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/ange.201304986

Cited by 14 publications

(24 citation statements)

References 48 publications

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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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Section: Resultsmentioning

confidence: 99%

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

et al. 2013

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“…The psoralen‐labelled U6 donor was therefore prepared by reaction of psoralen azide with the fully deprotected phosphorylated donor in solution. Cy3‐labeled ACA45 and biotin‐U6 acceptors were prepared in a straightforward manner by conjugation of the labels on solid support, as previously described . The two pairs of labelled RNA were then ligated following the protocol reported herein to yield Cy5/Cy3 ACA45 (ORN‐6) and biotin/psoralen U6 (ORN‐8) with good purity (>80 %).…”

Section: Figurementioning

confidence: 99%

“…Hence, we prepared ACA45 and U6, bis‐labelled with Cy5/Cy3 and biotin/trioxsalen (psoralen), respectively (Figure ). We used our previously optimized methods for site‐specific labelling, whereby groups are conjugated to 2′‐ O ‐propargyl nucleosides either on solid support or in solution …”

Section: Figurementioning

confidence: 99%

“…Hence, we prepared ACA45 and U6, bis-labelled with Cy5/Cy3 and biotin/trioxsalen (psoralen), respectively ( Figure 2). We used our previously optimizedm ethods for site-specific labelling, whereby groups are conjugated to 2'-O-propargyl nucleosides either on solid support [20] or in solution. [18] The target RNA was constructed from two smaller RNAs of similar lengths, one of which was5 '-phosphorylated (the donor).…”

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confidence: 99%

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Chemical synthesis of long RNAs with terminal 5′‐phosphate groups

Pradère

Halloy

Hall

2017

Chemistry A European J

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Long structured RNAs are useful biochemical and biological tools. They are usually prepared enzymatically, but this precludes their site‐specific modification with functional groups for chemical biology studies. One solution is to perform solid‐phase synthesis of multiple RNAs loaded with 5′‐terminal phosphate groups, so that RNAs can be concatenated using template ligation reactions. However, there are currently no readily available reagents suitable for the incorporation of the phosphate group into long RNAs by solid‐phase synthesis. Here we describe an easy‐to‐prepare phosphoramidite reagent suitable for the chemical introduction of 5′‐terminal phosphate groups into long RNAs. The phosphate is protected by a dinitrobenzhydryl group that serves as an essential lipophilic group for the separation of oligonucleotide by‐products. The phosphate is unmasked quantitatively by brief UV irradiation. We demonstrate the value of this reagent in the preparation of a library of long structured RNAs that are site‐specifically modified with functional groups.

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“…Therefore, to monitor the processing intermediates that are generated in the miRNA maturation step, the modifications of the dyes in pre-miRNA are required. Although the hetero-labeling of functional groups on RNA utilizing click-based methods have been attempted, there are still setbacks, including low yields and complicated preparation steps [ 29 , 30 , 31 ]. Here, we developed FRET-type pre-miRNAs by the D-threoninol technology [ 32 , 33 ].…”

Section: Introductionmentioning

confidence: 99%

Development and Modification of Pre-miRNAs with a FRET Dye Pair for the Intracellular Visualization of Processing Intermediates That Are Generated in Cells

Kamimoto

Zhu

Asanuma

2021

Sensors

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microRNAs (miRNAs) are small non-coding ribonucleic acids (RNAs), which regulate gene expression via the RNA interference (RNAi) system. miRNAs have attracted enormous interest because of their biological significance and disease relationship. In cell systems, the generation of miRNA is regulated by multiple steps: the transfer of primary miRNA from the nucleus to the cytosol, the generation of the precursor-miRNA (pre-miRNA), the production of double-stranded RNA from pre-miRNA by the Dicer, the interaction with protein argonaute-2 (AGO2), and the subsequent release of one strand to form miRISC with AGO2. In this study, we attempt to visualize the intermediates that were generated in the miRNA-maturation step in the cells to acquire a detailed understanding of the maturation process of miRNA. To achieve this, we developed pre-miRNAs labeling with a Dicer- or AGO2-responsible fluorescence resonance energy transfer (FRET) dye pair. We observed that modifications with the dye at suitable positions did not interfere with the biological activities of pre-miRNAs. Further, imaging analyses employing these pre-miRNAs demonstrated that the processing of pre-miRNA promoted the accumulation of miRNA at the specific foci in the cytosol. The FRET-labeled pre-miRNA would further elucidate the mechanisms of the RNAi process and provide the basis for development of nucleic acid drugs working in the RNAi system.

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

Cited by 14 publications

References 48 publications

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

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

Chemical synthesis of long RNAs with terminal 5′‐phosphate groups

Development and Modification of Pre-miRNAs with a FRET Dye Pair for the Intracellular Visualization of Processing Intermediates That Are Generated in Cells

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