2'-O-methyl-modified phosphorothioate antisense oligonucleotides have reduced non-specific effects in vitro

Abstract

Antisense oligodeoxynucleotides (ODNs) have biological activity in treating various forms of cancer. The antisense effects of two types of 20mer ODNs, phosphorothioate-modified ODNs (S-ODNs) and S-ODNs with 12 2'-O-methyl groups (Me-S-ODNs), targeted to sites 109 and 277 of bcl-2 mRNA, were compared. Both types were at least as effective as G3139 (Genta, Inc.) in reducing the level of Bcl-2 protein in T24 cells following a 4 h transfection at a dose of 0.1 micro M. Circular dichroism spectra showed that both types formed A-form duplexes with the complementary RNA, and the melting temperatures were in the order of Me-S-ODN.RNA > normal DNA.RNA > S-ODN.RNA. In comparison with the S-ODN, the Me-S-ODN had reduced toxic growth inhibitory effects, was less prone to bind the DNA-binding domain A of human replication protein A, and was as resistant to serum nucleases. Neither type of oligomer induced apoptosis, according to a PARP-cleavage assay. Hybrids formed with Me-S-ODN sequences were less sensitive to RNase H degradation than those formed with S-ODN sequences. Despite this latter disadvantage, the addition of 2'-O-methyl groups to a phosphorothioate-modified ODN is advantageous because of increased stability of binding and reduced non-specific effects.

Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity

“…The antisense oligonucleotides against pre-miR-886 were from Integrated DNA Technologies. They were 20-nt mixed oligonucleotides containing a backbone phosphorothioate and having 5 nt on each end substituted with 29-Omethyl ribonucleotides (Yoo et al 2004;Ideue et al 2009); the sequences are in Figure 5A. Synthetic pre-miR-886 and pre-miR-23a were made by in vitro transcription using MEGAscript high yield transcription kit (Applied Biosystems/Ambion).…”

“…To investigate pre-miR-886's biological roles, we observed cell proliferation upon its depletion by modified antisense oligonucleotides (anti-oligos) (Yoo et al 2004;Ideue et al 2009). We designed three anti-oligos (anti886 62_43, anti886 45_26, and anti886 75_56) in the middle portion of pre-miR-886 (Fig.…”

Impact of Targeting the Adenine- and Uracil-Rich Element ofbcl-2mRNA with Oligoribonucleotides on Apoptosis, Cell Cycle, and Neuronal Differentiation in SHSY-5Y Cells

“…The sequences of the asORNs are the following: asORN1 (27-mer), 5Ј-UGUCUU-AAAUAAAUAAAUCUUUUUUUC-3Ј; asORN2 (26-mer), 5Ј-UUAAUA-AUGUAAAAAAUAAAUGAUAU-3Ј; asORN3 (26-mer), 5Ј-UUC-CCUUUGGCAGUAAAUAGCUGAUU-3Ј; and degORN (26-mer), 5Ј-NNNNNNNNNNNNNNNNNNNNNNNNNN-3Ј, where N is any nucleotide. ORNs were 2Ј-O-methyl-modified, which increased their stability with respect to the natural RNA derivatives (Yoo et al, 2004). ORN cellular uptake and stability were increased by their vehiculation with the cationic lipid DOTAP (Roche, Mannheim, Germany), used as lipofection reagent, as reported previously (Luzi et al, 2003;Ghisolfi et al, 2005).…”

MicroRNAs in glioblastoma multiforme pathogenesis and therapeutics

“…These were followed by 2′‐ O ‐methyl‐modified oligonucleotides, which displayed increased binding stability and reduced nonspecific effects, but suboptimal efficiency 115. 2′‐ O ‐methyl‐modified oligonucleotides were further improved on by the development of locked nucleic acid (LNA)‐modified oligonucleotides, which have higher stability, efficacy, and specificity as well as lower toxicity 116, 117.…”