Site-selected introduction of modified purine and pyrimidine ribonucleosides into RNA by automated phosphoramidite chemistry

Abstract

The study of modified nucleoside contributions to RNA chemistry, structure and function has been hwarted by the lack of a site-selected method of incorporation which is both versatile and adaptable to present synthetic technologies. A reproducible and versatile site-selected incorporation of nine differently modified nucleosides into hepta- and octadecamer RNAs has been achieved with automated phosphoramidite chemistry. The 5′-O-4,4′-dimethoxytrityl-2′-O- tert-butyldimethylsilyl-ribonucleoside-3-′-O-(2-cyanoethyl-N -diisopropyl)phosphoramidite syntheses of m 5C, D, Ψ, riboT, s 2U, mnm 5U, m 1G and m 2A were designed for compatibility with the commercially available major and 2′OH methylated ribonucleoside phosphoramidites. The synthesis of the m 5C phosphoramidite was uniquely designed, and the first syntheses and incorporation of the two modified purine ribonucleosides are reported in detail along with that of Ψ, s 2U,, and mnm 5U. Cleavage of RNA product from the synthesis support column, deprotection of the RNA, its purification by HPLC and nucleoside composition analysis are described. Modified nucleoside-containing tRNA domains were synthesized and purified in μmol quantities required for biophysical, as well as biochemical, studies. The anticodon domain of yeast tRNA Phe was synhesized with modified nucleosides introduced at the native positions: Cm 32, Gm 34, m 1G 37 (precursor to Y), Ψ 39 and m 5C 40. The T loop and stem was synthesized with riboT 54 and the D loop and stem with D 16 and D 17. The E coli tRNA Glu 2 anticodon domain was synthesized with mnm 5U at wobble position 34, but an attempt at incorporating s 2U at the same position failed. The unprotected thio group was labile to the oxidation step of the cyclical process. Chemically synthesized anticodon and T domains have been used in assays of tRNA structure and function (Guenther et al (1994) Biochimie 76, 1143–1151).