Chemical synthesis of a biologically active natural tRNA with its minor bases

Gasparutto, Didier; Livache, Thierry; Hervé, Bazin; Duplaa, A.M.; Guy, A.; Khorlin, Alexander; Molko, D.; Roget, André; Téoule, R.

doi:10.1093/nar/20.19.5159

Cited by 137 publications

(87 citation statements)

References 28 publications

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“…However, no hydrolysis products were detected when slow-migrating oligomer was treated with 80% aqueous acetic acid for 4 hours at ambient temperature. In contrast, upon treatment with neat triethylamine trihydrofluoride for 1.5 hours at 65°C, conditions that are routinely used to remove 2Ј-O-tertbutyldimethylsilyl protecting groups during oligoribonucleotide synthesis (Gasparutto et al, 1992), specific and nearly quantitative cleavage of the phosphoramidate linkage occurred (Guerlavais Dagland et al, 2003). Two fragments were produced (Fig.…”

Section: Results and Dicussionmentioning

confidence: 99%

Formation of N-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

2006

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During the synthesis of oligonucleotides by the standard phosphoramidite method using 2-deoxycytidine-derivatized solid support, a side reaction was observed that gave rise to the formation of high molecular weight N-branched oligomers having two identical chains linked to the 3-terminal 2-deoxycytidine. Postsynthesis treatment with neat triethylamine trihydrofluoride selectively cleaved the phosphoramidate linkage and converted the N-branched oligomers back to the expected oligonucleotides.

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Section: Results and Dicussionmentioning

confidence: 99%

Formation of N-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

2006

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“…An excess of Bu4NF is commonly employed to ensure complete cleavage of the 2'-O-TBDMS groups, but this makes purification of the deprotected RNA difficult and time-consuming. A better reagent for the cleavage of TBDMS appears to be TEA.3HF (31,32). In N-methylpyrrolidone as solvent (30), this reagent gives complete deprotection in 1.5 h at 65°C.…”

Section: Introductionmentioning

confidence: 99%

“…However, the use of 5-ethylthio-lH-tetrazole as the activator allows the coupling time to be reduced to 5 min (30). Alternatively, the coupling time can be reduced to 2 min if nucleoside N-ethyl-N-methyl phosphoramidites are used (31).…”

Section: Introductionmentioning

confidence: 99%

A new 2′-hydroxyl protecting group for the automated synthesis of oligoribonucleotides

Rastogi

Usher

1995

Nucl Acids Res

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We have developed a new type of 2'-hydroxyl protecting group for the automated machine synthesis of RNA oligomers: a 2-hydroxyisophthalate formaldehyde acetal (HIFA). The unique feature of this protecting group is that, as the bis ester, it is relatively stable to the acidic conditions that are used for repeated removal of dimethoxytrityl groups during chain elongation, but the final deprotection step in alkali, which cleaves the chain from the support and removes the base and phosphate protecting groups, converts it to the bis carboxylate and this can be removed relatively rapidly by treatment with mild acid. Conversion of the bis ester to the bis carboxylic acid increases the rate of acid-catalyzed hydrolysis' of the acetal by 42-fold at pH 1, and, possibly, by 1320-fold at pH 3. The bis ester is 112 times more stable than the 1 -(2-fluorophenyl)-4-methoxypiperidin-4-yl group (Fpmp) towards hydrolysis at pH 1, while the bis acid is only 2.35 times more stable than Fpmp at pH 3. In synthesis of the dimers UpU and UpG, with a coupling time of 5 min, the dimethoxytrityl cation assay indicated coupling yields of >98%.

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“…The development of phosphoramidite chemistry resulted in an automated synthesis of whole-unmodified tRNA [5]. Later, chemical synthesis of modified tRNA containing −simple× modifications (dihydrouridine, ribothymidine, and pseudouridine) was reported [6]. The enzymatic ligation of chemically synthesized RNA fragments is an alternative approach in the preparation of tRNA [7 ± 9].…”

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confidence: 99%

Synthesis of RNA ContainingO-β-D-Ribofuranosyl-(1″2′)-adenosine-5″-phosphate and 1-Methyladenosine, Minor Components of tRNA

Mikhailov

Efimtseva

Rodionov

et al. 2005

C&B

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tRNA is best known for its function as amino acid carrier in the translation process, using the anticodon loop in the recognition process with mRNA. However, the impact of tRNA on cell function is much wider, and mutations in tRNA can lead to a broad range of diseases. Although the cloverleaf structure of tRNA is wellknown based on X-ray-diffraction studies, little is known about the dynamics of this fold, the way structural dynamics of tRNA is influenced by the modified nucleotides present in tRNA, and their influence on the recognition of tRNA by synthetases, ribosomes, and other biomolecules. One of the reasons for this is the lack of good synthetic methods to incorporate modified nucleotides in tRNA so that larger amounts become available for NMR studies. Except of 2'-O-methylated nucleosides, only one other sugar-modified nucleoside is present in tRNA, i.e., 2'-O-b-d-ribofuranosyl nucleosides. The T loop of tRNA often contains charged modified nucleosides, of which 1-methyladenosine and phosphorylated disaccharide nucleosides are striking examples. A protecting-group strategy was developed to introduce 1-methyladenosine and 5''-O-phoshorylated 2'-O-(b-dribofuranosyl)-b-d-ribofuranosyladenine in the same RNA fragment. The phosphorylation of the disaccharide nucleoside was performed after the assembly of the RNA on solid support. The modified RNA was characterized by mass-spectrometry analysis from the RNase T1 digestion fragments. The successful synthesis of this T loop of the tRNA of Schizosaccharomyces pombe initiator tRNA Met will be followed by its structural analysis by NMR and by studies on the influence of these modified nucleotides on dynamic interactions within the complete tRNA.

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Chemical synthesis of a biologically active natural tRNA with its minor bases

Cited by 137 publications

References 28 publications

Formation of N-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

Formation of N-Branched Oligonucleotides as By-products in Solid-Phase Oligonucleotide Synthesis

A new 2′-hydroxyl protecting group for the automated synthesis of oligoribonucleotides

Synthesis of RNA ContainingO-β-D-Ribofuranosyl-(1″2′)-adenosine-5″-phosphate and 1-Methyladenosine, Minor Components of tRNA

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