Structural properties and gene-silencing activity of chemically modified DNA-RNA hybrids with parallel orientation.

We report, herein, a new class of RNAi trigger molecules based on the unconventional parallel hybridization of two oligonucleotide chains. We have prepared and studied several parallel stranded (ps) duplexes, in which the parallel orientation is achieved through incorporation of isoguanine and isocytosine to form reverse Watson-Crick base pairs in ps-DNA:DNA, ps-DNA:RNA, ps-(DNA-2'F-ANA):RNA, and ps-DNA:2'F-RNA duplexes. The formation of these duplexes was confirmed by UV melting experiments, FRET and CD studies. In addition, NMR structural studies were conducted on a ps-DNA:RNA hybrid for the first time. Finally, we provide evidence for the unprecedented finding that ps-DNA:RNA and ps-DNA:2'F-RNA hybrids can engage the RNAi pathway to silence gene expression in vitro.

Dramatic effect of furanose C2' substitution on structure and stability: directing the folding of the human telomeric quadruplex with a single fluorine atom.

Human telomeric DNA quadruplexes can adopt different conformations in solution. We have found that arabinose, 2'F-arabinose, and ribose substitutions stabilize the propeller parallel G-quadruplex form over competing conformers, allowing NMR structural determination of this particularly significant nucleic acid structure. 2'F-arabinose substitution provides the greatest stabilization as a result of electrostatic (F-CH---O4') and pseudo-hydrogen-bond (F---H8) stabilizing interactions. In contrast, 2'F-rG substitution provokes a dramatic destabilization of the quadruplex structure due to unfavorable electrostatic repulsion between the phosphate and the 2'-F.

The solution structure of double helical arabino nucleic acids (ANA and 2'F-ANA): effect of arabinoses in duplex-hairpin interconversion.

We report here the first structure of double helical arabino nucleic acid (ANA), the C2'-stereoisomer of RNA, and the 2'-fluoro-ANA analogue (2'F-ANA). A chimeric dodecamer based on the Dickerson sequence, containing a contiguous central segment of arabino nucleotides, flanked by two 2'-deoxy-2'F-ANA wings was studied. Our data show that this chimeric oligonucleotide can adopt two different structures of comparable thermal stabilities. One structure is a monomeric hairpin in which the stem is formed by base paired 2'F-ANA nucleotides and the loop by unpaired ANA nucleotides. The second structure is a bimolecular duplex, with all the nucleotides (2'F-ANA and ANA) forming Watson-Crick base pairs. The duplex structure is canonical B-form, with all arabinoses adopting a pure C2'-endo conformation. In the ANA:ANA segment, steric interactions involving the 2'-OH substituent provoke slight changes in the glycosidic angles and, therefore, in the ANA:ANA base pair geometry. These distortions are not present in the 2'F-ANA:2'F-ANA regions of the duplex, where the -OH substituent is replaced by a smaller fluorine atom. 2'F-ANA nucleotides adopt the C2'-endo sugar pucker and fit very well into the geometry of B-form duplex, allowing for favourable 2'F···H8 interactions. This interaction shares many features of pseudo-hydrogen bonds previously observed in 2'F-ANA:RNA hybrids and in single 2'F-ANA nucleotides.