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ABSTRACT: Anti-miRs are oligonucleotide inhibitors complementary to miRNAs that have been used extensively as tools to gain understanding of specific miRNA functions and as potential therapeutics. We showed previously that peptide nucleic acid (PNA) anti-miRs containing a few attached Lys residues were potent miRNA inhibitors. Using miR-122 as an example, we report here the PNA sequence and attached amino acid requirements for efficient miRNA targeting and show that anti-miR activity is enhanced substantially by the presence of a terminal-free thiol group, such as a Cys residue, primarily due to better cellular uptake. We show that anti-miR activity of a Cys-containing PNA is achieved by cell uptake through both clathrin-dependent and independent routes. With the aid of two PNA analogues having intrinsic fluorescence, thiazole orange (TO)-PNA and [bis-o-(aminoethoxy)phenyl]pyrrolocytosine (BoPhpC)-PNA, we explored the subcellular localization of PNA anti-miRs and our data suggest that anti-miR targeting of miR-122 may take place in or associated with endosomal compartments. Our findings are valuable for further design of PNAs and other oligonucleotides as potent anti-miR agents.
Nucleic Acids Research 11/2011; 40(5):2152-67. · 8.03 Impact Factor
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ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs.
RNA 03/2011; 17(5):933-43. · 5.09 Impact Factor
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ABSTRACT: Design of site-selective artificial ribonucleases (aRNases) is one of the most challenging tasks in RNA targeting. Here, we designed and studied oligonucleotide-based aRNases containing multiple imidazole residues in the catalytic part and systematically varied structure of cleaving constructs. We demonstrated that the ribonuclease activity of the conjugates is strongly affected by the number of imidazole residues in the catalytic part, the length of a linker between the catalytic imidazole groups of the construct and the oligonucleotide, and the type of anchor group, connecting linker structure and the oligonucleotide. Molecular modeling of the most active aRNases showed that preferable orientation(s) of cleaving constructs strongly depend on the structure of the anchor group and length of the linker. The inclusion of deoxyribothymidine anchor group significantly reduced the probability of cleaving groups to locate near the cleavage site, presumably due to a stacking interaction with the neighbouring nucleotide residue. Altogether the obtained results show that dynamics factors play an important role in site-specific RNA cleavage. Remarkably high cleavage activity was displayed by the conjugates with the most flexible and extended cleaving construct, which presumably provides a better opportunity for imidazole residues to be correctly positioned in the vicinity of scissile phosphodiester bond.
Journal of nucleic acids 01/2011; 2011:748632.
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ABSTRACT: MicroRNAs (miRNAs) play an important role in diverse physiological processes and are potential therapeutic agents. Synthetic oligonucleotides (ONs) of different chemistries have proven successful for blocking miRNA expression. However, their specificity and efficiency have not been fully evaluated. Here, we show that peptide nucleic acids (PNAs) efficiently block a key inducible miRNA expressed in the haematopoietic system, miR-155, in cultured B cells as well as in mice. Remarkably, miR-155 inhibition by PNA in primary B cells was achieved in the absence of any transfection agent. In mice, the high efficiency of the treatment was demonstrated by a strong overlap in global gene expression between B cells isolated from anti-miR-155 PNA-treated and miR-155-deficient mice. Interestingly, PNA also induced additional changes in gene expression. Our analysis provides a useful platform to aid the design of efficient and specific anti-miRNA ONs for in vivo use.
Nucleic Acids Research 03/2010; 38(13):4466-75. · 8.03 Impact Factor
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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)/2'-O-methyl 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 2'-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 R6-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.
RNA 03/2008; 14(2):336-46. · 5.09 Impact Factor
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ABSTRACT: Serum-stabilized PNA-internalization peptides (Pip) conjugated to PNA complementary to the 705 aberrant beta-globin splice site are able to correct splicing and increase luciferase production in Hela pLuc705 cells with sub microM EC(50) in the absence of a transfection agent. Inhibition of microRNA-122 in liver cells is achieved by treatment with complementary PNA containing just a few attached Lys residues, again without need of a transfection agent.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: The recent availability of a wider range of oligonucleotide analogs has stimulated renewed interest in their application as potential antiviral agents through a variety of mechanisms. These mechanisms include RNase H-mediated antisense, steric block antisense and small interfering RNA targeting viral RNAs, but also other mechanisms, including blockage of virus uptake by cells and the stimulation of a Toll-like receptor-9-dependent immune response by CpG oligonucleotides.
Current opinion in molecular therapeutics 05/2006; 8(2):108-14. · 3.68 Impact Factor
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ABSTRACT: Antisense oligonucleotide conjugates, bearing constructs with two imidazole residues, were synthesized using a precursor-based technique employing post-synthetic histamine functionalization of oligonucleotides bearing methoxyoxalamido precursors at the 5'-termini. The conjugates were assessed in terms of their cleavage activities using both biochemical assays and conformational analysis by molecular modelling. The oligonucleotide part of the conjugates was complementary to the T-arm of yeast tRNA(Phe) (44-60 nt) and was expected to deliver imidazole groups near the fragile sequence C61-ACA-G65 of the tRNA. The conjugates showed ribonuclease activity at neutral pH and physiological temperature resulting in complete cleavage of the target RNA, mainly at the C63-A64 phosphodiester bond. For some constructs, cleavage was completed within 1-2 h under optimal conditions. Molecular modelling was used to determine the preferred orientation(s) of the cleaving group(s) in the complexes of the conjugates with RNA target. Cleaving constructs bearing two imidazole residues were found to be conformationally highly flexible, adopting no preferred specific conformation. No interactions other than complementary base pairing between the conjugates and the target were found to be the factors stabilizing the 'active' cleaving conformation(s).
Nucleic Acids Research 02/2004; 32(13):3887-97. · 8.03 Impact Factor
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ABSTRACT: Towards the development of oligonucleotide analogues and siRNA as drugs, one potential alternative to the use of liposomal transfection agents is the covalent conjugation of a cell-penetrating peptide (CPP), with the intention of imparting on the oligonucleotide or siRNA an enhanced ability to enter mammalian cells and reach the appropriate RNA target. We have developed robust methods for the chemical synthesis of disulfide-linked conjugates of oligonucleotide analogues, siRNA and peptide nucleic acids (PNA) with a range of cationic and other CPPs. In a HeLa cell assay with integrated plasmid reporters of Tat-dependent trans-activation at the TAR RNA target in the cell nucleus, we were unable to obtain steric block inhibition of gene expression for conjugates of CPPs with a 12-mer oligonucleotide mixmer of 2'-O-methyl and locked nucleic acids units. By contrast, we were able to obtain some reductions in expression of P38alpha MAP kinase mRNA in HeLa cells using microM concentrations of Penetratin or Tat peptides conjugated to the 3'-end of the sense strand of siRNA. However, the most promising results to date have been with a 16-mer PNA conjugated to the CPP Transportan or a double CPP R(6)-Penetratin, where we have demonstrated Tat-dependent trans-activation inhibition in HeLa cells. Results to date suggest the possibility of development of CPP-PNA conjugates as anti-HIV agents as well as other potential applications involving nuclear cell delivery, such as the redirection of splicing.
Blood Cells Molecules and Diseases 38(1):1-7. · 2.35 Impact Factor
