Specificity of Phage Display Selected Peptides for Modified Anticodon Stem and Loop Domains of tRNA

Abstract

Protein recognition of RNA has been studied using Peptide Phage Display Libraries, but in the absence of RNA modifications. Peptides from two libraries, selected for binding the modified anticodon stem and loop (ASL) of human tRNA(LyS3) having 2-thiouridine (s(2)U34) and pseudouridine (psi39), bound the modified human ASL(Lys3)(s(2)U34;psi39) preferentially and had significant homology with RNA binding proteins. Selected peptides were narrowed to a manageable number using a less sensitive, but inexpensive assay before conducting intensive characterization. The affinity and specificity of the best binding peptide (with an N-terminal fluorescein) were characterized by fluorescence spectrophotometry. The peptide exhibited the highest binding affinity for ASL(LYS3)(s(2)U34; psi39), followed by the hypermodified ASL(Lys3) (mcm(5)s(2) U34; ms(2)t(6)A37) and the unmodified ASL(Lys3), but bound poorly to singly modified ASL(Lys3) constructs (psi39, ms(2)t(6)A37, s(2)34), ASL(Lys1,2) (t(6)A37) and Escherichia coli ASL(Glu) (s(2)U34). Thus, RNA modifications are potentially important recognition elements for proteins and can be targets for selective recognition by peptides.

Strategies for the Design of Rna-Binding Small Molecules

“…This method has identified a set of peptides that bind to the modified anticodon stem-loop (ASL) domain of human tRNA -lys3 [89]. This RNA holds potential as an HIV target because of its apparent role as a primer for viral replication by reverse transcriptase.…”

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

“…By analogy with the protection of the t 6 A carboxyl group (Boudou et al 2000;Sundaram et al 2000;Eshete et al 2007;Bilbille et al 2009), variously substituted N-Boc taurine 2-phenylethyl esters (Fig. 2, 3a-3g) removable via the β-elimination process (10% DBU/MeCN) have been prepared (Leszczynska et al 2013).…”

Functional Recognition of the Modified Human tRNALys3UUU Anticodon Domain by HIV's Nucleocapsid Protein and a Peptide Mimic

“…47,48 Peptides that bound the hASL Lys3 UUU -ms 2 t 6 A 37 ;mcm 5 s 2 U 34 ;ψ 39 were selected using two different phage display libraries and two different conditions for elution from microplates. 49 This triply modified hASL Lys3 UUU was chemically synthesized with biotin at the 3′-end, and bound to streptavidin-coated, high capacity microplates. The first round of screening was conducted with the unmodified hASL Lys3 UUU bound to streptavidin plates, followed by multiple rounds of screening with the triply modified hASL Lys3 UUU .…”

“…Phages were eluted from the plates using both alkaline and acid conditions. 49 Phage that demonstrated an affinity to bind the unmodified hASL Lys3 UUU after four rounds of selection were catalogued and eliminated from further screening. In the subsequent step, phage that did not bind hASL Lys3 UUU were selected using hASL Lys3 UUU -mcm 5 s 2 U 34 ; ms 2 t 6 A 37 ;ψ 39.…”