Higher Efficiency of Thymine–Adenine Clamp-Modified Single-Stranded Oligonucleotides in Targeted Nucleotide Sequence Correction Is Not Correlated with Lower Intracellular Degradation

Abstract

Specific single-stranded oligonucleotides can induce targeted nucleotide sequence correction in eukaryotic genes in vitro and in vivo. Our model for investigating the reasons for the low correction rates achieved by this method is the correction of a point mutation in the hypoxanthine-guanine phosphoribosyltransferase gene (hprt) in the cell line V79-151. Using single-stranded phosphorothioate-modified oligonucleotides, the correction rates of this hprt mutation were low but always reproducible. One reason for low exchange rates may be fast intracellular degradation of the oligonucleotides. Therefore we compared the exchange rates of different 3' and 5' end-modified oligonucleotides with their degradation rates. Thymine-adenine (TA) repeat (clamp)-modified oligonucleotides showed higher correction rates than those with a guanine-cytosine (GC) clamp and 5' clamps induced higher correction rates than clamps at the 3' end. Experiments on the stability of the most effective 5'-TA and 3'-TA clamp-modified oligonucleotide indicated rapid cleavage and the occurrence of shortened oligonucleotides in the presence of cytoplasmic and nuclear extracts. The phosphorothioate-modified oligonucleotides were more stable, but their correction rates were lower. We suggest that there is no direct correlation between the biological stability of the full-length oligonucleotides and the exchange rates achieved.