miR-122 targeting with LNA/2′-O-methyl oligonucleotide mixmers, peptide nucleic acids (PNA), and PNA–peptide conjugates

Abstract

MicroRNAs are small noncoding RNAs that regulate many cellular processes in a post-transcriptional mode. MicroRNA knockdown by antisense oligonucleotides is a useful strategy to explore microRNA functionality and as potential therapeutics. MicroRNA-122 (miR-122) is a liver-specific microRNA, the main function of which has been linked with lipid metabolism and liver homeostasis. Here, we show that lipofection of an antisense oligonucleotide based on a Locked Nucleic Acids (LNA)/29-Omethyl mixmer or electroporation of a Peptide Nucleic Acid (PNA) oligomer is effective at blocking miR-122 activity in human and rat liver cells. These oligonucleotide analogs, evaluated for the first time in microRNA inhibition, are more effective than standard 29-O-methyl oligonucleotides in binding and inhibiting microRNA action. We also show that microRNA inhibition can be achieved without the need for transfection or electroporation of the human or rat cell lines, by conjugation of an antisense PNA to the cell-penetrating peptide R 6 -Penetratin, or merely by linkage to just four Lys residues, highlighting the potential of PNA for future therapeutic applications as well as for studying microRNA function.

miRNAs link metabolic reprogramming to oncogenesis

“…Other important intermediate steps in the glycolysis pathway may be regulated by or regulate microRNAs. Aldolase A is a direct target of miR-122 in liver cells [39]. Phosphoglucose isomerase (PGI) has been associated with invasion and metastasis of cancer cells.…”

Enhanced splice correction by 3′, 5′-serinol and 2′-(ω-O-methylserinol) guarded OMe-RNA/DNA mixmers in cells

“…By using these cells, various AONs have been evaluated by measuring induced luciferase activity as a tool to verify splice switching efficiency and specificity. 28 OMe-RNASb and OMe-RNA4-dTSb were used in this study in comparison with OMe-RNA, and also with an LNA-OMe mixmer, previously used in recent steric blocking applications 17,30 and the detection was of the expression of functional luciferase in the form of RLU/ μg protein expression.…”

“…3,10 In spite of being diastereomeric mixtures at each phosphorus atom, the PS linkages have not yet found replacement because of their favorable pharmacological properties such as increasing half-life and improved binding to serum proteins in vivo, allowing greater availability of AONs to biological targets. 11 Several of these chemistries are being mixed in the recent years to gain maximum advantages in terms of reducing off-target effects, increasing specificity and potency of the AONs in various strategies such as RNase-H dependent antisense, 3,10,12,13 siRNA, 14,15 miRNA 16,17 or splice switching antisense applications. 18 The recent literature also again points out the necessity to protect 3′-5′ ends by enzymatically stable capping of AONs.…”

Antisense, RNAi, and gene silencing strategies for therapy: Mission possible or impossible?

“…It is also possible to use antisense oligonucleotides to knockdown expression of miRNAs, inhibiting their function in vivo. One research group demonstrated that the use of a PNA (peptide nucleic acid) or LNA/2-O-methyl oligonucleotide could decrease expression of miRNA-122 in human and rodent cell lines [90]. These findings suggest that miRNA can be successfully targeted or can be used to regulate gene expression.…”