Publications (71)299.97 Total impact
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Article: Dramatic Effect of Furanose C2′ Substitution on Structure and Stability: Directing the Folding of the Human Telomeric Quadruplex with a Single Fluorine Atom
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ABSTRACT: 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 determi-nation of this particularly significant nucleic acid structure. 2′F-arabinose substitution provides the greatest stabiliza-tion 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 destabi-lization of the quadruplex structure due to unfavorable electrostatic repulsion between the phosphate and the 2′-F. G -quadruplexes are nucleic acid structures occurring in human telomers and oncogene-promoter regions, and they have garnered considerable attention due to their involvement in telomere maintenance and gene regulation. 1−3 They have also emerged as novel building blocks for supramolecular assemblies, 4 DNA-based nanodevices, 5−9 and potential antiviral agents, and as a new class of molecular targets for anti-cancer drugs. 10,11 G-quadruplexes exhibit significant structural diversity. 3,12,13 High resolution structural studies on human telomeric DNA have mainly focused on short DNA sequences able to form individual quadruplexes. In many cases, these oligonucleotide sequences can adopt and interconvert between different conformations, obscuring structural analysis. Among the observed telomeric G-quad-ruplex topologies, it has been suggested that the simple folds of the parallel "propeller" and "hybrid" (3+1) 14,15 structural models provide more adequate scaffolds for the compact-stacking multi-quadruplex structure of human telomeric DNA than other antiparallel models. 12,13 Interestingly, human telomeric RNA (TERRA), which is known to play a crucial role in telomere maintenance in vivo, 16,17 always adopts propeller-like parallel structures. 18,19 The tendency of TERRA and other RNA quadruplexes to form parallel structures is related to the strong preference of guanosine ribonucleosides to adopt the anti glycosidic bond conformation. 20 The syn/anti preference of dG vs rG has been used to modulate G-quadruplex folding, and different nucleo-side modifications have been proposed following this strategy. 20−26 In particular, 2′-deoxy-2′-fluoro-arabinonucleo-sides (e.g., 2′F-araG) favor the anti orientation in order to avoid steric interaction between guanine and the top-face fluorine of the arabinose sugar. 22,27 The 2′F-arabinose modification is especially interesting since it can modulate the structure and stability of quadruplexes, 22,23 and unlike ribose, it does not disrupt the conformation of the furanose ring. 28−30 Thus, replacement of dG's adopting the anti glycosidic bond conformation with 2′F-araG's stabilizes the thrombin binding aptamer 22 and the "hybrid (3+1)" quadruplex 23 up to +3.4 °C per 2′F-araG modification. To expand upon these findings and to investigate the structural basis of the effect of C2′ modifications in G-quadruplexes, we have studied the impact of several substitutions, 2′F-araG (AFtel), 2′F-rG (RFtel), araG (Atel), and rG (Rtel), in the human telomeric quadruplex (Figure 1). In this study, we have focused on the two-repeat telomeric sequence, d(TAGGGTTAGGGT)(Dtel), which offers a convenient model for high-resolution studies by either NMR 31 or crystallography. 14 In solution this sequence exists as a mixture of two interconverting forms: a parallel propeller-like fold and an antiparallel fold. 31 We have now found that a single dG-to-2′F-araG modification at the ninth position provides the propeller-like conformation exclusively, allowing for detailed structural analysis. Our rationale for substitution at G9 d(TAGGGTTAGGGT) stems from the observation that in antiparallel quadruplexes the guanine at the 3′-side of the first TTA loop tends to adopt a syn glycosidic bond conformation, 13 which is disfavored in 2′F-arabinonucleosides. The nucleotide structures and oligonucleotide sequences studied are shown in Table 1 and Figure 1. G-quadruplex formation and melting were monitored by circular dichroism (CD) and NMR spectroscopy in K + -containing solution (Figure 1 and Figure S1 in the Supporting Information (SI)). The spectra of the unmodified sequence, Dtel, were characteristic of a mixture of parallel and antiparallelJournal of the American Chemical Society 03/2013; · 9.91 Impact Factor -
Article: The 5' Binding MID Domain of Human Argonaute2 Tolerates Chemically Modified Nucleotide Analogues.
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ABSTRACT: Small interfering RNAs (siRNAs) can trigger potent gene silencing through the RNA interference (RNAi) pathway. The RNA-induced silencing complex (RISC) is key to this targeted mRNA degradation, and the human Argonaute2 (hAGO2) endonuclease component of RISC is responsible for the actual mRNA cleavage event. During RNAi, hAGO2 becomes loaded with the siRNA guide strand, making several key nucleic acid-enzyme interactions. Chemically modified siRNAs are now widely used in place of natural double-stranded RNAs, and understanding the effects chemical modifications have on guide strand-hAGO2 interactions has become particularly important. Here, interactions between the 5' nucleotide binding domain of hAGO2, MID, and chemically modified nucleotide analogues are investigated. Measured dissociation constants reveal that hAGO2 does not discriminate between nucleotide analogues during binding, regardless of the preferred sugar conformation of the nucleotide analogues. These results correlate well with cell-based gene silencing results employing siRNAs with 5'-modified guide strands. Additionally, chemical modification with 2'-deoxy-2'-fluoroarabino nucleic acid (2'F-ANA) and 2'-deoxy-2'-fluororibonucleic acid (2'F-RNA) at the passenger strand cleavage site of siRNAs has been shown to prevent hAGO2-mediated strand cleavage, an observation that appears to have little impact on overall gene silencing potency.Nucleic acid therapeutics. 01/2013; -
Article: Designing chemically modified oligonucleotides for targeted gene silencing.
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ABSTRACT: Oligonucleotides (ONs), and their chemically modified mimics, are now routinely used in the laboratory as a means to control the expression of fundamentally interesting or therapeutically relevant genes. ONs are also under active investigation in the clinic, with many expressing cautious optimism that at least some ON-based therapies will succeed in the coming years. In this review, we will discuss several classes of ONs used for controlling gene expression, with an emphasis on antisense ONs (AONs), small interfering RNAs (siRNAs), and microRNA-targeting ONs (anti-miRNAs). This review provides a current and detailed account of ON chemical modification strategies for the optimization of biological activity and therapeutic application, while clarifying the biological pathways, chemical properties, benefits, and limitations of oligonucleotide analogs used in nucleic acids research.Chemistry & biology 08/2012; 19(8):937-54. · 6.52 Impact Factor -
Article: The solution structure of double helical arabino nucleic acids (ANA and 2'F-ANA): effect of arabinoses in duplex-hairpin interconversion.
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ABSTRACT: 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.Nucleic Acids Research 07/2012; 40(18):9329-39. · 8.03 Impact Factor -
Article: The interactions of amphiphilic antisense oligonucleotides with serum proteins and their effects on in vitro silencing activity.
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ABSTRACT: Antisense oligonucleotides (AONs) are a class of compounds with high therapeutic potential. One of the challenges facing this platform is the development of effective techniques to achieve cellular delivery. AON conjugates, in which traditional AONs are attached to certain biomolecules, can exhibit improved intracellular bioavailability in the absence of delivery systems. In this study, the lipophilic moieties docosahexaenoic acid, cholesterol, and docosanoic acid (DSA) were conjugated to various phosphorothioated DNA and chemically-modified 2'-fluoro-arabinonucleic acid AONs via an amino-hexanol-linker added to the 5'-end of the molecule. The gene silencing potential of these compounds was evaluated in vitro in the absence or presence of a transfecting agent (polyion complex micelle). Incubation with sub-micromolar concentration of DSA-conjugates could, in the absence of serum proteins, downregulate more than 60% of the targeted mRNA under carrier-free and carrier-loaded delivery methods. Gene silencing activity of carrier-free DSA-conjugates was, however, decreased in a dose-dependent fashion by adding albumin in the transfection medium. Supplementing the medium with free fatty acid prevented the interaction of the DSA-conjugate with albumin, and restored its silencing activity. These findings suggest that strategies aiming at preventing the association of hydrophobized AONs to serum proteins at the site of action may improve their activity.Biomaterials 05/2012; 33(25):5955-65. · 7.40 Impact Factor -
Article: Phenylpyrrolocytosine as an unobtrusive base modification for monitoring activity and cellular trafficking of siRNA.
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ABSTRACT: 6-Phenylpyrrolocytosine (PhpC) is a cytosine mimic with excellent base-pairing fidelity, thermal stability, and high fluorescence. In this work, PhpC-containing small interfering RNAs (siRNAs) are shown to possess thermal stability and gene silencing activity that is virtually identical to that of natural siRNA. The emissive properties of PhpC allow the cellular trafficking of PhpC-containing siRNAs to be monitored by fluorescence microscopy. Accumulation in the cytoplasm of HeLa cells was observed using real time imaging. These findings demonstrate that PhpC-modified siRNAs retain the properties of natural siRNAs while allowing for fluorescence-based detection and monitoring, providing an ideal system for probing siRNA uptake and trafficking.ACS Chemical Biology 06/2011; 6(9):912-9. · 6.45 Impact Factor -
Article: New light labile linker for solid phase synthesis of 2'-O-acetalester oligonucleotides and applications to siRNA prodrug development.
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ABSTRACT: We report on the synthesis and properties of oligonucleotides containing 2'-O-(levulinic acid) and 2'-O-(amino acid) acetalesters. Given that esters serve as promoieties in several therapeutic prodrugs, we believe that these derivatives will have potential use as nucleic acid prodrugs. In addition, we report on the synthesis of a novel solid support with a photolabile linker that not only allows for the synthesis of oligonucleotides containing various 2'-O-acetalesters, but can be generally adopted to the synthesis of base-sensitive oligoribonucleotides. The release of oligonucleotides from this support is faster than with conventional linkers.Bioorganic & medicinal chemistry letters 06/2011; 21(12):3721-5. · 2.65 Impact Factor -
Article: Energetically important C-H···F-C pseudohydrogen bonding in water: evidence and application to rational design of oligonucleotides with high binding affinity.
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ABSTRACT: 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.Journal of the American Chemical Society 02/2011; 133(4):728-31. · 9.91 Impact Factor -
Article: siRNA nanocarriers based on methacrylic acid copolymers.
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ABSTRACT: Poly(ethylene glycol)-b-poly(propyl methacrylate-co-methacrylic acid) (PEG-b-P(PrMA-co-MAA) can be complexed with poly(amido amine) (PAMAM) dendrimers and nucleic acids to form pH-responsive nanosized core-shell type polyion complex micelles (PICMs). These PICMs have the ability to lose their shell and release the PAMAM/nucleic acid core under mildly acidic conditions such as those encountered in the endosomal compartment. In this work, pH-sensitive PICMs composed of PEG-b-P(PrMA-co-MAA), different PAMAMs, and siRNAs were prepared and characterized. These micelles had mean diameters ranging from 50 to 100 nm depending on the structure of the polycationic component. In order to trigger PICM uptake by receptor-mediated endocytosis, the micelles were decorated with an antibody fragment directed against the transferrin receptor (anti-CD71). The targeting ligand was stably conjugated to a semi-telechelic amino-PEG-b-P(PrMA-co-MAA) via a maleimide/activated ester bifunctional linker, yielding up to 60%-80% functionalization of the maleimide groups. The cellular uptake of the micelles was assessed on human prostate cancer cells (PC-3) via flow cytometry. Native PICMs and micelles bearing a non-specific antibody fragment were taken up to the same extent with a low efficiency, whereas anti-CD71 Fab'-decorated PICMs exhibited significantly higher uptake. The capacity of the targeted, siRNA-loaded, PICMs to downregulate the expression of the Bcl-2 anti-apoptotic oncoprotein was investigated using the appropriate unmodified or 2'-modified (2'F-RNA and 2'F-ANA) siRNA sequence. Bcl-2 mRNA and protein levels were greatly reduced when the cells were transfected with anti-CD71 decorated PICMs. Optimal silencing was achieved with the chemically modified siRNA. These data suggest that combining optimized siRNA chemistry with an effective delivery system can potentiate the activity of siRNA, thereby potentially reducing the total dose of carrier required to achieve a pharmacological effect.Journal of Controlled Release 12/2010; 152(1):159-67. · 5.73 Impact Factor -
Article: Differential stability of 2'F-ANA*RNA and ANA*RNA hybrid duplexes: roles of structure, pseudohydrogen bonding, hydration, ion uptake and flexibility.
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ABSTRACT: Hybrids of RNA with arabinonucleic acids 2'F-ANA and ANA have very similar structures but strikingly different thermal stabilities. We now present a thorough study combining NMR and other biophysical methods together with state-of-the-art theoretical calculations on a fully modified 10-mer hybrid duplex. Comparison between the solution structure of 2'F-ANA*RNA and ANA*RNA hybrids indicates that the increased binding affinity of 2'F-ANA is related to several subtle differences, most importantly a favorable pseudohydrogen bond (2'F-purine H8) which contrasts with unfavorable 2'-OH-nucleobase steric interactions in the case of ANA. While both 2'F-ANA and ANA strands maintained conformations in the southern/eastern sugar pucker range, the 2'F-ANA strand's structure was more compatible with the A-like structure of a hybrid duplex. No dramatic differences are found in terms of relative hydration for the two hybrids, but the ANA*RNA duplex showed lower uptake of counterions than its 2'F-ANA*RNA counterpart. Finally, while the two hybrid duplexes are of similar rigidities, 2'F-ANA single strands may be more suitably preorganized for duplex formation. Thus the dramatically increased stability of 2'F-ANA*RNA and ANA*RNA duplexes is caused by differences in at least four areas, of which structure and pseudohydrogen bonding are the most important.Nucleic Acids Research 04/2010; 38(7):2498-511. · 8.03 Impact Factor -
Article: Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing.
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ABSTRACT: We report that combining a DNA analog (2'F-ANA) with rigid RNA analogs [2'F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1-1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells.Nucleic Acids Research 04/2010; 38(13):4547-57. · 8.03 Impact Factor -
Article: Chemical modification of siRNA.
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ABSTRACT: The ability to manipulate the RNA interference (RNAi) machinery to specifically silence the expression of target genes could be a powerful therapeutic strategy. Since the discovery that RNAi can be triggered in mammalian cells by short double-stranded RNAs (small interfering RNA, siRNA), there has been a tremendous push by researchers, from academia to big pharma, to move siRNAs into clinical application. The challenges facing siRNA therapeutics are significant. The inherent properties of siRNAs (polyanionic, vulnerable to nuclease cleavage) make clinical application difficult due to poor cellular uptake and rapid clearance. Side effects of siRNAs have also proven to be a further complication. Fortunately, numerous chemical modification strategies have been identified that allow many of these obstacles to be overcome. This unit will present an overview of (1) the chemical modifications available to the nucleic acid chemist for modifying siRNAs, (2) the application of chemical modifications to address specific therapeutic obstacles, and (3) the factors that must be considered when assessing the activity of modified siRNAs.Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.] 12/2009; Chapter 16:Unit 16.3. -
Article: A single-label phenylpyrrolocytidine provides a molecular beacon-like response reporting HIV-1 RT RNase H activity.
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ABSTRACT: 6-Phenylpyrrolocytidine (PhpC), a structurally conservative and highly fluorescent cytidine analog, was incorporated into oligoribonucleotides. The PhpC-containing RNA formed native-like duplex structures with complementary DNA or RNA. The PhpC-modification was found to act as a sensitive reporter group being non-disruptive to structure and the enzymatic activity of RNase H. A RNA/DNA hybrid possessing a single PhpC insert was an excellent substrate for HIV-1 RT Ribonuclease H and rapidly reported cleavage of the RNA strand with a 14-fold increase in fluorescence intensity. The PhpC-based assay for RNase H was superior to the traditional molecular beacon approach in terms of responsiveness, rapidity and ease (single label versus dual). Furthermore, the PhpC-based assay is amenable to high-throughput microplate assay format and may form the basis for a new screen for inhibitors of HIV-RT RNase H.Nucleic Acids Research 11/2009; 38(3):1048-56. · 8.03 Impact Factor -
Article: Delivery of Nucleic Acids through the Controlled Disassembly of Multifunctional Nanocomplexes
Advanced Functional Materials 10/2009; 19(24):3862 - 3867. · 10.18 Impact Factor -
Article: Acetal levulinyl ester (ALE) groups for 2'-hydroxyl protection of ribonucleosides in the synthesis of oligoribonucleotides on glass and microarrays.
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ABSTRACT: We describe a synthetic strategy that permits both the growth and deprotection of RNA chains that remain attached to a solid polymer support or chip surface. The key synthons for RNA synthesis are novel 5'-O-DMTr 2'-acetal levulinyl ester (2'-O-ALE) ribonucleoside 3'-phosphoramidite derivatives. In the presence of 4,5-dicyanoimidazole (DCI) as the activator, these monomers coupled to Q-CPG solid support with excellent coupling efficiency (approximately 98.7%). The method was extended to the light directed synthesis of poly rU and poly rA on a microarray through the use of a 5'-O-(2-(2-nitrophenyl)propoxycarbonyl)-2'-O-ALE-3'-phosphoramidite derivative. A two-stage deprotection strategy was employed to fully deblock the RNA directly on the Q-CPG or microarray support without releasing it from the support's surface: phosphate group deblocking with NEt(3) in acetonitrile (ACN) (2:3 v/v; 1 h, r.t.) followed by removal of the 2'-O-ALE groups under mild hydrazinolysis conditions (0.5-4 h, r.t.). This last treatment also removed the levulinyl (Lv) group on adenine (N(6)) and cytosine (N(4)) and the dimethylformamidine (dmf) group on guanine (N(2)). The chemistry and methods described here pave the way to the fabrication of microarrays of immobilized RNA probes for analyzing molecular interactions of biological interest.Journal of the American Chemical Society 07/2009; 131(24):8496-502. · 9.91 Impact Factor -
Article: Studies on the hydrolytic stability of 2'-fluoroarabinonucleic acid (2'F-ANA).
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ABSTRACT: The stability of 2'-deoxy-2'-fluoroarabinonucleic acid (2'F-ANA) to hydrolysis under acidic and basic conditions was compared to that of DNA, RNA and 2'F-RNA. In enzyme-free simulated gastric fluid (pH approximately 1.2), 2'F-ANA was found to have dramatically increased stability (virtually no cleavage observed after 2 days) with respect to both DNA (t(1/2) approximately 2 min) and RNA (t(1/2) approximately 3 h (PO) or 3 days (PS)). These results were observed for both phosphodiester and phosphorothioate backbones and with multiple mixed-base sequences. Under basic conditions, 2'F-ANA also showed good stability. In 1 M NaOH at 65 degrees C, 2'F-ANA had a t(1/2) of approximately 20 h, while RNA was entirely degraded in a few minutes. Furthermore, the nuclease cleavage of phosphorothioate 2'F-ANA and DNA by snake venom phosphodiesterase was studied in detail. One diastereomer of the PS-2'F-ANA linkage was found to be much more vulnerable to enzymatic cleavage than the other, which is parallel to the properties observed for PS-DNA. Additional studies of 2'F-ANA-containing oligonucleotides are warranted based on the excellent stability properties described here.Organic & Biomolecular Chemistry 06/2009; 7(9):1904-10. · 3.70 Impact Factor -
Article: Synthesis and biophysical characterization of oligonucleotides containing a 4'-selenonucleotide.
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ABSTRACT: The first synthesis of oligonucleotides containing 4'-selenium-modified ribonucleotides (4'-Se-rN) is described. Four sequences containing 4'-Se-rT were successfully synthesized and compared with DNA and RNA oligonucleotides containing a dT, rT, or LNA insert in place of the 4'-Se-rT. The 4'-Se-rT behaved more like rT than dT in its effects on binding affinity, despite the DNA-like structure previously observed for the nucleoside, suggesting that a conformational switch occurs upon incorporation into an oligonucleotide. Incorporation of 4'-Se-rT into A-RNA and hybrid duplexes led to increased binding affinity, while incorporation into B-DNA destabilized the duplex to the same extent as an rT nucleotide.Journal of the American Chemical Society 08/2008; 130(27):8578-9. · 9.91 Impact Factor -
Article: Chemically modified siRNA: tools and applications.
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ABSTRACT: Chemical modification provides solutions to many of the challenges facing siRNA therapeutics. This review examines the various siRNA modifications available, including every aspect of the RNA structure and siRNA duplex architecture. The applications of chemically modified siRNA are then examined, with a focus on specificity (elimination of immune effects and hybridization-dependent off-target effects) and delivery. We also discuss improvement of nuclease stability and potency.Drug Discovery Today 08/2008; 13(19-20):842-55. · 6.83 Impact Factor -
Article: 2′F-Arabinonucleic acids (2′F-ANA) — History, properties, and new frontiers
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ABSTRACT: The development of arabinonucleosides and oligoarabinonucleotides is described, focusing especially on 2′-deoxy-2′-fluoroarabinonucleosides (araF-N) and -oligonucleotides (2'F-ANA). In addition to their chemical and enzymatic synthesis, we discuss various properties of 2′F-ANA: hydrolytic stability (to nucleases, acids, and bases), binding affinity to complementary strands, structure and conformation, and optimization of RNase H activity. We also discuss the use of 2′F-ANA in gene-silencing approaches (antisense, siRNA), and in the stabilization of higher-order structures (such as triplexes and quadruplexes) including aptamers. Finally, we examine several other oligonucleotide derivatives based on 2′F-ANA and look ahead to the future of 2′-fluoroarabinonucleosides and -oligonucleotides.Key words: arabinonucleic acids, 2′F-ANA, antisense oligonucleotides, siRNA, modified oligonucleotides.On décrit le développement d'arabinonucléosides et d'oligoarabino nucléotides, en portant une attention particulière aux 2′-désoxy-2-fluoroarabinonucléosides (araF-N) et -oligonucléotides (2'F-ANA). En plus de leur synthèse chimique et enzymatique, on discute de diverses propriétés des 2'F-ANA, telles la stabilité hydrolytique (des nucléases, des acides et des bases), l'affinité de fixation à des brins complémentaires, la structure et la conformation et l'optimisation de l'activité RNase H. On discute aussi de l'utilité des 2′F-ANA dans les approches tendant à rendre les gènes silencieux (antisens, siARN) et dans la stabilisation des structures d'ordres supérieurs (triplexes et quadruplexes), y compris les aptamères. Enfin, on examine plusieurs autres dérivés oligonucléotidiques dérivés du 2′F-ANA ainsi que l'avenir des fluoroarabinonucléosides et -oligonucléotides.Mots-clés : acides arabinonucléiques, 2′F-ANA, oligonucléotides antisens, siARN, oligonucléotides modifiés.[Traduit par la Rédaction]Canadian Journal of Chemistry 06/2008; 86(7):641-656. · 1.24 Impact Factor -
Article: An intact unfolded protein response in Trpt1 knockout mice reveals phylogenic divergence in pathways for RNA ligation.
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ABSTRACT: Unconventional mRNA splicing by an endoplasmic reticulum stress-inducible endoribonuclease, IRE1, is conserved in all known eukaryotes. It controls the expression of a transcription factor, Hac1p/XBP-1, that regulates gene expression in the unfolded protein response. In yeast, the RNA fragments generated by Ire1p are ligated by tRNA ligase (Trl1p) in a process that leaves a 2'-PO4(2-) at the splice junction, which is subsequently removed by an essential 2'-phosphotransferase, Tpt1p. However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin the cleaved Xbp-1 mRNA fragments. We report that inactivation of the Trpt1 gene, encoding the only known mammalian homolog of Tpt1p, eliminates all detectable 2'-phosphotransferase activity from cultured mouse cells but has no measurable effect on spliced Xbp-1 translation. Furthermore, the relative translation rates of tyrosine-rich proteins is unaffected by the Trpt1 genotype, suggesting that the pool of (normally spliced) tRNA(Tyr) is fully functional in the Trpt1-/- mouse cells. These observations argue against the presence of a 2'-PO4(2-) at the splice junction of ligated RNA molecules in Trpt1-/- cells, and suggest that Xbp-1 and tRNA ligation proceed by distinct pathways in yeast and mammals.RNA 03/2008; 14(2):225-32. · 5.09 Impact Factor
Top co-authors
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Institutions
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2002–2013
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McGill University
- Department of Chemistry
Montréal, Quebec, Canada
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2006
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Hôpital Notre-Dame
Montréal, Quebec, Canada
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