30 research outputs found
2'-Deoxy-3-isoadenosine Forms Hoogsteen-Type Base Pairs with Thymidine in the d(CG[iA]TCG)_2 Duplex^1
Present-day genetic material, DNA and RNA, consists of five major nucleic acid bases (A, G, T, C, and U) and the sugar-phosphate backbone. In addition to the question of their
selection from prebiotic matter, there are more subtle issues, including, in the case of purines, the site of attachment of ribose/deoxyribose at N9 versus N3. The reaction of adenine with protected sugar halides gives a mixture containing more or less equal amounts of N9 and N3 derivatives
Therapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor model
MicroRNAs (miRNAs) are increasingly implicated in the regulation of metastasis. Despite their potential as targets for anti-metastatic therapy, miRNAs have only been silenced in normal tissues of rodents and nonhuman primates. Therefore, the development of effective approaches for sequence-specific inhibition of miRNAs in tumors remains a scientific and clinical challenge. Here we show that systemic treatment of tumor-bearing mice with miR-10b antagomirsa class of chemically modified anti-miRNA oligonucleotidesuppresses breast cancer metastasis. Both in vitro and in vivo, silencing of miR-10b with antagomirs significantly decreases miR-10b levels and increases the levels of a functionally important miR-10b target, Hoxd10. Administration of miR-10b antagomirs to mice bearing highly metastatic cells does not reduce primary mammary tumor growth but markedly suppresses formation of lung metastases in a sequence-specific manner. The miR-10b antagomir, which is well tolerated by normal animals, appears to be a promising candidate for the development of new anti-metastasis agents
Potent inhibition of microRNA in vivo without degradation
Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2ā²-fluoro/2ā²-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies
Competition for RISC binding predicts in vitro potency of siRNA
Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2ā²-O-methyl and 2ā²-deoxy-2ā²-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity
Solution-state structure of a fully alternately 2ā²-F/2ā²-OMe modified 42-nt dimeric siRNA construct
A high-resolution solution structure of a stable 42-nt RNA dimeric construct has been derived based on a high number of NMR observables including nuclear overhauser effects (NOEs), J-coupling constants and residual dipolar couplings (RDCs), which were all obtained with isotopically unlabeled molecules. Two 21-nt siRNA that efficiently hybridize consist of ribose units that were alternately substituted by 2ā²-fluoro or 2ā²-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2ā²-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2ā²-F/2ā²-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 WatsonāCrick base pairs with additional 3ā² UU overhangs and a 5ā² phosphate group on the antisense strand. NMR data suggest that the stability of individual base pairs is not uniform throughout the construct. While most of the double helical segment exhibits well dispersed imino resonances, the last three base pairs either display uncharacteristic chemical shifts of imino protons or absence of imino resonances even at lower temperatures. Accessibility of imino protons to solvent exchange suggests a difference in stability of duplex ends, which might be of importance for incorporation of the guide siRNA strand into a RISC
Deoxyadenosine and Thymidine Bases Held Proximal and Distal by Means of a Covalently-Linked Dimensional Analogue of dAĀ·dT: Intramolecular vs Intermolecular Hydrogen Bonding 1
Structure Activity Relationships of Ī±-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals
We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with Ī±-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. Ī±-L-LNA represents the Ī±-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with Ī±-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the Ī±-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing Ī±-L-LNA nucleotides in the 3ā²- and 5ā²-flanks with R-5ā²-Me-Ī±-L-LNA but not R-6ā²-Me- or 3ā²-Me-Ī±-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5ā²-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5ā²-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs