Abstract
Triplex-forming oligonucleotides (TFOs) bind specifically to duplex DNA and provide a strategy for site-directed modification of genomic DNA. Recently we demonstrated TFO-mediated targeted gene knockout following systemic administration in animals. However, a limitation to this approach is the requirement for a polypurine tract (typically 15-30 base pairs (bp)) in the target DNA to afford high affinity third strand binding, thus restricting the number of sites available for effective targeting. To overcome this limitation, we have investigated the ability of chemically modified TFOs to target a short (10 bp) site in a chromosomal locus in mouse cells and induce site-specific mutations. We report that replacement of the phosphodiester backbone with cationic phosphoramidate linkages, either N,N-diethylethylenediamine or N,N-dimethylaminopropylamine, in a 10-nucleotide, psoralen-conjugated TFO confers substantial increases in binding affinity in vitro and is required to achieve targeted modification of a chromosomal reporter gene in mammalian cells. The triplex-directed, site-specific induction of mutagenesis in the chromosomal target was charge- and modification-dependent, with the activity of N,N-diethylethylenediamine > N,N-dimethylaminopropylamine phosphodiester, resulting in 10-, 6-, and <2-fold induction of target gene mutagenesis, respectively. Similarly, N,N-diethylethylenediamine and N,N-dimethylaminopropylamine TFOs were found to enhance targeting at a 16-bp G:C bp-rich target site in a chromatinized episomal target in monkey COS cells, although this longer site was also targetable by a phosphodiester TFO. These results indicate that replacement of phosphodiester bonds with positively charged N,N-diethylethylenediamine linkages enhances intracellular activity and allows targeting of relatively short polypurine sites, thereby substantially expanding the number of potential triplex target sites in the genome.
Highlights
§ Supported in part by a fellowship from the Anna Fuller Fellowship fund and National Research Service Award CA75723 from the National Institutes of Health
The binding specificity afforded by Triplex-forming oligonucleotides (TFOs) is obtained from the Hoogsteen or reverse Hoogsteen hydrogen bonds formed in the major groove between the TFO and the purine-rich strand of the underlying target duplex [33]
We have compared the effects of backbone composition and charge on triplex formation in vitro under physiologic conditions, and we have examined the ability of the modified TFOs to target mutations within a chromosomal supF reporter gene at a 10-bp homopurine site and within an episomal reporter at a 16-bp site
Summary
Genomic DNA was isolated from the cells and incubated with in vitro packaging extracts for shuttle vector rescue as described [30]. Episome Mutagenesis Protocol—An SV40-based shuttle vector plasmid pSupF10, similar to pSupFG1 [17], was used as a target. It was obtained from Dr Michael Seidman (National Institute on Aging, Baltimore, MD). This vector contains a modified supF gene, supF10, containing a 16-bp G:C-rich target site at the 3Ј end of the gene. The rescued vector DNA was used to transform indicator bacteria to ampicillin resistance, and supF10 gene function was determined based on colony color, as described [17]
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.