title: RNA-driven genetic changes in bacteria and in human cells.
abstract: As recently demonstrated in the yeast Saccharomyces cerevisiae model organism using synthetic RNA-containing oligonucleotides (oligos), RNA can serve as a template for DNA synthesis at the chromosomal level during the process of double-strand break (DSB) repair. Herein we show that the phenomenon of RNA-mediated DNA modification and repair is not limited to yeast cells. A tract of six ribonucleotides embedded in single-strand DNA oligos corresponding to either lagging or leading strand sequences could serve as a template to correct a defective lacZ marker gene in the chromosome of the bacterium Escherichia coli. In order to test the capacity of RNA to modify DNA in mammalian cells, we utilized DNA oligos containing an embedded tract of six ribonucleotides, as well as oligos mostly made of RNA. These oligos were designed to repair a chromosomal break generated within a copy of the green fluorescent protein (GFP) gene randomly integrated into the genome of human HEK-293 cells. We show that these RNA-containing oligos can serve as templates to repair a DSB in human cells and can introduce base changes into genomic or plasmid DNA. In both E. coli and human cells, the strand bias of chromosomal gene correction by the single-strand RNA-containing oligos was the same as that obtained for the corresponding DNA molecules. Therefore, the RNA-containing oligos are not converted into a cDNA before annealing with complementary DNA. Overall, we demonstrate that in both bacterial and human cells, as in yeast, RNA sequences can have a direct role in DNA genetic modification and remodeling.
authors.affiliation: School of Biology, Georgia Institute of Technology, Atlanta, GA, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Shen
authors.forename: Pavan
authors.initials: P
authors.lastname: Nandi
authors.forename: Matthew B
authors.initials: MB
authors.lastname: Taylor
authors.forename: Samantha
authors.initials: S
authors.lastname: Stuckey
authors.forename: Hershel P
authors.initials: HP
authors.lastname: Bhadsavle
authors.forename: Bernard
authors.initials: B
authors.lastname: Weiss
authors.forename: Francesca
authors.initials: F
authors.lastname: Storici
mesh_terms: D001483:Base Sequence; D002875:Chromosomes / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004926:Escherichia coli / Q000166:cytology* / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism*
chemical_list: D009841:Oligonucleotides; D012313:RNA; D004247:DNA