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Synthesis of RNA Containing O ‐ β ‐ D ‐Ribofuranosyl‐(1″2′)‐adenosine‐5″‐phosphate and 1‐Methyladenosine, Minor Components of tRNA
Author(s) -
Mikhailov Sergey N.,
Efimtseva Ekaterina V.,
Rodionov Andrei A.,
Shelkunova Alexandra A.,
Rozenski Jef,
Emmerechts Gert,
Schepers Guy,
Van Aerschot Arthur,
Herdewijn Piet
Publication year - 2005
Publication title -
chemistry and biodiversity
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.427
H-Index - 70
eISSN - 1612-1880
pISSN - 1612-1872
DOI - 10.1002/cbdv.200590085
Subject(s) - transfer rna , pseudouridine , chemistry , rna , nucleoside , aminoacylation , nucleotide , ribosome , ribose , biochemistry , nucleic acid structure , stereochemistry , enzyme , gene
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 well‐known 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 ‐ β ‐ 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 ‐( β ‐ D ‐ribofuranosyl)‐ β ‐ 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.