Chemical Synthesis of Mono‐ and Bis‐Labeled Pre‐MicroRNAs

Pradère, Ugo; Brunschweiger, Andreas; Gebert, Luca F. R.; Lucic, Matije; Roos, Martina; Hall, Jonathan

doi:10.1002/anie.201304986

Cited by 32 publications

(49 citation statements)

References 48 publications

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“…We then transfected syn-pre-34a into HeLa cells. We have shown recently that a Cy3-labeled pre-miRNA is efficiently delivered into HeLa cells using the same protocol as for miRNA mimics (Pradère et al 2013). We detected a concentration-dependent production of miR-34a-5p and miR-34a-3p strands by stem-loop quantitative real time PCR (qPCR) and Northern blotting (Fig.…”

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

confidence: 90%

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: 90%

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

Guennewig

Roos

Dogar

et al. 2013

RNA

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“…All other oligoribonucleotides were synthesized via standard solid-phase synthesis using 2΄- O -TBDMS phosphoramidites on a 50 nmol scale (27). The dimethylamino group of oligoribonucleotides 2585_DMA-Py and 2586_DMA-Py was introduced using the convertible nucleoside approach as described previously (28,29).…”

Section: Methodsmentioning

confidence: 99%

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain

Koch

Willi

Pradère

et al. 2017

Nucleic Acids Research

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The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and co-translational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson–Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation.

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“…24 Here, we intended to create a fast and simple access to azide labeled RNA even if restrictions with respect to positioning of the azide group were encountered. For many applications, in particular, for multiple, specific labeling of DNA 25,26 or RNA, 8,9,12 3′-end azide anchors would be a major asset, provided the approach is facile and applicable to standard phosphoramidite chemistry.…”

Section: Resultsmentioning

confidence: 99%

Efficient Access to 3′-Terminal Azide-Modified RNA for Inverse Click-Labeling Patterns

et al. 2013

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Labeled RNA becomes increasingly important for molecular diagnostics and biophysical studies on RNA with its diverse interaction partners, which range from small metabolites to large macromolecular assemblies, such as the ribosome. Here, we introduce a fast synthesis path to 3′-terminal 2′-O-(2-azidoethyl) modified oligoribonucleotides for subsequent bioconjugation, as exemplified by fluorescent labeling via Click chemistry for an siRNA targeting the brain acid-soluble protein 1 gene (BASP1). Importantly, the functional group pattern is inverse to commonly encountered alkyne-functionalized “click”-able RNA and offers increased flexibility with respect to multiple and stepwise labeling of the same RNA molecule. Additionally, our route opens up a minimal step synthesis of 2′-O-(2-aminoethyl) modified pyrimidine nucleoside phosphoramidites which are of widespread use to generate amino-modified RNA for N-hydroxysuccinimide (NHS) ester-based conjugations.

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Chemical Synthesis of Mono‐ and Bis‐Labeled Pre‐MicroRNAs

Cited by 32 publications

References 48 publications

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

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

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain

Efficient Access to 3′-Terminal Azide-Modified RNA for Inverse Click-Labeling Patterns

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