Effect of 2'-O-methyl/thiophosphonoacetate-modified antisense oligonucleotides on huntingtin expression in patient-derived cells.

Optimizing oligonucleotides as therapeutics will require exploring how chemistry can be used to enhance their effects inside cells. To achieve this goal it will be necessary to fully explore chemical space around the native DNA/RNA framework to define the potential of diverse chemical modifications. In this report we examine the potential of thiophosphonoacetate (thioPACE)-modified 2'-O-methyl oligoribonucleotides as inhibitors of human huntingtin (HTT) expression. Inhibition occurred, but was less than with analogous locked nucleic acid (LNA)-substituted oligomers lacking the thioPACE modification. These data suggest that thioPACE oligonucleotides have the potential to control gene expression inside cells. However, advantages relative to other modifications were not demonstrated. Additional modifications are likely to be necessary to fully explore any potential advantages of thioPACE substitutions.

Synthesis and biological activity of phosphonoacetate- and thiophosphonoacetate-modified 2'-O-methyl oligoribonucleotides.

Chimeric 2'-O-methyl oligoribonucleotides (2'-OMe ORNs) containing internucleotide linkages which were modified with phosphonoacetate (PACE) or thiophosphonoacetate (thioPACE) were prepared by solid-phase synthesis. The modified 2'-OMe ORNs contained a central phosphate or phosphorothioate sequence with up to 4 PACE or thioPACE modifications, respectively, at either end of the ORN in a "gapmer" motif. Both PACE and thioPACE 2'-OMe ORNs formed stable duplexes with complementary RNA. The majority of these duplexes had higher thermal melting temperatures than an unmodified RNA:RNA duplex. The modified 2'-OMe ORNs were effective passenger strands with complementary, unmodified siRNAs, for inducing siRNA activity in a dual luciferase assay in the presence of a lipid transfecting agent. As single strands, thioPACE 2'-OMe ORNs were efficiently taken up by HeLa cells in the absence of a lipid transfecting agent. Furthermore, thioPACE modifications greatly improved the potency of a 2'-OMe phosphorothioate ORN as an inhibitor of microRNA-122 in Huh7 cells, without lipid transfection.

Potent and sustained cellular inhibition of miR-122 by lysine-derivatized peptide nucleic acids (PNA) and phosphorothioate locked nucleic acid (LNA)/2'-O-methyl (OMe) mixmer anti-miRs in the absence of transfection agents.

Efficient cell delivery of antisense oligonucleotides (ONs) is a key issue for their potential therapeutic use. It has been shown recently that some ONs can be delivered into cells without the use of transfection agents (gymnosis), but this generally requires cell incubation over several days and high amounts of ONs (micromolar concentrations). Here we have targeted microRNA 122 (miR-122), a small non-coding RNA involved in regulation of lipid metabolism and in the replication of hepatitis C virus, with ONs of different chemistries (anti-miRs) by gymnotic delivery in cell culture. Using a sensitive dual-luciferase reporter assay, anti-miRs were screened for their ability to enter liver cells gymnotically and inhibit miR-122 activity. Efficient miR-122 inhibition was obtained with cationic PNAs and 2'-O-methyl (OMe) and Locked Nucleic Acids (LNA)/OMe mixmers containing either phosphodiester (PO) or phosphorothioate (PS) linkages at sub-micromolar concentrations when incubated with cells for just 4 hours. Furthermore, PNA and PS-containing anti-miRs were able to sustain miR-122 inhibitory effects for at least 4 days. LNA/OMe PS anti-miRs were the most potent anti-miR chemistry tested in this study, an ON chemistry that has been little exploited so far as anti-miR agents towards therapeutics.

Foldamers derived from nucleoside beta-amino acids: a new twist on the DNA helix.

Peptides derived from a thymidine beta-amino acid have been prepared by solid-phase synthesis and their conformation investigated by NMR. Interestingly, NMR and modelling studies indicate that the tetramer and octamer form an unusual 8-helical conformation. Studies are currently underway to investigate the synthesis of peptides derived from the other deoxyribonucleosides with the intention of examining the association between helices capable of nucleobase-pairing.

Towards fluorous-phase synthesis of nucleoside beta-peptides.

We have recently shown that peptides derived from nucleoside beta-amino acids adopt an unusual 8-helical conformation in solution(1). These beta-peptide homooligomers were constructed using Fmoc solid-phase peptide synthesis protocols (SPPS); however, we found these procedures to be somewhat limiting in terms of scale and lacking a quantitative method of monitoring reactions. Preliminary investigations into the use of fluorous dendrons as an alternative to SPPS have been conducted and show that coupling reactions with modified nucleoside (1) and fluorous dendrimer (2) are possible in the presence of a glycine linker. Significantly, success of the coupling reactions could be monitored by NMR without loss of materials or termination of the synthetic sequence and this promises much for the future of both oligonucleotide and polypeptide synthesis.

Peptides derived from nucleoside beta-amino acids form an unusual 8-helix.

Peptides of varying length (dimers to octamers) were prepared from nucleoside beta-amino acids and conformational studies, based on NOE observations, show that the beta-peptides form an unusual 8-helix.

Foldamers derived from nucleoside beta-amino acids: PNA Or DNA? Can we have both in one place?

The synthesis of a modified thymidine (nucleoside beta-amino acid) monomer and preliminary investigations into the solid phase peptide synthesis of PNA/DNA chimeras containing a neutral, internucleoside amide linkage are described.