Publications (36)126.89 Total impact
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Article: Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals.
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ABSTRACT: We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs) modified with α-L-locked nucleic acid (LNA) and related modifications targeting phosphatase and tensin homologue (PTEN) messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT) levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3'- and 5'-flanks with R-5'-Me-α-L-LNA but not R-6'-Me- or 3'-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5'-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5'-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.Molecular Therapy - Nucleic Acids (2012) 1, e47; doi:10.1038/mtna.2012.34; published online 18 September 2012.Molecular therapy. Nucleic acids. 01/2012; 1:e47. -
Article: Synthesis, improved antisense activity and structural rationale for the divergent RNA affinities of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides.
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ABSTRACT: The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F···H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py)···H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.Journal of the American Chemical Society 09/2011; 133(41):16642-9. · 9.91 Impact Factor -
Patent: Preparation of 5'-substituted bicyclic nucleosides and RNA duplexes and their binding affinity to gene phosphatase PTEN
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ABSTRACT: Provided herein are novel 5'-(S)-CH3 substituted bicyclic nucleosides I, wherein B is nucleobase; one of T1 and T2 is H or a hydroxy protecting group and the other of T1 and T2 is H, a hydroxy protecting group or a reactive phosphorus group; G is a diradical moiety selected from -[C(R1R2)]n-, -C(R1)=C(R2)-, -C(R1)=C(R2)-C(R3R4)-, -C[=C(R1R2)]-C(R3R4)-, -C(R1R2)-C[=C(R3R4)]-, -C(O)-N(R7)-C(R1R2)-, -C(R1R2)-N(R7)-C(R3R4)-, -C(R1R2)-N(R7)-O-, -C(R1R2)-O-N(R7)-, -C(R1R2)-N(OR3)-, -C(R1R2)-C(R3R4)-N(R7)-, -C(R1R2)-O-OC(R3R4)-, -C(R1R2)-O-C(R3R4)-O-, -C(R1R2)-C(R3R4)-O-, -C(R1R2)-C(R3R4)-C(R5R6)-O-; R1-R6 are each independently, H, halogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl; OJ1, SJ1,SOJ1, SO2J1, CN, NJ1J2, N3, C(O)J1, C(O)OJ1, C(O)NJ1J2, protecting group;each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ3, N(J3)(J4), =NJ3, SJ3, N33, CN, OC(=L)J3, OC(=L)N(J3)(J4) and C(=L)N(J3)(J4); L is O, S, NJ5; each J1-J5 is independently, H or C1-C6 alkyl; and is from 1 to 3, and oligomeric compds. prepd. therefrom and methods of using the oligomeric compds. More particularly, the furanose ring of each of the novel 5'-(S)-CH3 substituted bicyclic nucleosides includes a 2' to 4' bridging group. The 5'-(S)-CH3 substituted bicyclic nucleosides are expected to be useful for enhancing one or more properties of the oligomeric compds. they are incorporated into such as for example increasing the binding affinity. In certain embodiments, the oligomeric compds. provided herein hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. Thus, nucleoside II was prepd. and incorporated into RNA duplexes. [on SciFinder(R)]Year: 01/2011 -
Article: Configuration of the 5'-Methyl Group Modulates the Biophysical and Biological Properties of Locked Nucleic Acid (LNA) Oligonucleotides.
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ABSTRACT: As part of a program aimed at exploring the structure- activity relationships of 2',4'-bridged nucleic acid (BNA) containing antisense oligonucleotides (ASOs), we report the synthesis and biophysical and biological properties of R- and S-5'-Me LNA modified oligonucleotides. We show that introduction of a methyl group in the (S) configuration at the 5'-position is compatible with the high affinity recognition of complementary nucleic acids observed with LNA. In contrast, introduction of a methyl group in the (R) configuration reversed the stabilization effect of LNA. NMR studies indicated that the R-5'-Me group changes the orientation around torsion angle γ from the +sc to the ap range at the nucleoside level, and this may in part be responsible for the poor hybridization behavior exhibited by this modification. In animal experiments, S-5'-Me-LNA modified gapmer antisense olignucleotides showed slightly reduced potency relative to the sequence matched LNA ASOs while improving the therapeutic profile.Journal of Medicinal Chemistry 11/2010; · 4.80 Impact Factor -
Article: Solution-state structure of a fully alternately 2'-F/2'-OMe modified 42-nt dimeric siRNA construct.
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ABSTRACT: A high-resolution solution structure of a stable 42-nt RNA dimeric construct has been derived based on a high number of NMR observables including nuclear overhauser effects (NOEs), J-coupling constants and residual dipolar couplings (RDCs), which were all obtained with isotopically unlabeled molecules. Two 21-nt siRNA that efficiently hybridize consist of ribose units that were alternately substituted by 2'-fluoro or 2'-methoxy groups. Structure calculations utilized a set of H-F RDC values for all 21 2'-fluoro modified nucleotides under conditions of weak alignment achieved by Pf1 phages. A completely 2'-F/2'-OMe modified dimeric RNA construct adopts an antiparallel double-helical structure consisting of 19 Watson-Crick base pairs with additional 3' UU overhangs and a 5' phosphate group on the antisense strand. NMR data suggest that the stability of individual base pairs is not uniform throughout the construct. While most of the double helical segment exhibits well dispersed imino resonances, the last three base pairs either display uncharacteristic chemical shifts of imino protons or absence of imino resonances even at lower temperatures. Accessibility of imino protons to solvent exchange suggests a difference in stability of duplex ends, which might be of importance for incorporation of the guide siRNA strand into a RISC.Nucleic Acids Research 11/2010; 38(20):7298-307. · 8.03 Impact Factor -
Article: Antisense oligonucleotides containing conformationally constrained 2',4'-(N-methoxy)aminomethylene and 2',4'-aminooxymethylene and 2'-O,4'-C-aminomethylene bridged nucleoside analogues show improved potency in animal models.
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ABSTRACT: To identify chemistries and strategies to improve the potency of MOE second generation ASOs, we have evaluated gapmer antisense oligonucleotides containing BNAs having N-O bonds. These modifications include N-MeO-amino BNA, N-Me-aminooxy BNA, 2',4'-BNA(NC)[NMe], and 2',4'-BNA(NC) bridged nucleoside analogues. These modifications provided increased thermal stability and improved in vitro activity compared to the corresponding ASO containing the MOE modification. Additionally, ASOs containing N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] modifications showed improved in vivo activity (>5-fold) compared to MOE ASO. Importantly, toxicity parameters, such as AST, ALT, liver, kidney, and body weights, were found to be normal for N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] ASO treated animals. The data generated in these experiments suggest that N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] are useful modifications for applications in both antisense and other oligonucleotide based drug discovery efforts.Journal of Medicinal Chemistry 02/2010; 53(4):1636-50. · 4.80 Impact Factor -
Article: Synthesis and biophysical evaluation of 2',4'-constrained 2'O-methoxyethyl and 2',4'-constrained 2'O-ethyl nucleic acid analogues.
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ABSTRACT: We have recently shown that combining the structural elements of 2'O-methoxyethyl (MOE) and locked nucleic acid (LNA) nucleosides yielded a series of nucleoside modifications (cMOE, 2',4'-constrained MOE; cEt, 2',4'-constrained ethyl) that display improved potency over MOE and an improved therapeutic index relative to that of LNA antisense oligonucleotides. In this report we present details regarding the synthesis of the cMOE and cEt nucleoside phosphoramidites and the biophysical evaluation of oligonucleotides containing these nucleoside modifications. The synthesis of the cMOE and cEt nucleoside phosphoramidites was efficiently accomplished starting from inexpensive commercially available diacetone allofuranose. The synthesis features the use of a seldom used 2-naphthylmethyl protecting group that provides crystalline intermediates during the synthesis and can be cleanly deprotected under mild conditions. The synthesis was greatly facilitated by the crystallinity of a key mono-TBDPS-protected diol intermediate. In the case of the cEt nucleosides, the introduction of the methyl group in either configuration was accomplished in a stereoselective manner. Ring closure of the 2'-hydroxyl group onto a secondary mesylate leaving group with clean inversion of stereochemistry was achieved under surprisingly mild conditions. For the S-cEt modification, the synthesis of all four (thymine, 5-methylcytosine, adenine, and guanine) nucleobase-modified phosphoramidites was accomplished on a multigram scale. Biophysical evaluation of the cMOE- and cEt-containing oligonucleotides revealed that they possess hybridization and mismatch discrimination attributes similar to those of LNA but greatly improved resistance to exonuclease digestion.The Journal of Organic Chemistry 02/2010; 75(5):1569-81. · 4.45 Impact Factor -
Patent: Preparation of antisense DNA as modulators of splicing
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ABSTRACT: DNA consisting of 12 to 30 linked monomers were prepd., wherein the oligomeric compd. comprises at least 4 regions, wherein each monomer within each region comprises the same type of sugar moiety and wherein the sugar moieties of the monomers of adjacent regions are different from one another; and wherein: at least one region comprises 2-20 linked monomers and each of the other regions independently comprises 1-20 linked monomers; and wherein at least one region is a tetrahydropyran region, wherein each tetrahydropyran region independently comprises one or more tetrahydropyran nucleoside analog I, wherein Bx is a heterocyclic base moiety; T3 and T4 are independently internucleoside linking group or one of T3 and T4 is H, hydroxy protecting group, a linked conjugate group or a 5' or 3'-terminal group; q1-q7 are independently, H, alkyl, alkenyl, alkynyl; R3 and R4 are independently H, hydroxy, halogen, alkyl, alkoxy. Certain such antisense compds. are useful as RNase H antisense compds., as RNAi compds., and/or as modulators of splicing. Thus, nucleoside phosphoramidite II was prepd. and used in prepn. of antisense DNA as modulators of splicing and for treating a disease by inhibiting or altering gene expression. [on SciFinder(R)]Year: 01/2010 -
Article: Activity of siRNAs with 2-thio-2'-O-methyluridine modification in mammalian cells.
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ABSTRACT: In a search to identify chemical modifications to improve the properties of siRNA, we have investigated the effect of the 2 '-O-methyl-2-thiouridine modification on the biological activity of siRNA. Our results indicate that judicious placement of 2 '-O-methyl-2-thiouridine residues could lead to modified siRNA with activity in mammalian cells.Nucleosides Nucleotides & Nucleic Acids 10/2009; 28(10):902-10. · 0.90 Impact Factor -
Patent: process for the synthesis of bicyclic nucleic acid analogs via 3-O-protected furanosyl synthons
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ABSTRACT: A process for the prepn. of bicyclic nucleosides is presented such that intermediates I, wherein R is H or an acceptable hydroxyl protecting group; R1 and R2 are independently an (un)substituted C1-C6 alkyl group; and Q is an optionally substituted polycyclic arom. or heteroarom. moiety are characteristic synthons. Specifically, II and III were successfully employed toward the prepn. of bicyclic nucleic acid analog. The bicyclic nucleosides are useful for prepg. chem. modified oligomeric compds. Oligomeric compds. comprising these bicyclic nucleosides have enhanced properties such as increased nuclease resistance. [on SciFinder(R)]Year: 01/2009 -
Patent: Preparation and nuclease resistance of tetrahydropyran nucleic acid analogs
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ABSTRACT: The present disclosure describes tetrahydropyran nucleoside analogs I, wherein: B is a heterocyclic base moiety; R is a hydroxy protecting group; L1 is H, halogen, alkyl; Z1 is O or OE1; Z2 is OH, OE1 or N(E1)(E2); each E1l and E2 is independently alkyl or substituted alkyl; q1-q7 are each, independently, H, alkyl, , alkenyl, alkynyl; wherein each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1,, NJ1J2, SJ1, N3, OC(=X)J1, OC(=X)NJ1J2, NJ3C(=X)NJ1J2 and CN, wherein each J1-J3 is independently, H, alkyl; X is O, S or NJ1, and tetrahydropyran nucleic acid analogs were prepd. Thus, nucleoside II was prepd. and incorporated into tetrahydropyran nucleic acid analogs. More particularly, tetrahydropyran nucleoside analogs are provided, having one or more chiral substituents, that are useful for enhancing properties of oligomeric compds. including nuclease resistance and binding affinity. In some embodiments, the oligomeric compds. provided herein hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA. The use of title oligomeric compds. for the manuf. of a medicament for the treatment of a disease characterized by inhibiting gene expression, is reported. [on SciFinder(R)]Year: 01/2009 -
Article: Potent inhibition of microRNA in vivo without degradation.
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ABSTRACT: Chemically modified antisense oligonucleotides (ASOs) are widely used as a tool to functionalize microRNAs (miRNAs). Reduction of miRNA level after ASO inhibition is commonly reported to show efficacy. Whether this is the most relevant endpoint for measuring miRNA inhibition has not been adequately addressed in the field although it has important implications for evaluating miRNA targeting studies. Using a novel approach to quantitate miRNA levels in the presence of excess ASO, we have discovered that the outcome of miRNA inhibition can vary depending on the chemical modification of the ASO. Although some miRNA inhibitors cause a decrease in mature miRNA levels, we have identified a novel 2'-fluoro/2'-methoxyethyl modified ASO motif with dramatically improved in vivo potency which does not. These studies show there are multiple mechanisms of miRNA inhibition by ASOs and that evaluation of secondary endpoints is crucial for interpreting miRNA inhibition studies.Nucleic Acids Research 12/2008; 37(1):70-7. · 8.03 Impact Factor -
Article: Comparing in vitro and in vivo activity of 2'-O-[2-(methylamino)-2-oxoethyl]- and 2'-O-methoxyethyl-modified antisense oligonucleotides.
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ABSTRACT: A number of 2'- O-modified antisense oligonucleotides have been reported for their potential use in oligonucleotide-based therapeutics. To date, most of the in vivo data has been generated for 2'-O-MOE (2'-O-methoxyethyl)- and 2'-O-Me (2'-O-methyl)-modified ASOs (antisense oligonucleotides). We now report the synthesis and biological activity of another 2'-O-modification, namely 2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA). This modification resulted in an increase in the affinity of antisense oligonucleotides to complementary RNA similar to 2'-O-MOE-modified ASOs as compared to first-generation antisense oligodeoxynucleotides. The ASO modified with 2'-O-NMA reduced expression of PTEN mRNA in vitro and in vivo in a dose-dependent manner similar to 2'-O-MOE modified ASO. Importantly, toxicity parameters such as AST, ALT, organ weights, and body weights were found to be normal similar to 2'-O-MOE ASO-treated animal models. The data generated in these experiments suggest that 2'-O-NMA is a useful modification for potential application in both antisense and other oligonucleotide-based drug discovery efforts.Journal of Medicinal Chemistry 06/2008; 51(9):2766-76. · 5.25 Impact Factor -
Article: 2'-O-Methoxyethyl/2'-Fluoro Modified Oligonucleotides Result in More Potent Inhibition of micro RNA-122 in Vivo: A Target implicatedin HCV Replication.
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ABSTRACT: MicroRNAs are endogenous 20-24 nt small non-codingRNAs that have profound roles in multiple developmental and cellular processes. Dysregulation of microRNAs can lead to a host of pathologies suggesting that microRNAs could be important for therapeutic intervention in cancer, metabolic diseases, autoimmune disorders and viral diseases. Through recent studies,mir-122 has emerged as a potential target for metabolic diseases and HCY. Chemical modifications are essential to achieve clinically relevant potency and efficacy andtherapeutic index of anti-miRNA oligonucleotides. Wehave evaluated more than 65 chemically-modified ASOs for their ability to inhibit the activity of miR-122 inmice. Inhibition of miR-122 with ASOs resulted inincreased levels of miR-122 target gene mRNAs in theliver, as well as lowering of plasma cholesterol in a dose dependant manner. The current investigation led to the identification of a chimeric 2'Fluoro/2'-O-methoxyethyI(2'OME) modified motif with improved efficacy and 5-10 fold improvement in potency compared to LNA/DNAmodified and uniform 2'-MOE-PS compounds. Theseefforts have identified significantly improved anti-miR-122 ASOs for further evaluation as anti-HCV therapeutic agents.Nucleic Acids Symposium Series 02/2008; -
Article: Design, synthesis and evaluation of constrained methoxyethyl (cMOE) and constrained ethyl (cEt) nucleoside analogs.
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ABSTRACT: Antisense drug discovery technology is a powerful method to modulate gene expression in animals and represents a novel therapeutic platform.(1) We have previously demonstrated that replacing 2'O-methoxyethyl (MOE, 2) residues in second generation antisense oligonucleotides (ASOs) with LNA (3) nucleosides improves the potency of some ASOs in animals. However, this was accompanied with a significant increase in the risk for hepatotoxicity.(2) We hypothesized that replacing LNA with novel nucleoside monomers that combine the structural elements of MOE and LNA might mitigate the toxicity of LNA while maintaining potency. To this end we designed and prepared novel nucleoside analogs 4 (S-constrained MOE, S-cMOE) and 5 (R-constrained MOE, R-cMOE) where the ethyl chain of the 2'O-MOE moiety is constrained back to the 4' position of the furanose ring. As part of the SAR series, we also prepared nucleoside analogs 7 (S-constrained ethyl, S-cEt) and 8 (R-constrained Ethyl, R-cEt) where the methoxymethyl group in the cMOE nucleosides was replaced with a methyl substituent. A highly efficient synthesis of the nucleoside phosphoramidites with minimal chromatography purifications was developed starting from cheap commercially available starting materials. Biophysical evaluation revealed that the cMOE and cEt modifications hybridize complementary nucleic acids with the same affinity as LNA while greatly increasing nuclease stability. Biological evaluation of oligonucleotides containing the cMOE and cEt modification in animals indicated that all of them possessed superior potency as compared to second generation MOE ASOs and a greatly improved toxicity profile as compared to LNA.Nucleic Acids Symposium Series 02/2008; -
Article: 2'-Modified oligonucleotides for antisense therapeutics.
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ABSTRACT: Chemically modified antisense oligonucleotides are currently progressing in multiple clinical trials. Among several chemical modifications made, modification of the 2'-position has proved most successful. Second generation antisense oligonucleotides incorporating these 2'-modifications exhibit high binding affinity to target RNA, enhanced metabolic stability, and improved pharmacokinetic and toxicity profiles. This is, in part, due to the enhanced biophysical properties of second generation antisense oligonucleotides. 2'-Modifications that influence the sugar to adopt a 3'-endo sugar pucker can improve properties such as affinity. 2'-Modifications that provide a gauche effect and/or a charge effect can play a significant role in the level of nuclease resistance. The heterocyclic base modifications such as 2-thiothymine provides additive effect on the affinity of 2'-F and 2'-O-MOE modifications. This review summarizes the structural and biophysical properties of selected 2'-modified nucleosides which are candidates for use in oligonucleotide therapeutics.Current topics in medicinal chemistry 02/2007; 7(7):641-9. · 4.47 Impact Factor -
Article: RNA interference by 2',5'-linked nucleic acid duplexes in mammalian cells.
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ABSTRACT: Synthetic small interfering RNA (siRNA) mediated silencing of a specific gene is emerging as a powerful tool for gene regulation. However, their utility is limited for therapeutic applications primarily due to poor stability. The 2',5'-linked oligonucleotides are known to be more stable to nucleolytic degradation than 3',5'-linked oligonucleotides. The 2',5'-linkage is tolerated in the sense strand of the siRNA duplex. However, the 2',5'-linkage is not tolerated in the antisense strand of the siRNA duplex.Bioorganic & Medicinal Chemistry Letters 07/2006; 16(12):3238-40. · 2.55 Impact Factor -
Article: Improving RNA interference in mammalian cells by 4'-thio-modified small interfering RNA (siRNA): effect on siRNA activity and nuclease stability when used in combination with 2'-O-alkyl modifications.
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ABSTRACT: A systematic structure-activity relationship study of 4'-thioribose containing small interfering RNAs (siRNAs) has led to the identification of highly potent and stable antisense constructs. To enable this optimization effort for both in vitro and in vivo applications, we have significantly improved the yields of 4'-thioribonucleosides by using a chirally pure (R)-sulfoxide precursor. siRNA duplexes containing strategically placed regions of 4'-thio-RNA were synthesized and evaluated for RNA interference activity and plasma stability. Stretches of 4'-thio-RNA were well tolerated in both the antisense and sense strands. However, optimization of both the number and placement of 4'-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4'-thioribose with 2'-O-methyl and 2'-O-methoxyethyl modifications. These optimized 4'-thio-siRNAs may be valuable for developing stable siRNAs for therapeutic applications.Journal of Medicinal Chemistry 04/2006; 49(5):1624-34. · 5.25 Impact Factor -
Article: Competition for RISC binding predicts in vitro potency of siRNA.
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ABSTRACT: Short interfering RNAs (siRNA) guide degradation of target RNA by the RNA-induced silencing complex (RISC). The use of siRNA in animals is limited partially due to the short half-life of siRNAs in tissues. Chemically modified siRNAs are necessary that maintain mRNA degradation activity, but are more stable to nucleases. In this study, we utilized alternating 2'-O-methyl and 2'-deoxy-2'-fluoro (OMe/F) chemically modified siRNA targeting PTEN and Eg5. OMe/F-modified siRNA consistently reduced mRNA and protein levels with equal or greater potency and efficacy than unmodified siRNA. We showed that modified siRNAs use the RISC mechanism and lead to cleavage of target mRNA at the same position as unmodified siRNA. We further demonstrated that siRNAs can compete with each other, where highly potent siRNAs can compete with less potent siRNAs, thus limiting the ability of siRNAs with lower potency to mediate mRNA degradation. In contrast, a siRNA with low potency cannot compete with a highly efficient siRNA. We established a correlation between siRNA potency and ability to compete with other siRNAs. Thus, siRNAs that are more potent inhibitors for mRNA destruction have the potential to out-compete less potent siRNAs indicating that the amount of a cellular component, perhaps RISC, limits siRNA activity.Nucleic Acids Research 02/2006; 34(16):4467-76. · 8.03 Impact Factor -
Article: Positional effect of chemical modifications on short interference RNA activity in mammalian cells.
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ABSTRACT: A systematic study on the effect of 2'-sugar modifications (2'-F (2'-F-2'-deoxy-nucleoside residues), 2'-O-Me (2'-O-methyl-nucleoside residues), and 2'-O-MOE [2'-O-(2-methoxyethyl)]-nucleoside residues) in the antisense and sense strands of short interference RNA (siRNA) was performed in HeLa cells. The study of the antisense strand of siRNAs demonstrated that activity depends on the position of the modifications in the sequence. The siRNAs with modified ribonucleotides at the 5'-end of the antisense strand were less active relative to the 3'-modified ones. The 2'-F sugar was generally well-tolerated on the antisense strand, whereas the 2'-O-Me showed significant shift in activity depending on the position of modification. The 2'-O-MOE modification in the antisense strand resulted in less active siRNA constructs regardless of placement position in the construct. The incorporation of the modified residues, e.g., 2'-O-Me and 2'-O-MOE, in the sense strand of siRNA did not show a strong positional preference. These results may provide guidelines to design effective and stable siRNAs for RNA interference mediated therapeutic applications.Journal of Medicinal Chemistry 07/2005; 48(13):4247-53. · 5.25 Impact Factor
Top co-authors
Top Journals
Institutions
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1997–2012
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Isis Pharmaceuticals, Inc.
Carlsbad, CA, USA
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2011
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Vanderbilt University
- Department of Biochemistry
Nashville, MI, USA
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2003
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Merck
Whitehouse Station, NJ, USA
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