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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.

Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with three enzymes dedicated for the methylation of Guanine at different positions.

The nature and position of these modified bases are species-specific. Thus, there are several bases that are exclusive to eukaryotes or prokaryotes. For instance, thiolation of Adenine is only observed in prokaryotes, whereas methylation of cytosine is restricted to eukaryotes. Overall, eukaryotic tRNAs are modified to a greater extent than those from prokaryotes.

Although the nature of modifications may vary, some regions of the tRNA are always heavily modified. Each of the three stem-loop regions or "arms"of the tRNA have modified bases that serve unique purposes. The TΨC arm, named after the presence of the nucleotides, thymine, pseudouridine and cytosine, is recognized by the ribosome during translation. The DHU or D arm that contains the modified pyrimidine dihydrouracilserves as a recognition site for the aminoacyl-tRNA synthetase enzyme, that catalyzes the covalent addition of an amino acid to the tRNA. The anticodon loop often has a queuine base, which is a modified guanine. This base creates a Wobble pair with the codon sequence on the mRNA, i.e. it forms a base pair that does not follow Watson-Crick base pair rules. Usually, a tRNA binds the mRNA more “loosely” in the third position of the codon. This allows several types of non-Watson–Crick base pairing or Wobble bases at the third codon position. It has been observed that the presence of queuinein the first position of the anticodon, which pairs with the third position of the codon, improves the translation accuracy of the tRNA.

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