Selection of aminoacylated tRNAs from RNA libraries having randomized acceptor stem sequences: using old dogs to perform new tricks.

Publisher Summary Most systematic evolution of ligands by exponential enrichment (SELEX) schemes, involving nucleic acid ligands, require some type of postselection amplification of the nucleic acid sequence. This is usually accomplished by the polymerase chain reaction (PCR) that requires the use of short DNA oligonucleotide primers. When RNA ligands are the subject of study, PCR is also used to incorporate an RNA polymerase promoter into the DNA template for the subsequent transcription of more RNA ligand. Because the 5' and 3' termini of the polynucleotide serve as primer-binding sites, their sequence must be defined and invariant. Therefore, only internal stretches of the polynucleotide sequence can be randomized and studied, using typical SELEX schemes. Because this chapter is interested in using SELEX to explore whether unique combinations of acceptor stem nucleotides that can functionally mimic the conserved sequence of wild type Escherichi coli serine tRNAs exist, it is critical that the nucleotides at both termini be randomized. It, therefore, developed an approach that circumvents restrictions on the randomization of the transfer RNA (tRNA) acceptor stem nucleotides. This entailed extending the tRNA 5' and 3' termini to create defined and invariant primer-binding sites and, thus, allowing it to randomize the acceptor stem nucleotides so that their recognition by E. coli seryl-tRNA synthetase (SRS) could be studied, using a SELEX approach. The steps of the selection scheme outlined in this chapter, in total or in combination, are generally applicable to other problems that also involve selection for RNA ligands that are active for reversable chemical reactions and to situations, in which it would be advantageous to randomize the 3' terminus of a RNA.