial screening and rational optimization for hybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity. J. Biol. Chem. 272, 626–638 (1997).

    Article  CAS  Google Scholar 

  4. Birikh, K.R., Berlin, Y.A, Soreq, H. & Eckstein, F. Probing accessible sites for ribozymes on human acetylcholinesterase RNA. RNA 3, 429–437 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Ho, S.P. et al. Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA-accessible sites with oligonucleotide libraries. Nucleic Acids Res. 24, 1901– 1907 (1996).

    Article  CAS  Google Scholar 

  6. Ho, S.P. et al. Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries. Nat. Biotechnol. 16, 59– 63 (1998).

    Article  CAS  Google Scholar 

  7. Matveeva, O., Felden, B., Audlin, S., Gesteland, R.F. & Atkins, J.F. A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures. Nucleic Acids Res. 25, 5010–5016 (1997).

    Article  CAS  Google Scholar 

  8. Lieber, A. & Strauss, M. Selection of efficient cleavage sites in target RNAs by using a ribozyme expression library. Mol. Cell Biol. 15, 540–55 (1995).

    Article  CAS  Google Scholar 

  9. Campbell, T.B. & Cech, T.R. Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA. RNA 1, 598–609 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Sugimoto, N. et al. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34, 11211– 11216 (1995).

    Article  CAS  Google Scholar 

  11. Simons, M., Edelman, E.R. & Rosenberg, R.D. Antisense proliferating cell nuclear antigen oligonucleotides inhibit intimal hyperplasia in a rat carotid artery injury model. J. Clin. Invest. 93, 2351–2356 (1994).

    Article  CAS  Google Scholar 

  12. Lima, W.F., Monia, B.P., Ecker, D.J. & Freier, S.M. Implication of RNA structure on antisense oligonucleotide hybridisation kinetics. Biochemistry 31, 12055–12061 (1992).

    Article  CAS  Google Scholar 

  13. Ratmeyer, L., Vinayak, R., Zhong, Y.Y., Zon, G. & Wilson, W.D. Sequence specific thermodynamic and structural properties for DNA·RNA duplexes. Biochemistry 33, 5298–5304 (1994).

    Article  CAS  Google Scholar 

  14. Gyi, J.I., Lane, A.N., Conn, G.L. & Brown, T. Solution structures of DNA·RNA hybrids with purine-rich and pyrimidine rich strands: Comparison with homologous DNA and RNA duplexes. Biochemistry 37, 73–80 (1998).

    Article  CAS  Google Scholar 

  15. Inoue, H. et al. Synthesis and hybridization studies on two complementary nona(2´-O-methyl)ribonucleotides. Nucleic Acids Res. 15, 6131– 6148 (1987).

    Article  CAS  Google Scholar 

  16. Majlessi, M., Nelson, N.C. & Becker, M.M. Advantages of 2´-O-methylribonucleotide probes for detecting RNA targets. Nucleic Acids Res. 26, 2224–2229 (1998).

    Article  CAS  Google Scholar 

  17. Bielinska, A., Kukowska-Latallo, J.F., Johnson, J., Tomalia, D.A. & Baker, J.R. Jr. Regulation of in vitro gene expression using antisense oligonucleotides or antisense expression plasmids transfected using starburst PAMAM dendrimers. Nucleic Acids Res. 24, 2176–2182 (1996).

    Article  CAS  Google Scholar 

  18. Le Tinevez, R., Mishra, R.K. & Toulme, J.J. Selective inhibition of cell-free translation by oligonucleotides targeted to a mRNA hairpin structure. Nucleic Acids Res. 26, 2273–2278 (1998).

    Article  CAS  Google Scholar 

  19. Murray, V. Improved double-stranded DNA sequencing using the linear polymerase chain reaction. Nucleic Acids Res. 17, 8889 (1989).

    Article  CAS  Google Scholar 

  20. Hendry, P., McCall, M.J., Santiago, F.S. & Jennings, P.A. A ribozyme with DNA in the hybridising arms displays enhanced cleavage ability. Nucleic Acids Res. 21, 5737– 5741 (1992).

    Article  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Johnson and Johnson Research Laboratories, GPO Box 3331, Sydney, 2001, Australia

    Murray J. Cairns, Toni M. Hopkins, Craig Witherington, Li Wang & Lun-Quan Sun

Authors
  1. Murray J. Cairns
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Toni M. Hopkins
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Craig Witherington
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Li Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lun-Quan Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Lun-Quan Sun.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cairns, M., Hopkins, T., Witherington, C. et al. Target site selection for an RNA-cleaving catalytic DNA. Nat Biotechnol 17, 480–486 (1999). https://doi.org/10.1038/8658

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/8658

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing