Abstract
We have designed structure-based ligands for the guanidine-II riboswitch that bind with enhanced affinity, exploiting the twin binding sites created by loop–loop interaction. We synthesized diguanidine species, comprising two guanidino groups covalently connected by Cn linkers where n = 4 or 5. Calorimetric and fluorescent analysis shows that these ligands bind with a 10-fold higher affinity to the riboswitch compared to guanidine. We determined X-ray crystal structures of the riboswitch bound to the new ligands, showing that the guanidino groups are bound to both nucleobases and backbone within the binding pockets, analogously to guanidine binding. The connecting chain passes through side openings in the binding pocket and traverses the minor groove of the RNA. The combination of the riboswitch loop–loop interaction and our novel ligands has potential applications in chemical biology.
Highlights
RNA provides a versatile scaffold for binding small molecule ligands with high selectivity. This is well illustrated by the riboswitches (Roth and Breaker 2009; Serganov and Nudler 2013), cis-acting regulatory elements that occur in the 5′ noncoding regions of bacterial mRNA that are widely used to control gene expression
We have investigated the binding of guanidine, diguanidine-C4, and diguanidine-C5 to individual guanidine-II riboswitch stem–loops and a complete riboswitch using isothermal titration calorimetry (ITC) (Fig. 2; Supplemental Fig. S2)
We have further explored the binding of guanidine, diguanidine-C4, and diguanidine-C5 to a complete G. violaceous riboswitch with linked P1 and P2 stem loops using ITC (Fig. 2A–C)
Summary
RNA provides a versatile scaffold for binding small molecule ligands with high selectivity. This is well illustrated by the riboswitches (Roth and Breaker 2009; Serganov and Nudler 2013), cis-acting regulatory elements that occur in the 5′ noncoding regions of (mostly) bacterial mRNA that are widely used to control gene expression. Three ykkC types have been identified, called the guanidine-I (Nelson et al 2017), -II (Sherlock et al 2017), and -III (Sherlock and Breaker 2017) riboswitches. We have exploited a novel feature of the guanidine-II riboswitch structure to design a high-affinity ligand
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