Pseudo-cyclic oligonucleotides: In vitro and in vivo properties

We have designed and studied antisense oligodeoxynucleotides (oligonucleoti des; oligos) which we call 'pseudo-cyclic oligonucleotides' (PCOs). PCOs co ntain two oligonucleotide segments attached through their 3'-3'- or 5'-5'-e nds. One of the segments of the PCO is an antisense oligo complementary to a target mRNA, and the other is a short protective oligo that is 5-8 nucleo tides long and complementary to the 3'- or 5'-end of the antisense oligo. A s a result of complementarity between the antisense and protective oligo se gments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target RNA. The antisense oligo segment of PCOs used for the studies d escribed here is complementary to an 18-nucleotide-long site on the mRNA of the protein kinase A regulatory subunit RI alpha (PKA-RI alpha). Thermal m elting studies of PCOs in the absence and presence of the complementary RNA suggest that the pseudo-cyclic structures formed in the absence of the tar get RNA dissociate, bind to the target RNA, and form heteroduplexes. The re sults of RNase H cleavage assays suggest that PCOs bind to complementary RN A and activate RNase H in a manner similar to that of an 18-mer conventiona l antisense PS-oligo. In snake venom (a 3'-exonuclease) or spleen (a 5'-exo nuclease) phosphodiesterase digestion studies, PCOs are more stable than co nventional antisense oligos because of the presence of 3'-3'- or 5'-5'-link ages and the formation of intramolecular pseudo-cyclic structures. PCOs wit h a phosphorothioate antisense oligo segment inhibited cell growth of MDA-M B-468 and GEO cancer cell lines similar to that of the conventional antisen se PS-oligo, suggesting efficient cellular uptake and target binding. The n uclease stability studies in mice suggest that PCOs have higher in vivo sta bility than antisense PS-oligos. The studies in mice showed similar pharmac okinetic and tissue distribution profiles for PCOs to those of antisense PS -oligos in general, but rapid elimination from selected tissues. (C) 1999 E lsevier Science Ltd. All rights reserved.