Synthesis of atom-specific nucleobase and ribose labeled uridine phosphoramidite for NMR analysis of large RNAs

Olenginski, Lukasz T.; Becette, Owen B.; Beaucage, Serge L.; Dayie, T. Kwaku

doi:10.1007/s00706-021-02851-2

Cited by 3 publications

(5 citation statements)

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“…In principle, any nucleobase labeling scheme described in Section 3.1 can be coupled to any commercially available 13 C-or 2 H-labeled D-ribose (from Omicron Biochemicals or CIL) with the chemo-enzymatic method (Section 3.2) and built into an amidite with a variety of 2′-OH protecting groups (Section 3.3). Indeed, our group recently made [1′,8-13 C 2 ]-N 6 -Bz-adenosine 2′-O-tBDMS 144 and [1′,6-13 C 2 , 5-2 H]-uridine 2′-O-CEM 153 amidites via chemo-enzymatic synthesis, dephosphorylation with rSAP, and chemical synthesis. These amidites can then be used to make RNA via solid-phase synthesis.…”

Section: Synthesis Of Labeled Rna Phosphoramiditesmentioning

confidence: 99%

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

2022

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RNA is central to the proper function of cellular processes important for life on earth and implicated in various medical dysfunctions. Yet, RNA structural biology lags significantly behind that of proteins, limiting mechanistic understanding of RNA chemical biology. Fortunately, solution NMR spectroscopy can probe the structural dynamics of RNA in solution at atomic resolution, opening the door to their functional understanding. However, NMR analysis of RNA, with only four unique ribonucleotide building blocks, suffers from spectral crowding and broad linewidths, especially as RNAs grow in size. One effective strategy to overcome these challenges is to introduce NMR-active stable isotopes into RNA. However, traditional uniform labeling methods introduce scalar and dipolar couplings that complicate the implementation and analysis of NMR measurements. This challenge can be circumvented with selective isotope labeling. In this review, we outline the development of labeling technologies and their application to study biologically relevant RNAs and their complexes ranging in size from 5 to 300 kDa by NMR spectroscopy.

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Section: Synthesis Of Labeled Rna Phosphoramiditesmentioning

confidence: 99%

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

2022

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“…This problem can usually be circumvented if N 4 -acetyl instead of benzoyl protection is applied [ 22 , 23 ]. Moreover, for 2′-O protection we envisaged both 2′- O -TBDMS (to satisfy the most widespread concept of commercially available nucleoside building blocks) [ 24 – 27 ] and 2′- O -TOM protection [ 28 – 31 ]. The latter is conducive for the solid-phase synthesis of long RNAs (> 50–60 nt) due to reduced steric hindrance during the coupling step and hence very high coupling yields [ 28 , 29 ].…”

Section: Introductionmentioning

confidence: 99%

Synthesis of N4-acetylated 3-methylcytidine phosphoramidites for RNA solid-phase synthesis

2022

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The growing interest in 3-methylcytidine (m3C) originates from the recent discoveries of m3C modified tRNAs in humans as well as its intensively debated occurrence in mRNA. Moreover, m3C formation can be catalyzed by RNA without the assistance of proteins as has been demonstrated for a naturally occurring riboswitch fold using the methylated form of its cognate ligand as cofactor. Additionally, new RNA sequencing methods have been developed to detect this modification in transcriptome-wide manner. For all these reasons, an increasing demand for synthetic m3C containing oligoribonucleotides is emerging. Their chemical synthesis relies on RNA solid-phase synthesis using phosphoramidite building blocks. Here, we describe a facile synthetic path towards N4-acetylated 2′-O-TBDMS- and 2′-O-TOM m3C phosphoramidites to provide an optimal toolbox for solid-phase synthesis of m3C containing RNA. Graphical abstract

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“…Figures and some textual materials have been taken from work recently published in the literature (Olenginski, Becette, Beaucage, & Dayie, 2021) and adapted into step‐by‐step protocols to support facile reproduction of the original work.…”

Section: Introductionmentioning

confidence: 99%

“…This is commonly accomplished with T7 RNA polymerase-based in vitro transcription (Milligan & Uhlenbeck, 1989) using unlabeled and isotope-labeled ribonucleoside 5 -triphosphates (rNTPs). Some of these isotope-labeled rNTP precursors are commercially available (e.g., Cambridge Isotope Laboratories and Silantes) and the synthesis of many more exist in the Figures and some textual materials have been taken from work recently published in the literature (Olenginski, Becette, Beaucage, & Dayie, 2021) and adapted into step-by-step protocols to support facile reproduction of the original work.…”

Section: Introductionmentioning

confidence: 99%

“…Abbreviations: RK, ribokinase; dATP, deoxyadenosine 5 -triphosphate; dADP, deoxyadenosine 5 -diphosphate; dAMP, deoxyadenosine 5monophosphate; PRPPS, phosphoribosyl pyrophosphate synthetase; UPRT, uracil phosphoribosyl transferase; PPi, inorganic pyrophosphate; rSAP, recombinant shrimp alkaline phosphatase; NaPi, sodium inorganic phosphate. Adapted from Olenginski et al (2021).…”

Section: Introductionmentioning

confidence: 99%

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Synthesis of an Atom‐Specifically ²H/¹³C‐Labeled Uridine Ribonucleoside Phosphoramidite

et al. 2022

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A combined enzymatic and chemical synthesis of a 2 -O-cyanoethoxymethyl (CEM) protected [1 ,6-13 C 2 , 5-2 H]-uridine phosphoramidite is described herein. This is the first report of an atom-specific nucleobase and ribose labeled 2 -O-CEM protected ribonucleoside phosphoramidite. Importantly, the CEM 2 -OH protecting group permits the efficient solid-phase synthesis of large (>60 nucleotides) RNAs with good yield and purity. The new isotopelabeled phosphoramidite can therefore be applied to nuclear magnetic resonance (NMR) spectroscopy studies. Specifically, the [1 ,6-13 C 2 , 5-2 H]-uridine phosphoramidite can be used to make position-specifically labeled RNAs for NMR analysis without complications from resonance overlap and scalar and dipolar couplings.

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Synthesis of atom-specific nucleobase and ribose labeled uridine phosphoramidite for NMR analysis of large RNAs

Cited by 3 publications

References 38 publications

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

Synthesis of N4-acetylated 3-methylcytidine phosphoramidites for RNA solid-phase synthesis

Synthesis of an Atom‐Specifically ²H/¹³C‐Labeled Uridine Ribonucleoside Phosphoramidite

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Synthesis of atom-specific nucleobase and ribose labeled uridine phosphoramidite for NMR analysis of large RNAs

Cited by 3 publications

References 38 publications

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

Isotope Labels Combined with Solution NMR Spectroscopy Make Visible the Invisible Conformations of Small-to-Large RNAs

Synthesis of N4-acetylated 3-methylcytidine phosphoramidites for RNA solid-phase synthesis

Synthesis of an Atom‐Specifically 2H/13C‐Labeled Uridine Ribonucleoside Phosphoramidite

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Synthesis of an Atom‐Specifically ²H/¹³C‐Labeled Uridine Ribonucleoside Phosphoramidite