Efforts have been made to reduce the disadvantages associated with the natural oligonucleotides (all-PO) for antisense application by introducing phosphorothioate (PS) linkages into the molecule. A series of such oligodeoxynucleotide copolymers (17-mers) complementary to the coding region of the rabbit beta-globin mRNA, and containing different proportions and arrangements of PO and PS bonds, were synthesized and tested for their protein-binding properties, nuclease stability in vitro, hybridizing ability with the complementary DNA (cDNA), ability to form RNase H-sensitive substrates and antisense activity in cell-free systems. The melting temperatures (Tm) of the co-polymers were reduced by up to 6 degrees C relative to the all-PO oligo, compared to 11 degrees C for the all-PS compound, indicating intermediate hybridizing abilities of the co-polymers. The protein-binding studies with human serum albumin exhibited a linear correlation with the percentage of PS linkage present in the molecule. Nuclease susceptibilities of the co-polymers were also improved, but the number and position of the PS linkages played a significant role in such improvement. Translation inhibition by these oligonucleotides was only found in wheat germ agglutinin (WGA) extract, but not in rabbit reticulocyte lysate (RRL) cell-free system, suggesting the involvement of RNase H in their antisense activities. Provided they have > or = 50% PS linkages, the co-polymers produced almost the same increased inhibition in the WGA system as that of the all-PS oligo. The translation arrest in WGA extract is in good agreement with the in vitro cleavage found for rabbit globin mRNA in the oligo:mRNA duplex by RNase H alone. It is concluded that a copolymer of PO and PS might be preferable to either all-PO or all-PS for antisense applications.