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

Shao Y, Wu Y, Chan CY, McDonough K, Ding Y - Nucleic Acids Res. (2006)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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 beta-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.

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Thermodynamic cycle of free energy exchanges. ΔGdisruption is the target disruption energy, which measures the free energy cost to open the secondary structure at the target site; ΔGoligo is the self-folding minimal free energy (MFE) of the antisense oligo; ΔGhybrid is the binding energy (stability) for the hybrid formed between the antisense oligo and the target site.
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fig1: Thermodynamic cycle of free energy exchanges. ΔGdisruption is the target disruption energy, which measures the free energy cost to open the secondary structure at the target site; ΔGoligo is the self-folding minimal free energy (MFE) of the antisense oligo; ΔGhybrid is the binding energy (stability) for the hybrid formed between the antisense oligo and the target site.

Mentions: ΔGtotal is the total free energy change for the thermodynamic cycle of energy exchanges, as illustrated in Figure 1. It takes into account the intramolecular secondary structures of both the target and the antisense oligo, and the stability of the oligo:target hybrid. Thus, ΔGtotal is calculated byΔGtotal=ΔGhybrid−ΔGdisruption−ΔGoligo.


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

Shao Y, Wu Y, Chan CY, McDonough K, Ding Y - Nucleic Acids Res. (2006)

Thermodynamic cycle of free energy exchanges. ΔGdisruption is the target disruption energy, which measures the free energy cost to open the secondary structure at the target site; ΔGoligo is the self-folding minimal free energy (MFE) of the antisense oligo; ΔGhybrid is the binding energy (stability) for the hybrid formed between the antisense oligo and the target site.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636493&req=5

fig1: Thermodynamic cycle of free energy exchanges. ΔGdisruption is the target disruption energy, which measures the free energy cost to open the secondary structure at the target site; ΔGoligo is the self-folding minimal free energy (MFE) of the antisense oligo; ΔGhybrid is the binding energy (stability) for the hybrid formed between the antisense oligo and the target site.
Mentions: ΔGtotal is the total free energy change for the thermodynamic cycle of energy exchanges, as illustrated in Figure 1. It takes into account the intramolecular secondary structures of both the target and the antisense oligo, and the stability of the oligo:target hybrid. Thus, ΔGtotal is calculated byΔGtotal=ΔGhybrid−ΔGdisruption−ΔGoligo.

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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 beta-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.

Show MeSH
Related in: MedlinePlus