An assessment of the antisense properties of RNase H-competent and steric-blocking oligomers

Abstract

The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target.

Peptide nucleic acid delivery to human mitochondria

“…For example, extracellularly delivered fluorescent-labelled PNA remained within cytoplasmic endosomes, and did not localise within the nucleus. 7 By contrast, nuclear uptake was clearly demonstrated with the radiolabelled PNA molecules 24 and with both biotinylated PNA and biotinylated peptide-PNA in our study.…”

“…Different investigators have studied PNA uptake at lower concentrations and not in the cell types that we have studied. 7 Despite a sensitive semi-quantitative detection method, relatively high concentrations of PNA (Ͼ1 m) were needed to demonstrate uptake in our study. PNA uptake has been shown to occur in transformed and immortalised cell lines, 24 probably by fluid-phase endocytosis.…”

“…1,4 Despite their enormous potential as therapeutic agents, reports of poor cellular uptake, due to their low phospholipid permeability, dampened the initial enthusiasm. [5][6][7][8] However, early studies on PNA import were limited to a small number of different cell lines and import conditions. In addition, it is well recognised that oligodeoxynucleotide uptake can differ markedly between different cell types.…”

Tandem Mass Spectrometry of Modified and Platinated Oligoribonucleotides

“…The 2′-position of the sugar moiety can also serve as a modification site. 2′-methoxy (2′-O-methyl) substituted oligonucleotides were found to form very stable double strands and exhibit significant nuclease resistance, which further facilitates in vivo administration [14][15][16][17][18][19][20][21][22][23].…”

PNA interference mapping demonstrates functional domains in the noncoding RNA Xist

“…PNAs are not readily degraded and do not participate in or activate repair or degradative pathways for DNA or RNA, ensuring a very selective range of activity. In addition to the high thermal stability of complexes, PNA-RNA binding is highly sensitive to mismatches (27)(28)(29)(30). The PNAs used for P-IMP were conjugated to transportan via a cleavable disulfide linkage.…”