The genetic system contains several levels of information. Firstly, the sequence of the canonical bases A, C, G, and T/U in DNA and RNA encodes amino acids through specific base triplets. Secondly, the methylation status of the cytosine base in DNA imprints epigenetic information into the genetic system, thereby contributing to the division of genes into active and inactive elements. Both information layers are chemically well investigated. Less is known about a putative third level of information associated with the chemical modification of RNA nucleobases. Although RNA, and in particular tRNA, is known to contain more than 100 different modified nucleosides, the exact type of information added by base modification is largely unknown. A number of common modifications have been shown to improve the maintenance of the reading frame, influence RNA stability, and to be involved in proof-reading by tRNA synthetases.