<p>Supplementary Tables 1 and 2. Supplementary Table 1: Fold de-repression of miR-21 target genes after anti-miR-21 treatment. Supplementary Table 2: Taqman Primer and Probes</p>
<p>Supplementary Figure 3. HMBG1 and LDH are induced upon miR-21 inhibition. SKHep1, HepG2 and Hep3B cells were treated with anti-miR-21 or MM control and extracellular HMBG1 and LDH activity was quantified. (Mean, {plus minus} SEM, n=3)</p>
<p>Supplementary Figure 1. miR-21 mimic suppresses expression of ANKRD46, DDAH1 and RECK. SKHep1 cells were transfected with miR-21 or negative mimic. RNA was isolated and expression of ANKRD46, DDAH1 and RECK was assessed by qPCR. (Mean, {plus minus} SD, n=3).</p>
The association of proteins with the branch site region during pre-mRNA splicing was probed using a novel methodology to site-specifically modify the pre-mRNA with the photo-reagent benzophenone. Three sets of proteins were distinguished by the kinetics of their associations with pre-mRNAs, by their association with discrete splicing complexes, and by their differing factor requirements. An early U1 snRNP-dependent cross-link of the branch region to a p80 species was followed by cross-links to p14, p35, and p150 polypeptides associated with the U2 snRNP-pre-mRNA complex. Concomitant with formation of the spliceosome, a rearrangement of protein factors about the branch region occurred, in which the p35 and p150 cross-links were replaced by p220 and p70 species. These results establish that the branch region is recognized in a dynamic fashion by multiple distinct proteins during the course of spliceosomal assembly.
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 2'-O-methyl and 2'-deoxy-2'-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.
RNA-based therapeutics hold significant potential as promising treatment options for human disease. In the past 20 years, advances in the RNA field have identified several novel RNAbased therapies that are currently under clinical investigation, including antisense oligonucleotides, small interfering RNA (siRNA), and microRNA. By targeting RNA and modulating human biology at the molecular level, these new technologies have allowed drug-discovery efforts to focus on a broad range of disease targets once deemed to be “undruggable.” Leading RNA biotechnology companies have since expanded the target space and generated multiple clinical candidates characterized by improved target specificity, improved drug safety, and demonstrated efficacy in patients. These companies have traditionally focused on targeting specific genes relevant to the disease indication through the control of protein synthesis at the RNA level. More recently, drug discovery researchers are attempting to regulate entire networks of genes through the modulation of a single microRNA. Targeting microRNAs with either oligonucleotide inhibitors, namely anti-miRs, or miR-mimics (doublestranded oligonucleotides that replace microRNA function), provides a novel class of therapeutics and a unique approach to treating disease by modulating entire biological pathways (see Figure 1).
The synthesis, biophysical, structural, and biological properties of both isomers of 3′-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3′-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C–F···H–C pseudo hydrogen bond between F3′ of Ara-FHNA and C8–H of the nucleobase from the 3′-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H–C(Pu) pseudo hydrogen bonds in nucleic acid structures.
