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ABSTRACT: Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past 20 years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology.
The Journal of Pathology 09/2011; 226(2):365-79. · 6.32 Impact Factor
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ABSTRACT: Oligonucleotides and their derivatives are a proven chemical strategy for modulating gene expression. However, their negative charge remains a challenge for delivery and target recognition inside cells. Here we show that oligonucleotide-oligospermine conjugates (Zip nucleic acids or ZNAs) can help overcome these shortcomings by serving as effective antisense and antigene agents. Conjugates containing DNA and locked nucleic acid (LNA) oligonucleotides are active, and oligospermine conjugation facilitates carrier-free cell uptake at nanomolar concentrations. Conjugates targeting the CAG triplet repeat within huntingtin (HTT) mRNA selectively inhibit expression of the mutant huntingtin protein. Conjugates targeting the promoter of the progesterone receptor (PR) function as antigene agents to block PR expression. These observations support further investigation of ZNA conjugates as gene silencing agents.
Journal of the American Chemical Society 06/2011; 133(22):8404-7. · 9.91 Impact Factor
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ABSTRACT: Spinocerebellar ataxia-3 (also known as Machado-Joseph disease) is an incurable neurodegenerative disorder caused by expression of a mutant variant of ataxin-3 (ATX3) pro-tein. Inhibiting expression of ATX3 would provide a thera-peutic strategy, but indiscriminant inhibition of both wild-type and mutant ATX3 might lead to undesirable side effects. An ideal silencing agent would block expression of mutant ATX3 while leaving expression of wild-type ATX3 intact. We have previously observed that peptide nucleic acid (PNA) conjugates targeting the expanded CAG repeat within ATX3 mRNA block expression of both alleles. We have now identified additional PNAs capable of inhibiting ATX3 expression that vary in length and in the nature of the con-jugated cation chain. We can also achieve potent and selec-tive inhibition using duplex RNAs containing one or more mismatches relative to the CAG repeat. Anti-CAG antisense bridged nucleic acid oligonucleotides that lack a cationic domain are potent inhibitors but are not allele-selective. Allele-selective inhibitors of ATX3 expression provide insights into the mechanism of selectivity and promising lead compounds for further development and in vivo investigation.
Biological Chemistry 05/2011; 392:315-325. · 2.96 Impact Factor
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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
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ABSTRACT: Antigene RNAs (agRNAs) are small RNA duplexes that target non-coding transcripts rather than mRNA and specifically suppress or activate gene expression in a sequence-dependent manner. For many applications in vivo, it is likely that agRNAs will require chemical modification. We have synthesized agRNAs that contain different classes of chemical modification and have tested their ability to modulate expression of the human progesterone receptor gene. We find that both silencing and activating agRNAs can retain activity after modification. Both guide and passenger strands can be modified and functional agRNAs can contain 2'F-RNA, 2'OMe-RNA, and locked nucleic acid substitutions, or combinations of multiple modifications. The mechanism of agRNA activity appears to be maintained after chemical modification: both native and modified agRNAs modulate recruitment of RNA polymerase II, have the same effect on promoter-derived antisense transcripts, and must be double-stranded. These data demonstrate that agRNA activity is compatible with a wide range of chemical modifications and may facilitate in vivo applications.
Nucleic Acids Research 08/2010; 38(15):5242-59. · 8.03 Impact Factor
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ABSTRACT: Double-stranded RNA has become a ubiquitous tool for inhibition of gene expression in the laboratory. If similar success could be achieved in vivo, duplex RNA might provide a new class of therapeutics capable of treating a broad spectrum of disease. Chemists and biologists developing duplex RNA as a drug have made progress but continue to face challenges. This review presents the current status of duplex RNA in the clinic and comments on future prospects for the approach.
Bioorganic & medicinal chemistry letters 06/2010; 20(11):3203-7. · 2.65 Impact Factor
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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
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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
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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.
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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
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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
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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
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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
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ABSTRACT: The synthesis of oligonucleotides containing 2'-deoxy-2'-fluoro-4'-thioarabinonucleotides is described. 2'-Deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine (4'S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4'S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4'S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4'-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2'-deoxy-2'-fluoro-4'-thioarabinouridine (4'S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2'F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.
Nucleic Acids Research 02/2007; 35(5):1441-51. · 8.03 Impact Factor
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ABSTRACT: An improved synthesis of 2'-deoxy-2'-fluoro-5-methyl-4'-thioarabinouridine (4'S-FMAU) is described. Participation of the 3'-O-benzoyl protecting group in the thiosugar precursor influenced the stereochemistry of the N-glycosylation reaction in nonpolar solvents, permitting a higher beta/alpha ratio than previously observed for similar Lewis acid catalyzed glycosylations. Conformational analysis of the nucleoside using 3JHH and 3JHF NMR coupling constants together with the PSEUROT program showed that it adopted a predominantly northern conformation in contrast to 2'-deoxy-2'-fluoro-5-methylarabinouridine (FMAU), whose PSEUROT conformational analysis is presented here for the first time, which showed a dominantly southeast conformation. The sharp conformational switch attained by replacing the ring heteroatom is attributed to a decrease in relevant steric and stereoelectronic effects.
The Journal of Organic Chemistry 03/2006; 71(3):921-5. · 4.45 Impact Factor
