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Improvement of RNA-DNA oligonucleotide design by using mammalian nuclear extracts

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An RNA-DNA oligonucleotide (RDO) has been shown to either correct or cause a specific point mutation in episomal or genomic target DNA in mammalian cells. The original design of the RDO consists of a double-hairpin capped duplex comprising a 25 nucleotide-long DNA stretch (DNA-containing strand) paired to a fully complementary 2'-O-methyl RNA stretch with a pentameric DNA interruption that carries a mismatch to target DNA (RNA-containing strand). In order to improve the gene conversion activity of the RDO, several oligonucleotides with structural and chemical modifications were synthesized and compared in their gene correction activity. Previously, we established an in vitro system capable of RDO-mediated gene correction of a point mutation (G → A) in the E. coli β-galactosidase gene by using mammalian nuclear extracts. Conversion frequencies among six mammalian cell types and the chicken DT40 cell line were compared by using a convenient bacterial assay that score blue or white colonies. This in vitro reaction with DT40 nuclear extract is now used to study the structure and activity relationship of the RDO. Modifications of the original RDO design including a complete sequence complementarity of the RNA-containing strand to target DNA, a replacement of the central five DNA residues with 2'-O-methyl RNA, and chemical modification of the hairpin loops result in a ten-fold increase in gene correction activity. Moreover, we show that the single-base correction in the target DNA is preferentially driven by the DNA-containing strand of the RDO by comparing two RDOs that carry a mismatch to target DNA either on the RNA- or DNA-containing strand. Thus, the highly sensitive and convenient assay utilizing E. coli β-galactosidase is not only useful to compare the gene correction frequency among different cell types but also to optimize the RDO structure.

Affiliations: 1: Departments of Dermatology & Cutaneous Biology, and of Biochemistry & Molecular Pharmacology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University and Jefferson Medical College, 233 South 10th street, Philadelphia, PA19107, USA


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