Synthesis and Duplex-Stabilizing Properties of Fluorinated N

The efficient synthesis, antiviral activity, and duplex-stabilizing properties of both isomers of the 2'-fluoro analogue of Northern methanocarbathymidine (N-MCT), 2 and 3, are reported. We show that 2'-F incorporation on the N-MCT scaffold has a strong stabilizing effect on duplex thermal stability. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Structural studies and gene silencing activities of chemically modified nucleic acids: Effect of C2' sugar modifications and iso-nucleobases on the stability and polarity of duplex and tetraplex structures

The increasing number of promising applications for chemically modified nucleic acid analogues range from new oligonucleotide therapeutics to the design of DNA-based nanodevices, diagnostics, functional genomics and target validation. Among the many modified nucleic acids, and specifically those with modifications in their sugar moiety, arabinonucleic acid (ANA) and its 2'-fluorinated derivative (2'F-ANA) are particularly interesting to our group, and this thesis is primarily focused on understanding the structural basis for the interesting properties imparted by these analogues, with a focus on their structure, stability, and activity in cell based systems. Our work has led to the finding that the high binding affinity of 2'F-ANA for RNA or itself is, in part, due to a hydrogen bonding interaction between the organic fluorine of the 2'F-ANA nucleotides and the H8 of the proximal purine. The same conformational configuration leads to an unfavorable 2'-OH–nucleobase steric clash in the case of ANA:RNA and ANA:ANA duplexes. These results have been used as an additional design rule for the creation of therapeutically relevant 2'F-ANA modified antisense oligonucleotides. Our studies on substituted G-quadruplex structures have revealed that the same fluorine-mediated hydrogen bonding interactions observed in 2'F-ANA modified double helices also contribute to the stability of 2'F-ANA substituted G-quadruplex structures found in the human telomeric sequence. We have shown that a single 2'F-ANA substitution stabilizes only the propeller parallel G-quadruplex form over all competing conformers, and that this stabilization is in most part related to the formation of C-H8...2'F-C and 2'F-CH...O4' noncovalent interactions. Our studies showed that while stable ANA:ANA duplexes are too unstable to form via ANA+ANA bimolecular association, an appropriate combination of ANA and 2'F-ANA nucleotides into a gapmer containing a central segment (‘gap’) of ANA nucleotides flanked by two 2'F-ANA ‘wings’ will yield oligonucleotides that can adopt monomeric hairpin (with the unpaired ANA nucleotides on the loop) and bimolecular duplex structures of comparable thermal stabilities. This tendency of ANA nucleotide tracts to form loop structures offers a range of new applications for this modification, particularly in applications where a duplex/hairpin conformational switch is desirable. A screen of arabinose modified siRNA constructs revealed that ANA has a place among chemically modified oligonucleotides known to be compatible with siRNA-mediated gene silencing in mammalian cells. Taking advantage of ANA’s destabilizing effects, we have shown that ANA in combination with RNA and 2'F-ANA allows the siRNA duplex thermodynamics to be finely tuned to produce heavily-modified siRNA duplexes that are capable of potent silencing of Luciferase and DRR, a gene linked to malignant glioma. The impact of several sugar and nucleobase substitutions on the formation of parallel- stranded duplexes at neutral pH was investigated. Our results open up new perspectives for the development of parallel-stranded double helices at physiological-like conditions. We have demonstrated that rWC parallel hybridization incorporating iC/iG nucleobases occurs not only in ps-DNA:DNA, but also in ps-DNA:RNA, ps-DNA:2'F-RNA, and ps-DNA:2'F-ANA hybrids. This observation is of importance for potential future applications of parallel hybridization, and in particular in the design of novel oligonucleotide hybridization probes or oligonucleotide-based therapeutics.Les applications prometteuses des analogues d’acides nucléiques chimiquement modifiés, dont le nombre est en constante progression, couvrent un large champ s’étirant des thérapies à oligonucléotides aux nanotechnologies d’ADN et outils de diagnostic, à la génomique fonctionnelle et à la validation de cible. Parmi les très nombreuses variétés d’acides nucléiques modifiés qui ont été développées, et plus encore parmi celles dont le sucre central n’est pas naturel, l’acide arabinonucléique (ANA) et son dérivé fluoré en position 2' (2'F-ANA) nous sont particulièrement importants. La présente thèse se focalise en premier lieu sur la compréhension et l’élucidation des causes structurelles à l’origine des propriétés intéressantes qu’ont ces analogues nucléosidiques, et plus spécifiquement sur leurs structures, leurs stabilités et leur activité en milieu cellulaire.Notre travail a permis d’établir que la grande affinité de 2'F-ANA pour l’ARN ou pour lui-même est en partie du à des interactions de type liaison hydrogène entre l’atome de fluor sur le carbone 2' des nucléotides 2'F-ANA et l’atome d’hydrogène sur le carbone 8 d’une purine consécutive. Au contraire, pour des duplexes ANA:ARN ou ANA:ANA, une configuration équivalente aboutit à une gêne stérique entre l’hydroxyle 2' et la base purique. Ces résultats se sont traduits par la mise en place d’une règle additionnelle pour la conception d’oligonucléotides modifiés 2'F-ANA antisens à visée thérapeutique.Nos études sur des G-quadruplexes substitués ont révélé qu’une liaison hydrogène similaire impliquant l’atome de fluor de 2'F-ANA contribue de la même manière à la stabilité de ces structures secondaires présentes dans les séquences télomériques de l’homme. Nous avons montré qu’une seule incorporation d’un nucléotide 2'F-ANA suffit à stabiliser la forme en hélice de G-quadruplexes hybridés parallèlement uniquement, et ce parmi un certain nombre d’autres conformères structurels, et cette stabilisation est l’œuvre d’interactions non-covalentes entre C-H8..2'F-C et 2'F-CH..O4'.Bien que des duplexes stables ANA:ANA soient trop instables pour se former par association bimoléculaire, nos études ont montré qu’une combinaison adéquate de nucléotides ANA et 2'F-ANA selon laquelle le segment central de la séquence (gap) est composé d’ANA et les parties 5' et 3' terminales de 2'F-ANA (stratégie dite gapmer) permet d’obtenir des oligonucléotides qui peuvent adopter une structure monomérique en épingle (où les nucléotides ANA non-appariés se trouvent sur la boucle) ou s’apparier en duplexes, et ces structures bénéficient de stabilités thermiques comparables. Un criblage de différents siARN modifiés avec des arabinonucléotides a prouvé qu’ANA a sa place parmi les oligonucléotides chimiquement modifiés compatibles avec la machinerie cellulaire d’extinction de gène chez les mammifères.En prenant en compte l’effet déstabilisant provoqué par l’insertion de nucléotides ANA, il a été démontré que des combinaisons ANA, ARN et 2'F-ANA dans des siARN permettent de façonner avec précision la thermodynamique de ces duplexes et, ce faisant, des siARN largement modifiés ont été préparés qui se sont révélés capable de réduire l’expression des gènes de la luciférase et de DRR, un gène impliqué dans le développement de gliomes malins.L’effet provoqué par la substitution de sucres et de nucléobases sur la formation, à pH neutre, de duplexes dont les brins sont appariés parallèlement a également été analysé. Contrairement à la plupart des études publiées à ce jour sur l’interférence à l’ARN et sur les siARN chimiquement modifiés, nous avons prouvé que l’hybridation parallèle selon les règles d’appariement de type Watson-Crick inversé à l’aide de nucléobases iC et iG se produit non seulement pour des duplexes ps-DNA:DNA, mais aussi ps-DNA:RNA, ps-DNA:2'F-RNA et ps-DNA:2'F-ANA. Cette observation renforce le potentiel thérapeutique et diagnostique des oligonucléotides dont les brins sont hybridés parallèlement

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.Peer Reviewe

Energetically important C-H···F-C pseudohydrogen bonding in water: evidence and application to rational design of oligonucleotides with high binding affinity

It is controversial whether organic fluorine can form energetically important hydrogen bonds in aqueous environments. We previously showed by NMR and molecular modeling that the unexpectedly high binding affinity of 2?F-ANA is largely due to a C?H···F?C pseudohydrogen bond at pyrimidine?purine steps. Comparisons of the melting of duplexes with identical sequence composition but a rearranged sequence confirm that energetically important fluorine-mediated pseudohydrogen bonding is in operation in these sequences. The effect is of particular importance when the H-bond donor (purine H8) is activated by the presence of fluorine at its own 2?-position. These results provide a rational method to increase the binding affinity of antisense oligonucleotides by placement of 2?F-ANA modifications at pyrimidine?purine steps

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. © 2013 American Chemical Society.Peer Reviewe

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-2FANA): RNA, and ps-DNA:2F-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:2F-RNA hybrids can engage the RNAi pathway to silence gene expression in vitro.Peer Reviewe

Mapping the affinity landscape of Thrombin-binding aptamers on 2′F-ANA/DNA chimeric G-Quadruplex microarrays

Swiss National Science Foundation [PBBEP2-146174 to J.L.]; Natural Sciences and Engineering Research Council of Canada [to M.J.D.]; Austrian Science Fund [FWF P23797 and FWF P27275 to J.L. and M.M.S.]; Spanish MINECO [BFU2014-52864-R to C.G.]. Funding for open access charge: Austrian Science Fund [FWF P23797 and FWF P27275 to J.L. and M.M.S.].Peer Reviewe