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 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

Solution-state structure of a fully alternately 2′-F/2′-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 2′-fluoro or 2′-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2′-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2′-F/2′-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 Watson–Crick base pairs with additional 3′ UU overhangs and a 5′ 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

A high-capacity RNA affinity column for the purification of human IRP1 and IRP2 overexpressed in Pichia pastoris

Regulated expression of proteins involved in mammalian iron metabolism is achieved in part through the interaction of the iron regulatory proteins IRP1 and IRP2 with highly conserved RNA stem-loop structures, known as iron-responsive elements (IREs), that are located within the 5′ or 3′ untranslated regions of regulated transcripts. As part of an effort to determine the structures of the IRP–IRE complexes using crystallographic methods, we have developed an efficient process for obtaining functionally pure IRP1 and IRP2 that relies upon the improved overexpression (>10 mg of soluble IRP per liter of culture) of each human IRP in the yeast Pichia pastoris and large-scale purification using RNA affinity chromatography. Despite the utility of RNA affinity chromatography in the isolation of RNA-binding proteins, current methods for preparing RNA affinity matrices produce columns of low capacity and limited stability. To address these limitations, we have devised a simple method for preparing stable, reusable, high-capacity RNA affinity columns. This method utilizes a bifunctional linker to covalently join a 5′-amino tethered RNA with a thiol-modified Sepharose, and can be used to load 150 nmole or more of RNA per milliliter of solid support. We demonstrate here the use of an IRE affinity column in the large-scale purification of IRP1 and IRP2, and suggest that the convenience of this approach will prove attractive in the analysis of other RNA-binding proteins

Structure Activity Relationships of α-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 α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-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 α-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 α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3′- and 5′-flanks with R-5′-Me-α-L-LNA but not R-6′-Me- or 3′-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5′-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 5′-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

An IRP-like protein from Plasmodium falciparum binds to a mammalian iron-responsive element

This study cloned and sequenced the complementary DNA (cDNA) encoding of a putative malarial iron responsive element-binding protein (PflRPa) and confirmed its identity to the previously identified iron-regulatory protein (IRP)-like cDNA from Plasmodium falciparum. Sequence alignment showed that the plasmodial sequence has 47% identity with human IRP1. Hemoglobin-free lysates obtained from erythrocyte-stage P falciparum contain a protein that binds a consensus mammalian iron-responsive element (IRE), indicating that a protein(s) with iron-regulatory activity was present in the lysates. IRE-binding activity was found to be iron regulated in the electrophoretic mobility shift assays. Western blot analysis showed a 2-fold increase in the level of PfIRPa in the desferrioxamine-treated cultures versus control or iron-supplemented cells. Malarial IRP was detected by anti-PfIRPa antibody in the IRE-protein complex from P falciparum lysates. Immunofluorescence studies confirmed the presence of PfIRPa in the infected red blood cells. These findings demonstrate that erythrocyte P falciparum contains an iron-regulated IRP that binds a mammalian consensus IRE sequence, raising the possibility that the malaria parasite expresses transcripts that contain IREs and are iron-dependently regulated. © 2001 by The American Society of Hematology

Insights from Crystal Structures into the Opposite Effects on RNA Affinity Caused by the <i>S</i>- and <i>R</i>-6′-Methyl Backbone Modifications of 3′-Fluoro Hexitol Nucleic Acid

Locked nucleic acid (LNA) analogues with 2′,4′-bridged sugars show promise in antisense applications. <i>S</i>-5′-Me-LNA has high RNA affinity, and modified oligonucleotides show weakened immune stimulation in vivo. Conversely, an <i>R</i>-5′-methyl group dramatically lowers RNA affinity. To test the effects of <i>S</i>- and <i>R</i>-6′-methyl groups on 3′-fluoro hexitol nucleic acid (FHNA) stability, we synthesized <i>S</i>- and <i>R</i>-6′-Me-FHNA thymidine and incorporated them into oligo-2′-deoxynucleotides. As with LNA, <i>S</i>-6′-Me is stabilizing whereas <i>R</i>-6′-Me is destabilizing. Crystal structures of 6′<i>-</i>Me-FHNA-modified DNAs explain the divergent consequences for stability and suggest convergent origins of these effects by <i>S</i>- and <i>R</i>-6′-Me (FHNA) [-5′-Me (LNA and RNA)] substituents

Synthesis and antisense properties of fluoro cyclohexenyl nucleic acid (F-CeNA), a nuclease stable mimic of 2'-fluoro RNA

We report the design and synthesis of 2'-fluoro cyclohexenyl nucleic acid (F-CeNA) pyrimidine phosphoramidites and the synthesis and biophysical, structural, and biological evaluation of modified oligonucleotides. The synthesis of the nucleoside phosphoramidites was accomplished in multigram quantities starting from commercially available methyl-d-mannose pyranoside. Installation of the fluorine atom was accomplished using nonafluorobutanesulfonyl fluoride, and the cyclohexenyl ring system was assembled by means of a palladium-catalyzed Ferrier rearrangement. Installation of the nucleobase was carried out under Mitsunobu conditions followed by standard protecting group manipulations to provide the desired pyrimidine phosphoramidites. Biophysical evaluation indicated that F-CeNA shows behavior similar to that of a 2'-modified nucleotide, and duplexes with RNA showed slightly lower duplex thermostability as compared to that of the more rigid 3'-fluoro hexitol nucleic acid (FHNA). However, F-CeNA modified oligonucleotides were significantly more stable against digestion by snake venom phosphodiesterases (SVPD) as compared to unmodified DNA, 2'-fluoro RNA (FRNA), 2'-methoxyethyl RNA (MOE), and FHNA modified oligonucleotides. Examination of crystal structures of a modified DNA heptamer duplex d(GCG)-T*-d(GCG):d(CGCACGC) by X-ray crystallography indicated that the cyclohexenyl ring system exhibits both the (3)H(2) and (2)H(3) conformations, similar to the C3'-endo/C2'-endo conformation equilibrium seen in natural furanose nucleosides. In the (2)H(3) conformation, the equatorial fluorine engages in a relatively close contact with C8 (2.94 Å) of the 3'-adjacent dG nucleotide that may represent a pseudo hydrogen bond. In contrast, the cyclohexenyl ring of F-CeNA was found to exist exclusively in the (3)H(2) (C3'-endo like) conformation in the crystal structure of the modified A-form DNA decamer duplex [d(GCGTA)-T*-d(ACGC)](2.) In an animal experiment, a 16-mer F-CeNA gapmer ASO showed similar RNA affinity but significantly improved activity compared to that of a sequence matched MOE ASO, thus establishing F-CeNA as a useful modification for antisense applications.status: publishe