An RNA aptamer that induces suitable conformational changes upon binding to a user-defined ligand allows us to artificially construct a riboswitch, a ligand-dependent and -acting gene regulatory RNA. Although such an aptamer can be obtained through selection, it is still challenging to rationally expand the variety of orthogonal ligand/aptamer (ligand/riboswitch) pairs. To achieve this in a facile, selection-free way, we herein focused on a specific type of ligand, 6-nt nanosized DNA (nDNA) and its aptamer that was previously selected to construct a eukaryotic artificial riboswitch. Specifically, we merely mutated one or more possible Watson-Crick base pairs in the nDNA/aptamer (nDNA/riboswitch) interactions into another base pair or pairs. Using two sets that each had 16 comprehensive mutations, we obtained three groups of several orthogonal nDNA/riboswitch pairs. These pairs could be used to create complex gene circuits, including multiple simultaneous and/or multistep cascading regulations in synthetic biology.
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