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Nucleic Acids Research Advance Access originally published online on October 11, 2006
Nucleic Acids Research 2006 34(19):5660-5669; doi:10.1093/nar/gkl715
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Nucleic Acids Research, 2006, Vol. 34, No. 19 5660-5669
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Computational Biology

Rational design and rapid screening of antisense oligonucleotides for prokaryotic gene modulation

Yu Shao, Yan Wu, Chi Yu Chan, Kathleen McDonough* and Ye Ding*

Wadsworth Center, New York State Department of Health 150 New Scotland Avenue, Albany, NY 12208, USA

*To whom correspondence should be addressed. Tel: +518 486 1719; Fax: +518 402 4623; Email: yding{at}wadsworth.org

Received June 26, 2006. Revised August 18, 2006. Accepted September 15, 2006.

Antisense oligodeoxynucleotides (oligos) are widely used for functional studies of both prokaryotic and eukaryotic genes. However, the identification of effective target sites is a major issue in antisense applications. Here, we study a number of thermodynamic and structural parameters that may affect the potency of antisense inhibition. We develop a cell-free assay for rapid oligo screening. This assay is used for measuring the expression of Escherichia coli lacZ, the antisense target for experimental testing and validation. Based on a training set of 18 oligos, we found that structural accessibility predicted by local folding of the target mRNA is the most important predictor for antisense activity. This finding was further confirmed by a direct validation study. In this study, a set of 10 oligos was designed to target accessible sites, and another set of 10 oligos was selected to target inaccessible sites. Seven of the 10 oligos for accessible sites were found to be effective (>50% inhibition), but none of the oligos for inaccessible sites was effective. The difference in the antisense activity between the two sets of oligos was statistically significant. We also found that the predictability of antisense activity by target accessibility was greatly improved for oligos targeted to the regions upstream of the end of the active domain for ß-galactosidase, the protein encoded by lacZ. The combination of the structure-based antisense design and extension of the lacZ assay to include gene fusions will be applicable to high-throughput gene functional screening, and to the identification of new drug targets in pathogenic microbes. Design tools are available through the Sfold Web server at http://sfold.wadsworth.org.

*Correspondence may also be addressed to Kathleen McDonough. Tel: +518 486 4235; Fax:+518 402 4773; Email: mcdonoug{at}wadsworth.org

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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