Cellular Uptake and Degradation of Phosphorothioate Oligonucleotides by Marine and Freshwater Ciliates

Abstract

To date, no antisense studies have been reported with either ciliated protozoa or marine organisms. This study examines the feasibility of using antisene oligonucleotides to alter gene expression in marine and freshwater ciliates. Radiolabeled, phosphorothioate-modified oligonucleotides were used to investigate whether ciliates take up and degrade oligonucleotides present in the culture medium. With all three ciliates examined, Euplotes crassus, Tetrahymena thermophila, and Oxytricha nova, the oligonucleotide in the culture medium was degraded very rapidly (>90% in 8 h). The degradation probably occurred when the cells filtered the culture medium through the oral apparatus. Our results indicate that experiments involving the uptake of oligonucleotides from the culture medium are likely to be successful with ciliated protozoa. In studies designed to examine the uptake of fluorescent oligonucleotide by Euplotes cells, we found that dead or dying cells have a high affinity for fluorescein-labeled oligonucleotide. These results illustrate the importance of careful studies when only certain cell populations are found to have a high affinity for oligonucleotide. Although the seawater culture medium used to grow Euplotes causes some oligonucleotide to precipitate, this problem is not serious at concentrations ≥1 μM oligonucleotide. Thus, it should be possible to use antisense oligonucleotides to manipulate gene expression in other marine organisms.