More than 100 modified nucleosides have been found in naturally occurring RNA sequences. These modifications are involved in the correct folding, in the maintenance of the reading frame during translation and in the proper interaction with enzymes and protein complexes. For an exhaustive study of their function it would be necessary to incorporate by chemical synthesis such modifications at selected positions of RNA sequences. Here we present optimized protocols for the preparation of a large variety of 2'-O-TOM protected ribonucleoside phosphoramidite building blocks containing the most frequently encountered modified nucleobases m2G, m22G, m1A, m5U, D, m5C, ψ, m1I, i6A, m62A, m6A, m1G, t6A, I and imG. In addition, the non-natural ribonucleoside phosphoramidites containing isoG, isoC and 4-desmethyl-5-methylwyosine have been prepared (Chapters I, II and III). For the introduction of base-labile modifications into RNA sequences, modified deprotection conditions in combination with the new N2-methoxyacetyl protected guanosine phosphoramidite have been developed (Chapter V). For the first time, the sensitive modified nucleoside wyosine was incorporated into a 18mer RNA sequence and into the anticodon loop of a truncated tRNA. For this purpose, enzymatic ligation strategies, based on T4 RNA and T4 DNA ligase, were evaluated and optimized (Chapters III and V). The decoding properties of modified anticodons have been reviewed and new models, based on the formation of secondary hydrogen bonds have been proposed (Chapter IV). For the efficient preparation of tRNA analogues esterified with unnatural amino acids, a synthesis of modified RNA sequences containing a 3'-terminal 2'-deoxy-2'-thioadenosine was developed. In analogy to the "native chemical ligation" of oligopeptides, its spontaneous and site-specific aminoacylation with weakly activated amino acid thioesters occurred efficiently in buffered aqueous solutions and under a wide range of conditions. This concept could be employed for a straightforward aminoacylation of analogously modified tRNAs (Chapter VI).