Streamlined Process for the Chemical Synthesis of RNA Using 2′-<i>O</i>-Thionocarbamate-Protected Nucleoside Phosphoramidites in the Solid Phase

Dellinger, Douglas J.; Tímár, Zoltán; Myerson, Joel; Sierzchala, Agnieszka; Turner, John V.; Ferreira, Fernando; Kupihár, Zoltán; Dellinger, Geraldine; Hill, K. W.; Powell, James A.; Sampson, Jeffrey R.; Caruthers, Marvin H.

doi:10.1021/ja201561z

Cited by 65 publications

(56 citation statements)

References 47 publications

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“…Six GAC RNAs constructs were chemically synthesized[30] by Agilent Labs, each with a single 2AP substitution. Three substitutions were placed in the 5′ hairpin loop (A1067AP, A1069AP, and A1070AP) which, as shown in the cocrystal[2], arranges the first five bases (U1065 to A1069) in a U-turn conformation and extrudes the 3′ three bases (A1070/G1071/C1072) to create what we refer to here as a T+loop[31],[32].…”

Section: Resultsmentioning

confidence: 99%

Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding

Welty

Hall

2016

Journal of Molecular Biology

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The tertiary structure of the GTPase Center (GAC) of 23S rRNA as seen in cocrystals is extremely compact. It is stabilized by long-range hydrogen bonds and nucleobase stacking, and a triloop that forms within its 3-way junction. Its folding pathway from secondary structure to tertiary structure has not been previously observed, but it was shown in equilibrium experiments to require Mg2+ ions. The fluorescent nucleotide 2-aminopurine was substituted at selected sites within the 60 nucleotide GAC. Fluorescence intensity changes upon addition of MgCl2 were monitored over a time-course from 1 ms to 100 s as the RNA folds. The folding pathway is revealed here to be hierarchical through several intermediates. Observation of the nucleobases during folding provides a new perspective on the process and the pathway, revealing the dynamics of nucleobase conformational exchange during the folding transitions.

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Section: Resultsmentioning

confidence: 99%

Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding

Welty

Hall

2016

Journal of Molecular Biology

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“…RNAs were either transcribed using T7 RNA polymerase via run-off transcription from a plasmid[47] or chemically synthesized by Agilent labs [48] to insert 2-aminopurine (2AP) or 15 N-nucleotides. All RNAs in solution were dialyzed against 0.5 M EDTA, then two or three times against MilliQ water with the final exchange overnight.…”

Section: Methodsmentioning

confidence: 99%

Formation of Tertiary Interactions during rRNA GTPase Center Folding

Rau

Welty

Stump

et al. 2015

Journal of Molecular Biology

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The 60 nucleotide GTPase center (GAC) of 23S rRNA has a phylogenetically conserved secondary structure with two hairpin loops and a 3-way junction. It folds into an intricate tertiary structure upon addition of Mg2+ ions, which is stabilized by the L11 protein in cocrystal structures. Here we monitor the kinetics of its tertiary folding and Mg2+-dependent intermediate states by observation of selected nucleobases that contribute specific interactions to the GAC tertiary structure in the cocrystals. The fluorescent nucleobase 2-aminopurine (2AP) replaced three individual adenines, two of which make long-range stacking interactions, and one that also forms hydrogen bonds. Each site reveals a unique response to Mg2+ addition and temperature, reflecting its environmental change from secondary to tertiary structure. Stopped-flow fluorescence experiments revealed that kinetics of tertiary structure formation upon addition of MgCl2 are also site-specific, with local conformational changes occurring from 5 ms – 4 s, and with global folding from 1- 5 s. Site-specific substitution with 15N-nucleobases allowed observation of stable hydrogen bond formation by NMR experiments. Equilibrium titration experiments indicate that a stable folding intermediate is present at stoichiometric concentrations of Mg2+, and suggest that there are two initial sites of Mg2+ ion association.

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“…[10][11][12] The nucleoside building blocks require suitable protection of nucleobase amino and ribose 2′-hydroxy groups. Whereas the former are usually acyl protected, silyl protection (tBDMS or TOM) [13,14] of the latter are most widely applied, and orthoesters (ACE), [15] 2-cyanoethoxymethyl (CEM), [16] or thiocarbamates (TC) [17] also represent powerful alternatives. For all five approaches, the syntheses of the standard nucleoside phosphoramidites (A, C, G, U) have been very well optimized and are robust.…”

Section: Introductionmentioning

confidence: 99%

An Unconventional Acid‐Labile Nucleobase Protection Concept for Guanosine Phosphoramidites in RNA Solid‐Phase Synthesis

Jud

Micura

2017

Chemistry A European J

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We present an innovative O 6 -tert-butyl/N 2 -tert-butyloxycarbonyl protection concept for guanosine (G) phosphoramidites. This concept is advantageous for 2′-modified G building blocks because of very efficient synthetic access when compared with existing routes that usually employ O 6 -(4-nitrophenyl)ethyl/N 2 -acyl protection or that start from 2-aminoadenosine involving enzymatic transformation into guanosine later on in the synthetic path. The new phosphoramidites are fully compatible with 2′-O-tBDMS or TOM phosphoramidites in standard RNA solid-phase synthesis and deprotection, and provide excellent quality of tailored RNAs for the growing range of applications in RNA biophysics, biochemistry, and biology.

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Streamlined Process for the Chemical Synthesis of RNA Using 2′-O-Thionocarbamate-Protected Nucleoside Phosphoramidites in the Solid Phase

Cited by 65 publications

References 47 publications

Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding

Nucleobases Undergo Dynamic Rearrangements during RNA Tertiary Folding

Formation of Tertiary Interactions during rRNA GTPase Center Folding

An Unconventional Acid‐Labile Nucleobase Protection Concept for Guanosine Phosphoramidites in RNA Solid‐Phase Synthesis

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