Synthesis and characterization of the native anticodon domain of E. coli TRNA(Lys): simultaneous incorporation of modified nucleosides mnm(5)s(2)U, t(6)A, and pseudouridine using phosphoramidite chemistry.
J Org Chem
; 65(18): 5609-14, 2000 Sep 08.
Article
em En
| MEDLINE
| ID: mdl-10970299
ABSTRACT
The anticodon domain of E. coli tRNA(Lys) contains the hypermodified nucleosides mnm(5)s(2)U and t(6)A at positions 34 and 37, respectively, along with a more common psi at position 39. The combination of these three nucleotides represents one of the most extensively modified RNA domains in nature. 2-Cyanoethyl diisopropylphosphoramidites of the hypermodified nucleosides mnm(5)s(2)U and t(6)A were each synthesized with protecting groups suitable for automated RNA oligonucleotide synthesis. The 17 nucleotide anticodon stem-loop of E. coli tRNA(Lys) was then assembled from these synthons using phosphoramidite coupling chemistry. Coupling efficiencies for the two hypermodified nucleosides and for pseudouridine phosphoramidite were all greater than 98%. A mild deprotection scheme was developed to accommodate the highly functionalized RNA. High coupling yields, mild deprotection, and efficient HPLC purification allowed us to obtain 1. 8 mg of purified RNA from a 1 micromol scale RNA synthesis. Our efficient synthetic protocol will allow for biophysical investigation of this rather unique tRNA species wherein nucleoside modification has been shown to play a role in codon-anticodon recognition, tRNA aminoacyl synthetase recognition, and programmed ribosomal frameshifting. The human analogue, tRNA(Lys,3), is the specific tRNA primer for HIV-1 reverse transcriptase and has a similar modification pattern.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Pseudouridina
/
Tionucleotídeos
/
Anticódon
/
RNA Bacteriano
/
RNA de Transferência de Lisina
/
Escherichia coli
/
Nucleosídeos
Limite:
Humans
Idioma:
En
Revista:
J Org Chem
Ano de publicação:
2000
Tipo de documento:
Article
País de afiliação:
Estados Unidos