Cationic phosphoramidate  -oligonucleotides efficiently target single-stranded DNA and RNA and inhibit hepatitis C virus IRES-mediated translation

Abstract

A potential means to improve the efficacy of steric-blocking antisense oligonucleotides (ON) is to increase their affinity for a target RNA. The grafting of cationic amino groups to the backbone of the ON is one way to achieve this, as it reduces the electrostatic repulsion between the ON and its target. We have examined the duplex stabilising effects of introducing cationic phosphoramidate internucleoside linkages into ON with a non-natural alpha-anomeric configuration. Cationic alpha-ON bound with high affinity to single-stranded DNA and RNA targets. Duplex stabilisation was proportional to the number of cationic modifications, with fully cationic ON having particularly high thermal stability. The average stabilisation was greatly increased at low ionic strength. The duplex formed between cationic alpha-ON and their RNA targets were not substrates for RNase H. The penalty in T(m) inflicted by a single mismatch, however, was high; suggesting that they are well suited as sequence-specific, steric-blocking, antisense agents. Using a well-described target sequence in the internal ribosome entry site of the human hepatitis C virus, we have confirmed this potential in a cell-free translation assay as well as in a whole cell assay. Interestingly, no vectorisation was necessary for the cationic alpha-ON in cell culture.

Impact of the Guanidinium Group on Hybridization and Cellular Uptake of Cationic Oligonucleotides

“…[12,41] Here, we have demonstrated that guanidinium groups as cationic tethers significantly increase the affinity of phosphoramidate a-ONs toward nucleic acid targets, in particular toward RNA. This high affinity could be explained in terms of the dual recognition resulting from Watson-Crick or Hoogsteen base-pairing, combined with cationic/anionic backbone recognition between strands, involving H-bond formation and salt bridging.…”

“…The thermal stabilities of these duplexes were compared to those of the natural PO b-ON or of another previously studied cationic PNHDMAP a-ON 3 (Figure 1). [12] All tested cationic a-ONs, whatever the phosphoramidate backbone modification, formed much more stable duplexes than those formed with the natural PO b-ON with DNA III or RNA IV. The introduction of PNHBuNH 2 or PNHBuGua linkages into a-ONs 5 or 8 greatly increased the thermal stabilities of the DNA duplexes (DT m = + 29 8C).…”

“…The average stabilization of 2.2 8C per PNHBuGua modification, was the highest among all the phosphoramidate modifications tested on a-ONs. [12,40] Thus, with RNA targets, the duplex stability was significantly affected by the type of phosphoramidate linkages (neutral PNHME [40] or cationic PNHDMAP, [12] PNHBuNH 2 , PNHBuGua). Both the pendant tether (methoxyethyl, dimethylammonium, ammonium or guanidinium) connected to the phosphoramidate function through an alkyl chain and the length of the www.chembiochem.org alkyl chain (C3 or C4) seem to play important roles in the stabilization of duplexes with RNA.…”

“…Along the same lines, it was anticipated that ON analogues carrying a net positive charge should even be more beneficial in terms of binding efficiency and should also give hybridization with increased association rates. A few examples of cationic ONs (with positively charged backbones) [8][9][10][11][12] or zwitterionic ONs (with positively charged tethers attached to the nucleobases or to the 2'-position of the sugars) [13][14][15][16][17][18][19][20][21][22] have been described. Strategies have included the incorporation of amino groups prone to protonation under physiological conditions or of the guanidinium group (pK a 12.5), which is highly basic and positively charged over a wide pH range.…”

Inhibition of HIV-1 Replication by Oligonucleotide Analogues Directed to the Packaging Signal and Trans-Activating Response Region

“…ONs may act by inducing Ribonuclease H cleavage of a DNA-RNA hybrid, or by steric blocking. Steric-block ONs do not cause degradation of the target RNA, but bind to the target RNA at a high affinity, resulting in a gene expression block through mechanisms such as inhibiting elongation of translation (Michel et al, 2003), altering splicing pathways (Mercatante et al, 2002), and inhibiting RNA-protein interactions (Arzumanov et al, 2001b;Boulme et al, 1998;Darfeuille et al, 2002b;Darfeuille et al, 2004;Kaushik et al, 2002). These have been used to investigate gene function and have been explored as therapeutics against a variety of diseases, such as cytomegalovirus (CMV) retinitis (Leeds et al, 1997) and a range of cancers (Elayadi et al, 2002;Mercatante et al, 2002;Zhang et al, 2003).…”