Recent Advances in the Chemical Synthesis of RNA

Beaucage, Serge L.; Reese, Colin B.

doi:10.1002/0471142700.nc0216s38

Cited by 41 publications

(33 citation statements)

References 96 publications

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“…Synthesis was performed on the Gene World DNA synthesizer under the conditions recommended by the manufacturer. Oligonucleotides were cleaved from the solid support as 5′-DMT-derivatives, then deprotected and purified according to the described procedure [48]. Support-bound oligonucleotides were treated with 33% ethanolic methylamine (Sigma-Aldrich) and DMSO 1 : 1 (v : v) mixture at 65°C for 15 min and then with triethylamine-trihydrofluoride (Sigma-Aldrich) at 65°C for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

Sierant

Paduszynska

Kaźmierczak-Barańska

et al. 2011

International Journal of Alzheimer's Disease

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RNA interference (RNAi) technology provides a powerful molecular tool to reduce an expression of selected genes in eukaryotic cells. Short interfering RNAs (siRNAs) are the effector molecules that trigger RNAi. Here, we describe siRNAs that discriminate between the wild type and mutant (1174 C→G) alleles of human Presenilin1 gene (PSEN1). This mutation, resulting in L392V PSEN1 variant, contributes to early onset familial Alzheimer's disease. Using the dual fluorescence assay, flow cytometry and fluorescent microscopy we identified positions 8th–11th, within the central part of the antisense strand, as the most sensitive to mismatches. 2-Thiouridine chemical modification introduced at the 3′-end of the antisense strand improved the allele discrimination, but wobble base pairing adjacent to the mutation site abolished the siRNA activity. Our data indicate that siRNAs can be designed to discriminate between the wild type and mutant alleles of genes that differ by just a single nucleotide.

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Section: Methodsmentioning

confidence: 99%

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

Sierant

Paduszynska

Kaźmierczak-Barańska

et al. 2011

International Journal of Alzheimer's Disease

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“…A universally active heavily modified siRNA design has thus far proven elusive but remains an attractive goal. We have argued that siRNA designs comprising combinations of chemical modifications are more likely to achieve this goal (3,4). …”

Section: Introductionmentioning

confidence: 99%

“…A wide variety of nucleotide modifications can enhance potency, improve serum stability and reduce OTEs in certain siRNA sequence contexts (3,4). …”

Section: Introductionmentioning

confidence: 99%

Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing

Deleavey¹,

Watts²,

Alain³

et al. 2010

Nucleic Acids Research

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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.

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“…The absence of a vicinal hydroxyl function in 4 precluded the notorious migration of the TBDMS group (26) and formation of any isomeric side product when using this reagent. Phosphitylation of the propargylated diribonucleoside phosphate triester 6 when employing only one molar equivalent of commercial 2-cyanoethyl tetraisopropylphosphordiamidite led to the formation of 10 , for the first time through exclusively phosphoramidite chemistry.…”

Section: Resultsmentioning

confidence: 99%

Convenient Synthesis of a Propargylated Cyclic (3′-5′) Diguanylic Acid and Its “Click” Conjugation to a Biotinylated Azide

et al. 2010

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The ribonucleoside building block, N 2 -isobutyryl-2′-O-propargyl-3′-O-levulinyl guanosine (Scheme 1), was prepared from commercial N 2 -isobutyryl-5′-O-(4,4′-dimethoxytrityl)-2′-Opropargyl guanosine in a yield of 91%. The propargylated guanylyl(3′-5′)guanosine phosphotriester shown in Scheme 2 was synthesized from the reaction ofguanosine and isolated in a yield of 88% after P(III) oxidation, 3′-/5′-deprotection and purification. The propargylated guanylyl(3′-5′)guanosine phosphotriester was phosphitylated using 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole and was followed by an in situ intramolecular cyclization to give a propargylated c-di-GMP triester (Scheme 3), which was isolated in a yield of 40% after P(III) oxidation and purification. Complete N-deacylation of the guanine bases and removal of the 2-cyanoethyl phosphate protecting groups from the propargylated c-di-GMP triester were performed by treatment with aqueous ammonia at ambient temperature. The final 2′-desilylation reaction was effected by exposure to triethylammonium trihydrofluoride to give the desired propargylated c-di-GMP diester, the purity of which exceeded 95% ( Figure 2B). Biotinylation of the propargylated c-di-GMP diester was easily accomplished through its cycloaddition reaction with a biotinylated azide derivative (Scheme 4) under click conditions to produce the biotinylated c-di-GMP conjugate of interest in an isolated yield of 62%.

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Recent Advances in the Chemical Synthesis of RNA

Cited by 41 publications

References 96 publications

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

Specific Silencing of L392V PSEN1 Mutant Allele by RNA Interference

Synergistic effects between analogs of DNA and RNA improve the potency of siRNA-mediated gene silencing

Convenient Synthesis of a Propargylated Cyclic (3′-5′) Diguanylic Acid and Its “Click” Conjugation to a Biotinylated Azide

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