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| - Synthesis of the tRNALys,3 Anticodon Stem-Loop DomainContaining the Hypermodified ms2t6A Nucleoside
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| - The synthesis of a protected form of the hypermodified nucleoside, N-[(9-β-d-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, (ms2t6A) is reported. The hypermodified nucleoside wassubsequently elaborated to the protected nucleoside phosphophoramidite using a protecting groupstrategy compatible with standard RNA oligonucleotide chemistry. The phosphoramidite reagentwas then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodonstem-loop (ASL) domain of human tRNALys,3, the primer for HIV-1 reverse transcriptase.Introduction of the modification at position 37 of the tRNA ASL modestly decreases thethermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t6Anucleoside lacking the 2-methylthio substituent. 2D NOESY NMR spectra of the ms2t6A containingtRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in thesolution structure of the analogous t6A containing E. coli tRNALys, despite the presence of the bulkymethylthio group. This synthetic approach allows for site-specific incorporation of the hypermodifiednucleoside and should facilitate future studies directed at understanding the roles of nucleosidemodification in modulating the stability and specificity of biologically important RNA−RNAinteractions. Our synthesis of the ms2t6A containing RNAs demonstrates that this methodology issuitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.
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