Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2′-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate

Shiba, Yoshinobu; Masuda, Hirofumi; Watanabe, Naoki; Ego, Takeshi; Takagaki, Kazuchika; Ishiyama, Koichi; Ohgi, Tadaaki; Yano, Junichi

doi:10.1093/nar/gkm202

Cited by 89 publications

(75 citation statements)

References 34 publications

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“…Given these limitations, we turned to a chemical synthesis of pre-miRNAs (syn-premiRNA). The length of human premiRNAs (50-90 nt) is challenging for routine laboratory synthesis (Shiba et al 2007). Consequently, reports of the use of syn-pre-miRNAs as miRNA mimics are rare (see, e.g., Macrae et al 2008;Koscianska et al 2011;Starega-Roslan et al 2011) (see Siolas et al 2005 for a synthesis of shRNAs).…”

Section: Tgf-β1 Rnai Induces Mir-34a-5p and Mir-34a-3pmentioning

confidence: 99%

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α

Guennewig

Roos

Dogar

et al. 2013

RNA

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Functional microRNAs (miRNAs) are produced from both arms of their precursors (pre-miRNAs). Their abundances vary in contextdependent fashion spatiotemporarily and there is mounting evidence of regulatory interplay between them. Here, we introduce chemically synthesized pre-miRNAs (syn-pre-miRNAs) as a general class of accessible, easily transfectable mimics of premiRNAs. These are RNA hairpins, identical in sequence to natural pre-miRNAs. They differ from commercially available miRNA mimics through their complete hairpin structure, including any regulatory elements in their terminal-loop regions and their potential to introduce both strands into RISC. They are distinguished from transcribed pre-miRNAs by their terminal 5′ hydroxyl groups and their precisely defined terminal nucleotides. We demonstrate with several examples how they fully recapitulate the properties of pre-miRNAs, including their processing by Dicer into functionally active 5p-and 3p-derived mature miRNAs. We use syn-pre-miRNAs to show that miR-34a uses its 5p and 3p miRNAs in two pathways: apoptosis during TGF-β signaling, where SIRT1 and SP4 are suppressed by miR-34a-5p and miR-34a-3p, respectively; and the lipopolysaccharide (LPS)-activation of primary human monocyte-derived macrophages, where TNF (TNFα) is suppressed by miR-34a-5p indirectly and miR-34a-3p directly. Our results add to growing evidence that the use of both arms of a miRNA may be a widely used mechanism. We further suggest that syn-pre-miRNAs are ideal and affordable tools to investigate these mechanisms.

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Section: Tgf-β1 Rnai Induces Mir-34a-5p and Mir-34a-3pmentioning

confidence: 99%

Synthetic pre-microRNAs reveal dual-strand activity of miR-34a on TNF-α

Guennewig

Roos

Dogar

et al. 2013

RNA

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“…9B). We found that substitution of acetic anhydride with phenoxyacetic anhydride (59) eliminates this artifact as shown in Fig. 9C.…”

Section: A Potential Side Reaction During Oligonucleotide Synthesis Imentioning

confidence: 62%

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cui

Theruvathu

Farrel

et al. 2008

Analytical Biochemistry

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Oligonucleotides containing modified bases are commonly used for biochemical and biophysical studies to assess the impact of specific types of structural damage on DNA structure and function. In contrast to the synthesis of oligonucleotides with normal DNA bases, oligonucleotide synthesis with modified bases often requires modified synthetic or deprotection conditions. Furthermore, several modified bases of biological interest are prone to further damage during synthesis and oligonucleotide isolation. In this paper, we describe the application of MALDI-TOF-MS to the characterization of a series of modified synthetic oligonucleotides. The potential for and limits in obtaining high mass accuracy for confirming oligonucleotide composition are discussed. Examination of the isotope cluster is also proposed as a method for confirming oligonucleotide elemental composition. MALDI-TOF-MS analysis of the unpurified reaction mixture can be used to confirm synthetic sequence and to reveal potential problems during synthesis. Analysis during and after purification can yield important information on depurination and base oxidation. It can also reveal unexpected problems that can occur with non-standard synthesis, deprotection or purification strategies. Proper characterization of modified oligonucleotides is essential for the correct interpretation of experiments performed with these substrates, and MALDI-TOF-MS analysis provides a simple yet extensive method of characterization that can be used at multiple stages of oligonucleotide production and utilization.

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“…The phosphoramidite method is currently the most efficient method for the synthesis of oligonucleotides, and can produce long oligomers using an automatic synthesizer (62,63). However, the application of this method to the synthesis of glycosyl phosphate derivatives has not been well explored (64)(65)(66)(67)(68)(69).…”

Section: F Synthesis Of Glycosyl Phosphate Derivatives Using the Phomentioning

confidence: 99%