tRNAs are essential molecules in protein synthesis, responsible for translating the four-nucleotide genetic code into the corresponding amino acid sequence. RNA modifications play a crucial role in influencing tRNA folding, structure, and function. These modifications, ranging from simple methylations to complex hypermodified species, are distributed throughout the tRNA molecule. Depending on their type and position, they contribute to the accuracy and efficiency of decoding by participating in a complex network of interactions. The enzymatic processes introducing these modifications are equally intricate and diverse, adding further complexity. As a result, studying tRNA modifications faces limitations at multiple levels. This review addresses the challenges involved in manipulating and studying the function of tRNA modifications and discusses experimental strategies and possibilities to overcome these obstacles.
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