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ABSTRACT: The pivaloyloxymethyl (PivOM) group is a biolabile 2'-O-ribose protection that is under development in a prodrug-based approach for siRNA applications. Besides an expected cellular uptake, nucleic acid sequences carrying PivOM showed also increased nuclease resistance and, in most cases, an affinity for complementary RNA. The r(CGCU*ACGC)dT:r(GCGUAGCG)dT model duplex containing a single modified residue (U*) was synthesized and its solution structure was determined by NMR. The duplex showed a maintained A-RNA helix. In U*, both 2'-O-acetal ester side chain and ring pucker presented a notable rigid conformation. The PivOM moiety was oriented with the carbonyl group turned outside the minor groove and with trans, -ac and -ac torsion angles around the C2'-O2', O2'-CA and CA-OB1 bonds respectively. Gauche effects and dipolar interactions between the PivOM and the backbone appeared to be the predominant factors influencing the PivOM conformation and the orientation of the two supplementary H acceptors suggested that hydration could also play a role in the duplex stability.
Organic & Biomolecular Chemistry 03/2013; · 3.70 Impact Factor
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ABSTRACT: The dengue virus (DV) is an important human pathogen from the Flavivirus genus, whose genome- and antigenome RNAs start with the strictly conserved sequence pppAG. The RNA-dependent RNA polymerase (RdRp), a product of the NS5 gene, initiates RNA synthesis de novo, i.e., without the use of a pre-existing primer. Very little is known about the mechanism of this de novo initiation and how conservation of the starting adenosine is achieved. The polymerase domain NS5Pol(DV) of NS5, upon initiation on viral RNA templates, synthesizes mainly dinucleotide primers that are then elongated in a processive manner. We show here that NS5Pol(DV) contains a specific priming site for adenosine 5'-triphosphate as the first transcribed nucleotide. Remarkably, in the absence of any RNA template the enzyme is able to selectively synthesize the dinucleotide pppAG when Mn(2+) is present as catalytic ion. The T794 to A799 priming loop is essential for initiation and provides at least part of the ATP-specific priming site. The H798 loop residue is of central importance for the ATP-specific initiation step. In addition to ATP selection, NS5Pol(DV) ensures the conservation of the 5'-adenosine by strongly discriminating against viral templates containing an erroneous 3'-end nucleotide in the presence of Mg(2+). In the presence of Mn(2+), NS5Pol(DV) is remarkably able to generate and elongate the correct pppAG primer on these erroneous templates. This can be regarded as a genomic/antigenomic RNA end repair mechanism. These conservational mechanisms, mediated by the polymerase alone, may extend to other RNA virus families having RdRps initiating RNA synthesis de novo.
PLoS Pathogens 09/2012; 8(9):e1002912. · 9.13 Impact Factor
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ABSTRACT: The 5' end of eukaryotic mRNA carries a N(7)-methylguanosine residue linked by a 5'-5' triphosphate bond. This cap moiety ((7m)GpppN) is an essential RNA structural modification allowing its efficient translation, limiting its degradation by cellular 5' exonucleases and avoiding its recognition as "nonself" by the innate immunity machinery. In vitro synthesis of capped RNA is an important bottleneck for many biological studies. Moreover, the lack of methods allowing the synthesis of large amounts of RNA starting with a specific 5'-end sequence have hampered biological and structural studies of proteins recognizing the cap structure or involved in the capping pathway. Due to the chemical nature of N(7)-methylguanosine, the synthesis of RNAs possessing a cap structure at the 5' end is still a significant challenge. In the present work, we combined a chemical synthesis method and an enzymatic methylation assay in order to produce large amounts of RNA oligonucleotides carrying a cap-0 or cap-1. Short RNAs were synthesized on solid support by the phosphoramidite 2'-O-pivaloyloxymethyl chemistry. The cap structure was then coupled by the addition of GDP after phosphorylation of the terminal 5'-OH and activation by imidazole. After deprotection and release from the support, GpppN-RNAs or GpppN(2'-Om)-RNAs were purified before the N(7)-methyl group was added by enzymatic means using the human (guanine-N(7))-methyl transferase to yield (7m)GpppN-RNAs (cap-0) or (7m)GpppN(2'-Om)-RNAs (cap-1). The RNAs carrying different cap structures (cap, cap-0 or, cap-1) act as bona fide substrates mimicking cellular capped RNAs and can be used for biochemical and structural studies.
RNA 02/2012; 18(4):856-68. · 5.09 Impact Factor
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ABSTRACT: The cellular delivery of bioactive nucleic acid-based drugs such as small interfering RNA (siRNA) represents a major technical hurdle for their pharmaceutical application. Prodrug-like approaches provide an attractive concept to address the delivery problem. With the aim to prepare RNA-based prodrugs bearing biolabile protections which facilitate cellular uptake and are prone to be removed enzymatically inside cells in order to release functional RNA, we synthesized pro-RNA totally or partially masked in 2'-OH position with pivaloyloxymethyl (PivOM) groups. A suitable strategy has been developed to synthesize and to purify base-sensitive mixed 2'-OH/2'-O-PivOM oligoribonucleotides, and to include them in siRNA. In this strategy, the fluoride labile [(triisopropylsilyl)oxy]-benzyloxycarbonyl group (tboc) as nucleobase protection (for A and C), the TBS group as 2'-OH protection and the Q-linker to solid-support were compatible with the PivOM groups masking some 2'-OH. We have taken advantage of the specific stability of the PivOM group to apply selected acidic, basic, and fluoride ions treatment for the deprotection and release of pro-RNA. This kind of pro-siRNA was studied in a human cell culture-based RNAi assay and preliminary promising data are discussed.
The Journal of Organic Chemistry 06/2011; 76(14):5719-31. · 4.45 Impact Factor
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ABSTRACT: The efficiency of chemical RNA synthesis has been radically improved by the use of pivaloyloxymethyl (PivOM) groups as 2'-protection, containing an acetal spacer that minimizes the steric effect of the ester group on the neighboring amidite during the coupling. However, the major benefit of the base-labile PivOM groups is their simple removal upon standard basic conditions applied to deprotect the RNA and release it from solid supports. Combined with standard acyl groups for nucleobases, cyanoethyl groups for phosphates, and base-cleavable linkers, PivOM groups make RNA deprotection as simple as DNA deprotection. Thus, no additional deprotection step with tedious desalting workup procedures is required, and RNA synthesis becomes as convenient and efficient as DNA synthesis.
Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.] 12/2010; Chapter 3:Unit3.19.
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ABSTRACT: A robust, reproducible, and scalable method for the solid-phase synthesis of 5'-triphosphates of DNA, RNA, and their chemically modified analogues using 5'-H-phosphonate intermediates is described. 5'-Triphosphates of oligonucleotides with varying lengths and sequences containing different 5'-terminal nucleotides, with and without internal sugar-backbone modifications, were efficiently prepared as crude products or further purified by HPLC.
Organic Letters 05/2010; 12(10):2190-3. · 5.86 Impact Factor
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Chemistry & Biodiversity 03/2010; 7(3):494-535. · 1.80 Impact Factor
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ABSTRACT: New base-labile acyloxymethyl groups were evaluated to protect 2'-OH functions of ribonucleotides for regular RNA synthesis in order to shorten the deprotection procedure upon ammonia. These same acetalester groups were assessed in 2'-modified proRNA as biolabile 2'-protections removable by cell enzymes to generate parent RNA. Demasking of 2'-modified pro-uridylates was studied in cell extracts.
Bioorganic & medicinal chemistry letters 07/2009; 19(15):4046-9. · 2.65 Impact Factor
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ABSTRACT: With the aim to prepare biolabile oligoribonucleotides that are partially or fully base-sensitive 2′-O-pivaloyloxymethyl functionalized, we investigated the cleavage of a silyl-type linker and a Q-linker anchored to a solid support under several fluoride ion conditions. Thus, an original fluoride ion treatment was perfected to release oligonucleotides bearing base-sensitive modifications from solid supports without affecting them and with high efficiency. Chimeric oligouridylates containing both 2′-OH and 2′-O-PivOM groups were obtained in high yield and purity. Moreover, the conditions used to cleave the linker were extended to the release of a base-sensitive DNA-oligonucleotide with 4-N-acetylcytosine residues; the acetyl groups were maintained.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)
European Journal of Organic Chemistry 03/2009; · 3.33 Impact Factor
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Chemistry 10/2008; 14(30):9135-8. · 5.93 Impact Factor
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ABSTRACT: To synthesize oligoribonucleotides containing base-sensitive 2'-O-modifications, we used the Q-linker or a silyl linker anchored to CPG or polystyrene resin. A fluoride ions treatment was optimized to release oligoribonucleotides from the solid-support and to remove TBDMS groups without affecting 2'-O-acyloxymethyl groups.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: The use of base-labile groups for 2'-OH protection is a major challenge for RNA synthesis. A new method with acyloxymethyl groups has been developed. These groups were fully compatible with standard base-labile protections for nucleobases and phosphates and were removed in a short two-step all-base deprotection. Oligoribonucleotides were synthesized efficiently, rapidly and in high purity without chain rupture or isomerisation via this new whole base-labile strategy.
Nucleic Acids Symposium Series 02/2008;
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ABSTRACT: Incorporation of 5-propynylamino and 5-propynyl alpha-2'-deoxyuridine into alpha-oligonucleotides (alpha-ON) allows high-affinity targeting of complementary DNA for alpha-ON with anionic and neutral backbone but not for cationic alpha-ON, revealing clues on the role of the amino group of the propynylamino on the formation of DNA duplexes.
Bioorganic & Medicinal Chemistry Letters 03/2007; 17(4):951-4. · 2.55 Impact Factor
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ABSTRACT: Specific control of gene expression by synthetic oligonucleotides (ON) is now widely used for target validation but clinical applications are limited by ON bioavailability. Moreover, most currently used strategies for physical and chemical delivery cannot be easily implemented in vivo. This article reviews new strategies which appear promising for ON delivery. The first part deals with ON chemical modifications aiming at improving cellular uptake as for instance the grafting of cationic groups on the ON backbone. The second part concerns ON conjugation to cell penetrating peptides.
Current topics in medicinal chemistry 02/2007; 7(7):727-37. · 4.47 Impact Factor
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Allison A Johnson,
Jane M Sayer,
Haruhiko Yagi,
Sachindra S Patil, Françoise Debart,
Martin A Maier,
David R Corey,
Jean-Jacques Vasseur,
Terrence R Burke,
Victor E Marquez,
Donald M Jerina,
Yves Pommier
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ABSTRACT: Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase catalyzes two sequential reactions, 3'-processing (3'-P) and strand transfer (ST). The first integrase inhibitors are undergoing clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3'-processing site. 3'-Processing was blocked with substrates containing constrained sugars and alpha-anomeric residues, suggesting that integrase requires flexibility of the phosphodiester backbone at the 3'-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3'-P site inhibited 3'-P, suggesting the importance of the minor groove contacts for 3'-P. ST occurred in the presence of bulky BaP DE DNA adducts attached to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.
Journal of Biological Chemistry 11/2006; 281(43):32428-38. · 4.77 Impact Factor
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Allison A. Johnson,
Jane M. Sayer,
Haruhiko Yagi,
Sachindra S. Patil, Françoise Debart,
Martin A. Maier,
David R. Corey,
Jean-Jacques Vasseur,
Jr. Terrence R. Burke,
Victor E. Marquez,
Donald M. Jerina,
Yves Pommier
[show abstract]
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ABSTRACT: Integration of the viral cDNA into host chromosomes is required for viral replication. Human immunodeficiency virus integrase
catalyzes two sequential reactions, 3′-processing (3′-P) and strand transfer (ST). The first integrase inhibitors are undergoing
clinical trial, but interactions of inhibitors with integrase and DNA are not well understood in the absence of a co-crystal
structure. To increase our understanding of integrase interactions with DNA, we examined integrase catalysis with oligonucleotides
containing DNA backbone, base, and groove modifications placed at unique positions surrounding the 3′-processing site. 3′-Processing
was blocked with substrates containing constrained sugars and α-anomeric residues, suggesting that integrase requires flexibility
of the phosphodiester backbone at the 3′-P site. Of several benzo[a]pyrene 7,8-diol 9,10-epoxide (BaP DE) adducts tested, only the adduct in the minor groove at the 3′-P site inhibited 3′-P,
suggesting the importance of the minor groove contacts for 3′-P. ST occurred in the presence of bulky BaP DE DNA adducts attached
to the end of the viral DNA suggesting opening of the active site for ST. Position-specific effects of these BaP DE DNA adducts
were found for inhibition of integrase by diketo acids. Together, these results demonstrate the importance of DNA structure
and specific contacts with the viral DNA processing site for inhibition by integrase inhibitors.
Journal of Biological Chemistry 10/2006; 281(43):32428-32438. · 4.77 Impact Factor
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ABSTRACT: [reaction: see text] Short oligo-U sequences containing 2'-O-acyloxymethyl or acylthiomethyl groups as biolabile 2'-O-protections of RNA have been synthesized. These modified homouridylates are deprotected upon cellular esterase activation to release the parent RNA. They exhibit exceptional resistance to nuclease degradation, and the evaluation of their pairing properties shows that the 2'-acyloxymethyl groups do not prevent the duplex dsRNA formation. These biolabile 2'-modifications overcome the first hurdle to turn oligoribonucleotides into candidates for RNA interference drugs.
Organic Letters 09/2006; 8(17):3869-72. · 5.86 Impact Factor
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ABSTRACT: The grafting of cationic groups to synthetic oligonucleotides (ONs) in order to reduce the charge repulsion between the negatively charged strands of a duplex or triplex, and consequently to increase a complex's stability, has been extensively studied. Guanidinium groups, which are highly basic and positively charged over a wide pH range, could be an efficient ON modification to enhance their affinity for nucleic acid targets and to improve cellular uptake. A straightforward post-synthesis method to convert amino functions attached to ONs (on sugar, nucleobase or backbone) into guanidinium tethers has been perfected. In comparison to amino groups, such cationic groups anchored to alpha-oligonucleotide phosphoramidate backbones play important roles in duplex stability, particularly with RNA targets. This high affinity could be explained by dual recognition resulting from Watson-Crick or Hoogsteen base pairing combined with cationic/anionic backbone recognition between strands involving H-bond formation and salt bridging. Molecular-dynamics simulations corroborate interactions between the cationic backbones of the alpha-ONs and the anionic backbones of the nucleic acid targets. Moreover, ONs with guanidinium modification increased cellular uptake relative to negatively charged ONs. The cellular localization of these new cationic phosphoramidate ONs is mainly cytoplasmic. The uptake of these ON analogues might occur through endocytosis.
ChemBioChem 05/2006; 7(4):684-92. · 3.94 Impact Factor
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ABSTRACT: During the last decade, cell penetrating peptides (CPP) have been extensively used to mediate the cellular delivery of non-permeant biomolecules, including oligonucleotides (ONs). A covalent linkage between the CPP and the transported ON is required to mediate efficient cell internalization, and a disulfide bridge between the CPP and the ON has been shown to induce the most potent biological response. In this paper, we describe the activation. In a one step process of the sulfhydryl function from a synthon commercially available for ON synthesis. In addition, since the highly cationic nature of currently used CPP caused serious precipitation problems during the coupling step, we further improved the method by adsorbing the crude activated ON on an anion exchange matrix prior to specific peptide coupling.
Bioorganic & Medicinal Chemistry Letters 12/2005; 15(22):5084-7. · 2.55 Impact Factor
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ABSTRACT: The ability of cationic phosphoramidate pyrimidine alpha-oligonucleotides (ONs) to form triplexes with DNA duplexes was investigated by UV melting experiments, circular dichroism spectroscopy and gel mobility shift experiments. Replacement of the phosphodiester linkages in alpha-ONs with positively charged phosphoramidate linkages results in more efficient triplex formation, the triplex stability increasing with the number of positive charges. At a neutral pH and in the absence of magnesium ions, it was found that a fully cationic phosphoramidate alpha-TFO (triplex-forming oligonucleotide) forms a highly stable triplex that melts at a higher temperature than the duplex target. No hysteresis between the annealing and melting curves was noticed; this indicates fast association. Moreover, the recognition of a DNA duplex with a cationic alpha-TFO through Hoogsteen base pairing is highly sequence-specific. To the best of our knowledge, this is the first report of stable triplexes in the pyrimidine motif formed by cationic alpha-oligonucleotides and duplex targets.
ChemBioChem 08/2005; 6(7):1254-62. · 3.94 Impact Factor
