4 research outputs found
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'-OHânucleobase 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 (âgapâ) of ANA nucleotides flanked by two 2'F-ANA âwingsâ 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 ANAâs 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 dâacides nuclĂ©iques chimiquement modifiĂ©s, dont le nombre est en constante progression, couvrent un large champ sâĂ©tirant des thĂ©rapies Ă oligonuclĂ©otides aux nanotechnologies dâADN et outils de diagnostic, Ă la gĂ©nomique fonctionnelle et Ă la validation de cible. Parmi les trĂšs nombreuses variĂ©tĂ©s dâacides nuclĂ©iques modifiĂ©s qui ont Ă©tĂ© dĂ©veloppĂ©es, et plus encore parmi celles dont le sucre central nâest pas naturel, lâacide arabinonuclĂ©ique (ANA) et son dĂ©rivĂ© fluorĂ© en position 2' (2'F-ANA) nous sont particuliĂšrement importants. La prĂ©sente thĂšse se focalise en premier lieu sur la comprĂ©hension et lâĂ©lucidation des causes structurelles Ă lâorigine des propriĂ©tĂ©s intĂ©ressantes quâont ces analogues nuclĂ©osidiques, et plus spĂ©cifiquement sur leurs structures, leurs stabilitĂ©s et leur activitĂ© en milieu cellulaire.Notre travail a permis dâĂ©tablir que la grande affinitĂ© de 2'F-ANA pour lâARN ou pour lui-mĂȘme est en partie du Ă des interactions de type liaison hydrogĂšne entre lâatome de fluor sur le carbone 2' des nuclĂ©otides 2'F-ANA et lâatome dâhydrogĂšne sur le carbone 8 dâune purine consĂ©cutive. Au contraire, pour des duplexes ANA:ARN ou ANA:ANA, une configuration Ă©quivalente aboutit Ă une gĂȘne stĂ©rique entre lâhydroxyle 2' et la base purique. Ces rĂ©sultats se sont traduits par la mise en place dâune rĂšgle additionnelle pour la conception dâoligonuclĂ©otides modifiĂ©s 2'F-ANA antisens Ă visĂ©e thĂ©rapeutique.Nos Ă©tudes sur des G-quadruplexes substituĂ©s ont rĂ©vĂ©lĂ© quâune liaison hydrogĂšne similaire impliquant lâatome de fluor de 2'F-ANA contribue de la mĂȘme maniĂšre Ă la stabilitĂ© de ces structures secondaires prĂ©sentes dans les sĂ©quences tĂ©lomĂ©riques de lâhomme. Nous avons montrĂ© quâune seule incorporation dâun nuclĂ©otide 2'F-ANA suffit Ă stabiliser la forme en hĂ©lice de G-quadruplexes hybridĂ©s parallĂšlement uniquement, et ce parmi un certain nombre dâautres conformĂšres structurels, et cette stabilisation est lâĆuvre dâinteractions 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 bimolĂ©culaire, nos Ă©tudes ont montrĂ© quâune combinaison adĂ©quate de nuclĂ©otides ANA et 2'F-ANA selon laquelle le segment central de la sĂ©quence (gap) est composĂ© dâANA et les parties 5' et 3' terminales de 2'F-ANA (stratĂ©gie dite gapmer) permet dâobtenir des oligonuclĂ©otides qui peuvent adopter une structure monomĂ©rique en Ă©pingle (oĂč les nuclĂ©otides ANA non-appariĂ©s se trouvent sur la boucle) ou sâapparier en duplexes, et ces structures bĂ©nĂ©ficient de stabilitĂ©s thermiques comparables. Un criblage de diffĂ©rents siARN modifiĂ©s avec des arabinonuclĂ©otides a prouvĂ© quâANA a sa place parmi les oligonuclĂ©otides chimiquement modifiĂ©s compatibles avec la machinerie cellulaire dâextinction de gĂšne chez les mammifĂšres.En prenant en compte lâeffet dĂ©stabilisant provoquĂ© par lâinsertion de nuclĂ©otides ANA, il a Ă©tĂ© dĂ©montrĂ© que des combinaisons ANA, ARN et 2'F-ANA dans des siARN permettent de façonner avec prĂ©cision la thermodynamique de ces duplexes et, ce faisant, des siARN largement modifiĂ©s ont Ă©tĂ© prĂ©parĂ©s qui se sont rĂ©vĂ©lĂ©s capable de rĂ©duire lâexpression des gĂšnes de la lucifĂ©rase et de DRR, un gĂšne impliquĂ© dans le dĂ©veloppement de gliomes malins.Lâeffet provoquĂ© par la substitution de sucres et de nuclĂ©obases sur la formation, Ă pH neutre, de duplexes dont les brins sont appariĂ©s parallĂšlement a Ă©galement Ă©tĂ© analysĂ©. Contrairement Ă la plupart des Ă©tudes publiĂ©es Ă ce jour sur lâinterfĂ©rence Ă lâARN et sur les siARN chimiquement modifiĂ©s, nous avons prouvĂ© que lâhybridation parallĂšle selon les rĂšgles dâappariement de type Watson-Crick inversĂ© Ă lâaide de nuclĂ©obases 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 thĂ©rapeutique et diagnostique des oligonuclĂ©otides dont les brins sont hybridĂ©s parallĂšlement
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