Taking a DNA molecule into the vicinity of a homologous target gene by a DNA aptamer provides a many-fold enhancement of gene correction frequency at that genetic locus. Aptamer-guided gene targeting, or AGT, is a novel approach for genetic engineering developed by Patrick Ruff in Francesca Storici’s group.

Gene targeting is a genetic technique to modify an endogenous DNA sequence at will, by changing a mutant DNA sequence into a wild-type copy or vice versa in its genomic location via homologous recombination. Gene targeting is therefore a fundamental process not only for functional analysis of genes, proteins, and complex biological systems, but potentially also in molecular therapy for the prevention and cure of human genetic diseases originating from specific DNA alterations. However, editing of genetic information is a challenging task. The goal of gene correction goes far beyond the process of making a desired change in a chosen target gene in the most efficient way. It is essential that the product of the modified gene should then be functional, the DNA correction stable, and the engineering process accurate and restrained to the target in order to minimize unwanted DNA, cellular, and/or tissue damage.

In the most recent years a lot of progress has been made in activating cellular DNA repair and recombination machinery at the target sites for gene correction, mainly via the specific induction of DNA double-strand breaks (DSBs) at these sites. However, there has been much less focus on the other essential component for gene targeting: the donor DNA necessary to make the desired modification. To address the problem of donor DNA availability, Patrick Ruff, fresh PhD recipient in the lab of Francesca Storici from the School of Biology at Georgia Tech, developed a novel gene targeting approach, aptamer-guided gene targeting (AGT), in which he bound the homing endonuclease I-SceI by a DNA aptamer fused to the donor DNA of choice, to target the donor DNA to a desired genetic locus located next to an I-SceI cut site. DNA aptamers, which mimic antibodies, are sequences of DNA that are able to bind to a specific target with high affinity because of their unique secondary structure. Using a variant of capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) called “Non-SELEX”, Patrick obtained a DNA aptamer for the I-SceI endonuclease, and with the assistance of Storici lab graduate students Kyung Duk Koh and Havva Keskin, and the research scientist Rekha Pai, found that the AGT approach increases the efficiency of gene targeting by guiding an exogenous donor DNA into the vicinity of the site targeted for genetic modification. Dr. Storici said: "by utilizing DNA oligodeoxyribonucleotides that contained the I-SceI aptamer sequence as well as homology to repair the I-SceI DSB and correct a target gene, we were able to increase gene targeting frequencies up to 32-fold over a non-binding control in yeast and up to 16-fold over a non-binding control in human cells".

This study shows that DNA aptamers can be exploited to increase donor DNA availability, and thus promote the transfer of genetic information from a donor DNA molecule to a desired genetic locus. The AGT strategy offers a novel way to increase gene targeting efficiency, represents the first investigation to use aptamers in the context of gene correction, and provides a new direction to the field of genetic engineering.

The study is just published as an article in the journal Nucleic Acids Res (Wednesday February 5, 2014):

Ruff, P., Koh K.D., Keskin H., Pai R.B. and Storici, F. Aptamer-guided gene targeting in yeast and human cells, Nucleic Acids Res, Feb 5 2014 doi:10.1093/nar/gku101 http://nar.oxfordjournals.org/cgi/reprint/gku101?
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 This project was supported by the Georgia Tech Fund for Innovation in Research and Education (GTFIRE-021763), the NIH grant (R21EB9228), and the Georgia Cancer Coalition grant (award R9028).