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Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.

ABSTRACT: Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.

SUBMITTER: Lloyd BH

PROVIDER: S-EPMC55886 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

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Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.

Lloyd B H BH Giles R V RV Spiller D G DG Grzybowski J J Tidd D M DM Sibson D R DR

Nucleic acids research 20010901 17

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha ...[more]

PMID: 11522838

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Project description:It is widely accepted that most cell types efficiently exclude oligonucleotides in vitro and require specific delivery systems, such as cationic lipids, to enhance uptake and subsequent antisense effects. Oligonucleotides are not readily transfected into leukaemia cell lines using cationic lipid systems and streptolysin O (SLO) is used to effect their delivery. We wished to investigate the optimal oligonucleotide composition for antisense efficacy and specificity following delivery into leukaemia cells using SLO. For this study the well characterised chronic myeloid leukaemia cell line KYO-1 was selected and oligonucleotides (20mers) were targeted to an empirically identified accessible site of c- myc mRNA. The efficiency and specificity of antisense effect was measured 4 and 24 h after SLO-mediated delivery of the oligonucleotides. C5-propyne phosphodiester and phosphorothioate compounds were found to present substantial non-specific effects at 20 microM but were inactive at 0.2 microM. Indeed, no antisense-specific effect was noted at any concentration at either time. All of the other oligonucleotides tested induced some measurable antisense effect, except 7 (chimeric, all-phosphorothioate, 2'-methoxyethoxy termini) which was essentially inactive at 20 microM. The rank efficiency order of the remaining antisense compounds was 4 = 3 >> 9 >> 10 = 8 = 5 = 6 > 11. The efficient antisense effects induced by the chimeric methylphosphonate-phosphodiester compounds were found to be highly specific. Increased phosphorothioate content in the oligonucleotide backbone correlated with reduced antisense activity (efficacy: 2'-methoxyethoxy series 9 >> 8 >> 7, 2'-methoxytriethoxy series 10 > 11). No consistent evidence was obtained for increased activity correlating with increased oligonucleotide-mRNA heteroduplex thermal stability. In conclusion, the chimeric methylphosphonate-phosphodiester oligodeoxynucleotides present the most favourable characteristics of the compounds tested, for efficient and specific antisense suppression of gene expression following SLO-mediated delivery.

Dataset Information

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.

Publications

Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.

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