SEQUENCE DEPENDENCY OF THE INTERNALIZATION AND DISTRIBUTION OF PHOSPHOROTHIOATE OLIGONUCLEOTIDES IN VASCULAR SMOOTH-MUSCLE CELLS

Antisense studies imply the utilization of oligonucleotides (ODN) for sequence-specific down-regulation of genes. This usually consists in a ssessing antisense sequences versus control sequences (mismatched, inv erted, 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 neces sary but not a sufficient condition to demonstrate an antisense effect . Indeed, biochemical parameters such as stability, uptake and subcell ular compartmentalization of ODN in a given cellular system are most o ften sequence-dependent processes. In this work, a series of phosphoro thioate ODN of different lengths and sequences were evaluated as to th eir binding, internalization and subcellular distribution properties i n vascular smooth muscle cells. In addition to membrane binding and nu clear accumulation, the partition of ODN in the cytosol of cells was m easured by a method based upon controled permeabilization of the plasm a membrane, permitting the recovery of the cytosolic content with mini mal damage to the membranes of the endocytic vesicles and lysosomes. W e found that the tested ODN showed striking differences in their uptak e and distribution in smooth muscle cells. Our results gave rise to th e problem of validating the observed biological effects when different sequences of ODN were compared. Cellular studies such as the one pres ented in this work could help in choosing the proper control sequences among ODN exhibiting similar cell interactions as compared to the ant isense sequences. Moreover, this method could be useful for the select ion of antisense sequences that can be efficiently internalized and pr eferentially distributed in the appropriate compartments in cells for in vitro antisense studies. (C) 1998 Elsevier Science Inc.