Mode of action of RNA/DNA oligonucleotides: progress in the development of gene repair as a therapy for alpha(1)-antitrypsin deficiency.
We describe a technology developed for the site-specific correction of a single base carried on an episome or chromosome in prokaryotic and eukaryotic cells. Critical to the development of this technology as a therapeutic device for treating genetic disorders, like alpha(1)-antitrypsin deficiency, is the establishment of a standardized assay to study its mode of action and structure-activity relationships (SARs). To this end, a positive-selection system in Escherichia coli has been developed to assess RNA/DNA oligonucleotide (RDO)-directed repair activity. We demonstrate that RDO-directed repair requires the concerted action of the two following repair proteins: the pairing protein RecA; and the mismatch recognition protein, MutS. SAR studies demonstrate that the RDO molecule is functionally asymmetric. The RNA-containing strand enables strand-pairing and stabilization of the molecule, and the DNA-containing strand confers the information transfer.
Metz
Richard
R
ValiGen, Inc, Princeton, NJ, USA. janislmetz@aol.com
DiCola
Mike
M
Kurihara
Takao
T
Bailey
Austin
A
Frank
Bruce
B
Roecklein
Bryan
B
Blaese
Michael
M
D000251:Adenosine Triphosphatases; D001426:Bacterial Proteins / Q000235:genetics; D004260:DNA Repair; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D015316:Genetic Therapy; D006801:Humans; D051719:MutS DNA Mismatch-Binding Protein; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011656:Pulmonary Emphysema / Q000209:etiology / Q000235:genetics / Q000628:therapy; D011938:Rec A Recombinases / Q000235:genetics; D013329:Structure-Activity Relationship; D000515:alpha 1-Antitrypsin / Q000235:genetics; D019896:alpha 1-Antitrypsin Deficiency / Q000150:complications / Q000235:genetics / Q000628:therapy*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D000515:alpha 1-Antitrypsin; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli