Sequence Dependency of the Internalization and Distribution of Phosphorothioate Oligonucleotides in Vascular Smooth Muscle Cells

Abstract

Antisense studies imply the utilization of oligonucleotides (ODN) for sequence-specific down-regulation of genes. This usually consists in assessing antisense sequences versus control sequences (mismatched, inverted, scrambled, randomized or any sequence unrelated to the relevant target). Even though the investigated biological effect (knockdown of an unwanted protein) is observed only with the antisense sequence and weakly, if at all, with any of the control sequences, this is a necessary but not a sufficient condition to demonstrate an antisense effect. Indeed, biochemical parameters such as stability, uptake and subcellular compartmentalization of ODN in a given cellular system are most often sequence-dependent processes. In this work, a series of phosphorothioate ODN of different lengths and sequences were evaluated as to their binding, internalization and subcellular distribution properties in vascular smooth muscle cells. In addition to membrane binding and nuclear accumulation, the partition of ODN in the cytosol of cells was measured by a method based upon controled permeabilization of the plasma membrane, permitting the recovery of the cytosolic content with minimal damage to the membranes of the endocytic vesicles and lysosomes. We found that the tested ODN showed striking differences in their uptake and distribution in smooth muscle cells. Our results gave rise to the problem of validating the observed biological effects when different sequences of ODN were compared. Cellular studies such as the one presented in this work could help in choosing the proper control sequences among ODN exhibiting similar cell interactions as compared to the antisense sequences. Moreover, this method could be useful for the selection of antisense sequences that can be efficiently internalized and preferentially distributed in the appropriate compartments in cells for in vitro antisense studies.