Binding of phosphorothioate oligodeoxynucleotides to basic fibroblast growth factor, recombinant soluble CD4, laminin and fibronectin is P-chirality independent

Abstract

Antisense oligodeoxynucleotides can selectively inhibit the expression of individual genes and thus have potential applications in anticancer and antiviral therapy. A critical prerequisite to their use as therapeutic agents is the understanding of their non-specific interactions with biological structures, e.g. proteins. In this study we examined the interactions of P-chiral phosphorothioate oligodeoxynucleotides with several proteins. The Rp- and Sp- diastereomers, and racemic machine-made mixtures, or M-oligodeoxynucleotides were used independently as competitors of the binding of a probe, phosphodiester oligodeoxynucleotide bearing a 5' alkylating moiety, to rsCD4, bFGF and laminin. These oligodeoxynucleotides were also used as competitors of the binding of a non-alkylating probe M-phosphorothioate oligodeoxynucleotide, 5'-32P-SdT18 to fibronectin. The average values of and quantitative estimates for the IC50 of competition and the constant of competition (Kc) of Rp-, Sp- and M-stereoisomers of several homo- and heteropolymer oligodeoxynucleotides were determined and compared. Surprisingly, in the proteins we studied, the values of IC50 and Kc for the Rp-, Sp- and M-oligodeoxynucleotides were essentially identical. Thus, the ability of the phosphorothioate oligodeoxynucleotides we employed, to bind to the proteins studied in this work, is virtually independent of P-chirality. Our results also imply that the role of the purine and pyrimidine bases in oligodeoxynucleotide-protein interactions, as well as the nature of the contact points (sulfur versus oxygen) between the oligomer and the protein, may be relatively unimportant.

Phosphorothioate oligonucleotides, suramin and heparin inhibit DNA-dependent protein kinase activity

“…S-oligos act as heparin mimetics and they bind to heparin binding proteins such as bFGF (Benimetskaya et al, 1995). Furthermore, S-oligos inhibit HIV-1 DNA integration (Matsukura et al, 1987;Agrawal et al, 1988).…”

“…Marshall et al (1992) reported that the inhibitory effect of oligonucleotides on HIV-1 reverse transcriptase could be at least 30-fold greater with phosphorodithioate oligonucleotides, which have two sulphur at each site of internucleotide linkages, than with phosphorothioate oligonucleotides. Benimetskaya et al (1995) reported that binding of phosphorothioate oligonucleotides to proteins is independent on P-chirality at the internucleotide linkage sites.…”

“…Phosphorothioate oligonucleotides contain a sulphur atom at each phosphorus atom. Each n-mer phosphorothioate oligonucleotide has (n-1)-centres of asymmetry at phosphorous because each linkage can occur as either the Rp-of Sp-diastereomer (Benimetskaya et al, 1995). Phosphorothioate oligonucleotides used in this experiment contained a random mixture of diastereomers that would have a variety of three-dimensional structure.…”

“…Phosphorothioate oligonucleotides used in this experiment contained a random mixture of diastereomers that would have a variety of three-dimensional structure. Benimetskaya et al (1995) reported that binding of phosphorothioate oligonucleotides to basic fibroblast growth factor, recombinant soluble CD4, laminin and fibronectin is P-chirality independent. It is unknown what effect the three-dimensional structure may have on the interactions of phosphorothioate oligonucleotides with DNA-PK.…”

“…Thus both S-oligo and suramin act as heparin mimetics and they inhibit HIV-1 integration into host DNA Benimetskaya et al, 1995). In the present study, we investigated the effects of S-oligo, suramin and heparin on activity of DNAdependent protein kinase (DNA-PK) because DNA-PK is thought to be a heparin binding protein (Lees-Miller and Anderson, 1991) and because it is involved in the retroviral integration into host DNA (Daniel et al, 1999).…”