The contributions of the natural modified nucleosides to RNA identity in protein/RNA interactions are not understood. We had demonstrated that 15 amino acid long peptides could be selected from a random phage display library using the criterion of binding to a modified, rather than unmodified, anticodon domain of yeast tRNA^Phe (ASL^Phe). Affinity and specificity of the selected peptides for the modified ASL^Phe have been characterized by fluorescence spectroscopy of the peptides' tryptophans. One of the peptides selected, peptide t^F2, exhibited the highest specificity and most significant affinity for ASL^Phe modified with 2‘-O-methylated cytidine-32 and guanosine-34 (Cm₃₂ and Gm₃₄) and 5-methylated cytidine-40 (m⁵C₄₀) (K_d = 1.3 ± 0.4 μM) and a doubly modified ASL^Phe-Gm₃₄,m⁵C₄₀ and native yeast tRNA^Phe (K_d ≅ 2.3 and 3.8 μM, respectively) in comparison to that for the unmodified ASL^Phe (K_d = 70.1 ± 12.3 μM). Affinity was reduced when a modification altered the ASL loop structure, and binding was negated by modifications that disfavored hairpin formation. Peptide t^F2's higher affinity for the ASL^Phe-Cm₃₂,Gm₃₄,m⁵C₄₀ hairpin and fluorescence resonance energy transfer from its tryptophan to the hypermodified wybutosine-37 in the native tRNA^Phe placed the peptide across the anticodon loop and onto the 3‘-side of the stem. Inhibition of purified yeast phenylalanyl-tRNA synthetase (FRS) catalyzed aminoacylation of cognate yeast tRNA^Phe corroborated the peptide's binding to the anticodon domain. The phage-selected peptide t^F2 has three of the four amino acids crucial to G₃₄ recognition by the β-structure of the anticodon-binding domain of Thermus thermophilus FRS and exhibited circular dichroism spectral properties characteristic of β-structure. Thus, modifications as simple as methylations contribute identity elements that a selected peptide specifically recognizes in binding synthetic and native tRNA and in inhibiting tRNA aminoacylation.

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