We report the synthesis of a modified 8-mer RNA sequence, (C-C-C-C-A-C-C-(2'-thio)A)-RNA 5'-(dihydrogen phosphate) contg. a 3'-terminal 2'-thio-adenosine and its spontaneous and site-specific aminoacylation with the weakly activated amino acid thioester H-Phe-SPh. This reaction, designed in analogy to the 'native chem. ligation' of oligo-peptides, occurs efficiently in buffered aq. solns. and under a wide range of conditions. At pH values between 5.0 and 7.4, two products, the 3'-O-mono-acylated and the 3'-O,2'-S-diacylated RNA sequences are formed fast and quant. At pH 7.4 and 37 Deg, the 3'-O-mono-acylated product is formed as major product in situ by regioselective hydrolysis of the O,S-diacylated precursor. Addnl., the prepn. and isolation of the relevant 3'-O-mono-acylated product was optimized at pH 5. This concept could be employed for a straightforward aminoacylation of analogously modified tRNAs. [on SciFinder (R)]