As recently demonstrated in the yeast Saccharomyces cerevisiae model organism using synthetic RNAcontaining
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.