High-Purity Enzymatic Synthesis of Site-Specifically Modified tRNA

Transfer RNA (tRNA) molecules play the key role in adapting the genetic code sequences with amino acids. The execution of this key role is highly dependent on the presence of modified nucleotides in tRNA, each of which performs a distinct function. To better understand how individual modifications modulate tRNA function, a method to isolate and purify a site-specifically modified tRNA is essential. This chapter describes an enzymatic method to synthesize a site-specifically modified tRNA, followed by purification of this tRNA away from unmodified tRNA using a selective oligonucleotide-based hybridization approach. This method is broadly applicable to site-specific tRNA modifications that interfere with nucleic-acid base-pairing principles.