[1] Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis

Scaringe, Stephen A.

doi:10.1016/s0076-6879(00)17003-x

Cited by 56 publications

(31 citation statements)

References 18 publications

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“…Double-stranded RNAs (dsRNAs) targeting the diazepam binding inhibitor (DBI, NM_020548) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, NM_002046), and all other genes were synthesized using 29-ACE chemistry (Scaringe 2000) at Dharmacon, Inc. For the experiments showing dsRNAs of different lengths, the dsRNA molecules contained a duplex region (e.g., 19, 21, 23, 25, 27, 29, or 31 bp) and two nucleotide 39 overhangs on their sense strands. The duplexes were designed by extending sequence from Cold Spring Harbor Laboratory Press on April 28, 2019 -Published by rnajournal.cshlp.org Downloaded from the 59 end of the sense strand, thus preserving the thermodynamic stability and targeting region (or motif) of the 59 AS end of the molecule.…”

Section: Rnas Genes and Cell Linesmentioning

confidence: 99%

Induction of the interferon response by siRNA is cell type– and duplex length–dependent

Reynolds¹,

Anderson²,

Vermeulen³

et al. 2006

RNA

293

197

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Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell typespecific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.

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Section: Rnas Genes and Cell Linesmentioning

confidence: 99%

Induction of the interferon response by siRNA is cell type– and duplex length–dependent

Reynolds¹,

Anderson²,

Vermeulen³

et al. 2006

RNA

293

197

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“…The 59 substrate strand used for ligation was transcribed in vitro using T7 RNA polymerase. All other RNA strands were synthesized using 29-ACE-protected phosphoramidites (Scaringe 2000). Fully deprotected oligonucleotides were purified by electrophoresis in 20% polyacrylamide gels containing 7 M urea and recovered by electroelution.…”

Section: Synthesis Of Rnamentioning

confidence: 99%

Nucleobase catalysis in the hairpin ribozyme

Wilson

Ouellet²,

Zhong³

et al. 2006

RNA

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RNA catalysis is important in the processing and translation of RNA molecules, yet the mechanisms of catalysis are still unclear in most cases. We have studied the role of nucleobase catalysis in the hairpin ribozyme, where the scissile phosphate is juxtaposed between guanine and adenine bases. We show that a modified ribozyme in which guanine 8 has been substituted by an imidazole base is active in both cleavage and ligation, with ligation rates 10-fold faster than cleavage. The rates of both reactions exhibit bell-shaped dependence on pH, with pK a values of 5.7 6 0.1 and 7.7 6 0.1 for cleavage and 6.1 6 0.3 and 6.9 6 0.3 for ligation. The data provide good evidence for general acid-base catalysis by the nucleobases.

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“…Other oligoribonucleotides were synthesized using ACE chemistry (Scaringe 2000) implemented on a modified 394 synthesizer (Applied Biosystems). Phosphoramidites (Dharmacon) were supplied in the form of oils, and were diluted to a concentration of 0.1 M in dry acetonitrile (Glen Research) and filtered through a 0.45-mm filter (Aldrich) before use.…”

Section: Preparation Of a Fulgidus L7ae Proteinmentioning

confidence: 99%

Induced fit of RNA on binding the L7Ae protein to the kink-turn motif

Turner¹,

Melcher²,

Wilson³

et al. 2005

RNA

101

100

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The kink-turn is a widespread motif in RNA consisting of a three-nucleotide bulge flanked on one side by consecutive AÁG mismatches. Important examples are found in the ribosome, U4 RNA, and in snoRNAs involved in RNA modification. The motif is a common protein binding site, and the RNA has been found to adopt a tightly kinked conformation in crystal structures. However, in free solution there is a dynamic exchange between kinked and extended conformations, with the equilibrium driven toward the kinked form by the addition of metal ions. Here we used fluorescence resonance energy transfer (FRET) to show that the L7Ae protein of Archaeoglobus fulgidus binds to RNA containing a kink-turn with nanomolar affinity, and induces folding into the tightly kinked conformation even in the absence of metal ions. Thus this RNA may act as a relatively flexible hinge during RNA folding, until fixed into its ultimate kinked structure by the binding of L7 or related protein.

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[1] Advanced 5′-silyl-2′-orthoester approach to RNA oligonucleotide synthesis

Cited by 56 publications

References 18 publications

Induction of the interferon response by siRNA is cell type– and duplex length–dependent

Induction of the interferon response by siRNA is cell type– and duplex length–dependent

Nucleobase catalysis in the hairpin ribozyme

Induced fit of RNA on binding the L7Ae protein to the kink-turn motif

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