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Updated: May 21, 2025

Nanomanipulation of Single RNA Molecules by Optical Tweezers
Published on: August 20, 2014
Structural insights into lab-coevolved RNA-RBP pairs and applications of synthetic riboswitches in cell-free system
Keisuke Fukunaga1,2,3, Takamasa Teramoto4, Momoka Nakashima4
1Earth-Life Science Institute (ELSI), Institute of Science Tokyo, Tokyo 152 8550, Japan.
Abstract:
CS1-LS4 and CS2-LS12 are ultra-high affinity and orthogonal RNA-protein pairs that were identified by PD-SELEX (Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment). To investigate the molecular basis of the lab-coevolved RNA-RBP pairs, we determined the structures of the CS1-LS4 and CS2-LS12 complexes and the LS12 homodimer in an RNA-free state by X-ray crystallography. The structural analyses revealed that the lab-coevolved RNA-RBPs have acquired unique molecular recognition mechanisms, whereas the overall structures of the RNP complexes were similar to the typical kink-turn RNA-L7Ae complex. The orthogonal RNA-RBP pairs were applied to construct high-performance cell-free riboswitches that regulate translation in response to LS4 or LS12. In addition, by using the orthogonal protein-responsive switches, we generated an AND logic gate that outputs staphylococcal γ-hemolysin in cell-free system and carried out hemolysis assay and calcein leakage assay using rabbit red blood cells and artificial cells, respectively.
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