Solution-state structure of a fully alternately 2′-F/2′-OMe modified 42-nt dimeric siRNA construct

Abstract

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.

Interplay of LNA and 2′-O-Methyl RNA in the Structure and Thermodynamics of RNA Hybrid Systems: A Molecular Dynamics Study Using the Revised AMBER Force Field and Comparison with Experimental Results

“…Furthermore, base-pair-step analyses of slide, twist, stacking area, roll, and tilt do not show major differences between RNA 1H0Q and 2′- O -methyl RNA:RNA hybrid systems (Figures S8 and S9, Supporting Information). Different groups have determined solution and X-ray structures of RNA and DNA systems mixed with 2′- O -methyl RNA residues, and the results indicate that mixing of 2′- O -methyl RNA residues even into DNA systems will make the duplex gain A-form properties in line with our computational results. − …”

MicroRNA Quantitation During Dendritic Cell Endocytosis Using Imaging Flow Cytometry: Key Factors and Requirements

“…miR-590-5p-Cy3 was synthesized directly, but miR-mock-Fam-F was modified with 2-O-fluoro (2'-O-methoxyethyl/2'OME) , which was a common modification to increase RNA stability [32]. After incubation with miR-590-5p-Cy3 and miR-mock-Fam-F for 3 hours in basally treated DCs, the internalization ratio of Cy3 label in DCs increased significantly, but the internalization ratio of FAM label remained stable (Fig.…”

Small Interfering RNAs Containing Dioxane- and Morpholino-Derived Nucleotide Analogues Show Improved Off-Target Profiles and Chirality-Dependent In Vivo Knock-Down

“…Metabolic stability is mainly achieved by a high number of chemically modified nucleotides, of which the 2′-modified fluoro ( 1 ) and methoxy ( 2 ) ribose analogues are the most prominent examples (Figure ). − Additionally, phosphorothioate moieties ( 3 ) in the phosphate backbone are used to increase metabolic stability, although to a much lesser extent as in single-stranded antisense therapeutics. Non-hybridizing 3′-overhangs, as, for example, desoxy thymidine dinucleotides ( 4 ), can be attached to further increase the protection against exo-nucleases. − With such modifications, siRNAs could be optimized for high metabolic stabilities paired with reduced immunoinflammatory potential. ,− A screen of various chemically modified nucleotides and their influence on siRNA potencies, stabilities and toxicities was published in 2009 …”