Designed Arginine-Rich RNA-Binding Peptides with Picomolar Affinity

Abstract

Arginine-rich peptide motifs (ARMs) capable of binding unique RNA structures play critical roles in transcription, translation, RNA trafficking, and RNA packaging. Bacteriophage ARMs necessary for transcription antitermination bind to distinct boxB RNA hairpin sequences with a characteristic induced α-helical structure. Characterization of ARMs from lambdoid phages reveals that the dissociation constant of the P22 bacteriophage model−antitermination complex (P22_(N21)−P22boxB) is 200 ± 56 pM in free solution at physiologic concentrations of monovalent cation, significantly stronger than previously determined by gel mobility shift and polyacrylamide gel coelectophoresis, and 2 orders of magnitude stronger than the tightest known native ARM−RNA interaction at physiological salt. Here, we use a reciprocal design approach to enhance the binding affinity of two separate α-helical ARM−RNA interactions; one derived from the native λ phage antitermination complex and a second isolated using mRNA display selection experiments targeting boxB RNA.

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