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Silencing gene expression by targeting chromosomal DNA with antigene peptide nucleic acids and duplex RNAs

Nature Protocols volume 1, pages 436–443 (2006)Cite this article

Abstract

The value of recognizing cellular RNA sequences by short interfering RNAs (siRNAs) in mammalian cells is widely appreciated, but what might be learned if it were also possible to recognize chromosomal DNA? Recognition of chromosomal DNA would have many applications, such as inhibiting gene expression, activating gene expression, introducing mutations, and probing chromosome structure and function. We have shown that antigene peptide nucleic acids (agPNAs) and antigene duplex RNAs (agRNAs) block gene expression and probe chromosomal DNA. Here we describe a protocol for designing antigene agents and introducing them into cells. This protocol can also be used to silence expression with PNAs or siRNAs that target mRNA. From preparation of oligomers to analysis of data, experiments with agPNAs and agRNAs require ∼14 d and 9 d, respectively.

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Figure 1: Targeting a transcription start site with agPNAs or agRNAs.
Figure 2: Fluorescent confocal microscopy showing cellular uptake of PNA complexes.
Figure 3: Inhibition of human PR expression by agPNA-peptide conjugates.
Figure 4: Time course for inhibiting the expression of PR-A and PR-B by agPNAs delivered in complex with lipid and DNA.
Figure 5: Inhibiting expression of PR-A and PR-B isoforms by agRNAs.

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Acknowledgements

We thank S. Younger, D. Ly and B. Armitage for comments. This work was supported by the National Institutes of Health (grants NIGMS 60642 and 73042 to D.R.C.), the High-Q Foundation, Applied Biosystems and the Robert A. Welch Foundation (grant I-1244 to D.R.C.).

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Authors and Affiliations

  1. Departments of Pharmacology and Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, 75390-9041, Texas, USA

    Bethany A Janowski, Jiaxin Hu & David R Corey

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  1. Bethany A Janowski
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Contributions

D.R.C. wrote the manuscript and supervised the experiments. B.A.J. and J.H. developed the protocols and assisted in writing the manuscript.

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Correspondence to Bethany A Janowski or David R Corey.

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This work was partially funded by Applied Biosystems.

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Janowski, B., Hu, J. & Corey, D. Silencing gene expression by targeting chromosomal DNA with antigene peptide nucleic acids and duplex RNAs. Nat Protoc 1, 436–443 (2006). https://doi.org/10.1038/nprot.2006.64

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