Figure 3.
Design of the TAV2b-deried crosslinked helix fork (CHF) scaffold. (A) From wild-type TAV2b protein, the α1 helix was retained and truncated to an 8-residue sequence containing R26-R33 (yellow and blue ribbon). The individual α1-derived peptides were covalently crosslinked to obtain the CHF scaffold. (B) Side-chain interactions of R26-R33 with duplex RNA from the native complex, PDB 2zi0. Black dashes represent side-chain interaction with the RNA phosphate backbone, cyan. Red wedges represent Hoogsteen hydrogen bonds between amino acid side chain to nucleobases, red dotted box. (C) Binding affinities of TAV2b-α1Flu and CHF-1Flu for designed oligonucleotide hairpins, RNA 1 and DNA 1. TAV2b-α1Flu and CHF-1Flu sequences are listed in brackets. Yellow highlights on helices and amino acid sequences represent residues involved in Hoogsteen recognition. Nle = Norleucine. R1 = Fluorescein-β-Alanine. R2 = Fluorescein-β-Alanyl-glycine. R3 = Acetyl-tyrosine. All peptide C-termini are carboxamides. Designed hairpin oligonucleotides RNA1 and DNA1 are shown with the oligonucleotide sequences listed in Table S1. (D) Comparison of monomeric and dimeric peptide recognition of RNA1. Binding affinities of fluorescein-labeled peptides for RNA1 as measured in a fluorescence polarization assay. HBS-1 = hydrogen bond surrogate stabilized peptide −1. Ac = Acetyl. Flu = Fluorescein. X = 4-pentenoic acid. G* = N-allylglycine. Φ = melamine. All peptide C-termini are carboxamides. Error bars represent the standard deviation of three independent experiments.