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:

Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector

Gene Therapy volume 10, pages 1776–1780 (2003)Cite this article

Abstract

A set of single-stranded DNA (ssDNA) expression vectors, which can generate intracellularly any ssDNA or oligodeoxynucleotide (ODN) molecules, have been developed in our laboratory. Studies from our laboratory as well as our collaborators demonstrated that these ssDNA expression vectors are capable of producing: (1) 10-23 DNA enzyme for downregulating c-raf kinase gene expression and (2) triplex-forming oligodeoxynucleotide (TFO) for inducing genomic recombination. We report here the construction of a new version of ssDNA expression vector. A β-galactosidase (β-gal) reporter gene was used as a test target so that the alteration of gene expression can be easily measured using β-gal activity assay. We designed a 10-23 DNA enzyme molecule that specifically cleaves β-gal mRNA at protein translation starting site (ATG). Using a cell-free RNA cleavage assay, we confirmed that this DNA enzyme molecule could effectively cleave β-gal RNA. However, a single substitution from T to G in the catalytic domain of this DNA enzyme molecule abolished its RNA cleavage activity. We also constructed an expression vector that can generate DNA enzyme molecules in cells. A549 lung carcinoma cells were cotransfected with both DNA enzyme expression vector and the β-gal reporter gene. Compared to the cells that were transfected with the mutated DNA enzyme expression vector, significant reduction of β-gal gene expression (up to 76%) was observed in the cells transfected with DNA enzyme expression vector as indicated by the protein expression level as well as its enzyme activity. These results further suggest that the ssDNA expression vector has potential applications in the study of gene function and target validation.

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
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Uhlmann E . Oligonucleotide technologies: synthesis, production, regulations and application. Exp Opin Biol Ther 2001; 1: 319–328.

    Article  CAS  Google Scholar 

  2. Crooke ST . Molecular mechanisms of action of antisense drugs. Biochim Biophys Acta 1999; 1489: 31–44.

    Article  CAS  Google Scholar 

  3. Amarzguioui M, Prydz H . Hammerhead ribozymes design and application. Cell Mol Life Sci 1998; 54: 1175–1202.

    Article  CAS  Google Scholar 

  4. Knauert MP, Glazer PM . Triplex forming oligonucleotides: sequence-specific tools for gene targeting. Hum Mol Genet 2001; 10: 2243–2251.

    Article  CAS  Google Scholar 

  5. Breaker RR . Catalytic DNA: in training and seeking employment. Nat Biotechnol 1999; 17: 422–423.

    Article  CAS  Google Scholar 

  6. Santoro SW, Joyce GF . A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci USA 1997; 94: 4262–4266.

    Article  CAS  Google Scholar 

  7. Santoro SW, Joyce GF . Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry 1998; 37: 13330–13342.

    Article  CAS  Google Scholar 

  8. Cairns MJ, Saravolac EG, Sun LQ . Catalytic DNA: a novel tool for gene suppression. Curr Drug Targets 2002; 3: 269–279.

    Article  CAS  Google Scholar 

  9. Chen Y, Ji Y, Roxby R, Conrad C . In vivo expression of single-stranded DNA in mammalian cells with DNA enzyme sequences targeted to C-raf. Antisense Nucleic Acid Drug Dev 2000; 10: 415–422.

    Article  CAS  Google Scholar 

  10. Datta HJ, Glazer PM . Intracellular generation of single-stranded DNA for chromosomal triplex formation and induced recombination. Nucleic Acid Res. 2001; 29: 5140–5147.

    Article  CAS  Google Scholar 

  11. Chen Y, Ji Y, Conrad C . A novel system for the expression of single-stranded DNA in mammalian cells. Biotechniques 2003; 34: 167–171.

    Article  CAS  Google Scholar 

  12. Lau QC, Brusselback S, Muller R . Abrogation of C-raf expression induces apoptosis in tumor cells. Oncogene 1998; 16: 1899–1902.

    Article  CAS  Google Scholar 

  13. Lau QC et al. In vivo pro-apoptotic and antitumor efficacy of a c-Raf antisense phosphorothioate oligonucleotide: relationship to tumor size. Antisense Nucleic Acid Drug Dev. 2002; 12: 11–20.

    Article  CAS  Google Scholar 

  14. Tanase N, Goff SP . Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities. Proc Natl Acad Sci USA 1988; 85: 1777–1781.

    Article  Google Scholar 

  15. Shinnick TM, Lerner RA, Sutcliffe JG . Nucleotide sequence of Moloney murine leukaemia virus. Nature 1981; 293: 543–548.

    Article  CAS  Google Scholar 

  16. Marquet R, Isel C, Ehresmann C, Ehresmann B . tRNAs as primer of reverse transcriptase. Biochimie 1995; 77: 113–124.

    Article  CAS  Google Scholar 

  17. Wu Y et al. Inhibition of bcr-abl oncogene expression by novel deoxyribozymes (DNAzymes). Hum Gene Ther 1999; 10: 2847–2857.

    Article  CAS  Google Scholar 

  18. Akhtar S et al. The delivery of antisense therapeutics. Adv Drug Delivery Rev 2000; 44: 3–21.

    Article  CAS  Google Scholar 

  19. Miyata S, Ohshima A, Inouye S, Inouye M . In vivo production of a stable single-stranded cDNA in Saccharomyces cerevisiae by means of a bacterial retron. Proc Natl Acad Sci USA 1992; 89: 5735–5739.

    Article  CAS  Google Scholar 

  20. Mirochnitchenko O, Inouye S, Inouye M . Production of single-stranded DNA in mammalian cells by means of a bacterial retron. J Biol Chem 1994; 269: 2380–2383.

    CAS  PubMed  Google Scholar 

  21. Mao J, Shimada M, Inouye S, Inouye M . Gene regulation by antisense DNA produced in vivo. J Biol Chem 1995; 270: 19684–19687.

    Article  CAS  Google Scholar 

  22. Lampson B, Inouye M, Inouye S . The msDNAs of bacteria. Prog Nucleic Acid Res Mol Biol 2001; 67: 65–91.

    Article  CAS  Google Scholar 

  23. Kurreck J, Bieber B, Jahnel R, Erdmann VA . Comparative study of DNA enzymes and ribozymes against the same full-length messenger RNA of the vanilloid receptor subtype I. J Biol Chem 2002; 277: 7099–7107.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank PM Glazer, AM Gewirtz, M Skolnick and XX Tan for their critical reading of the manuscript. We also thank MP Knauert for technical assistance.

Author information

Authors and Affiliations

  1. CytoGenix, Inc., Houston, TX, USA

    Y Chen & H W McMicken

Authors
  1. Y Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. H W McMicken
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, Y., McMicken, H. Intracellular production of DNA enzyme by a novel single-stranded DNA expression vector. Gene Ther 10, 1776–1780 (2003). https://doi.org/10.1038/sj.gt.3302068

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3302068

Keywords

This article is cited by

Search

Advanced search

Quick links