Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ₁riboswitches inE.coli

Abstract

For this study, we utilized class-I and class-II preQ1-sensing riboswitches as model systems to decipher the structure-activity relationship of rationally designed ligand derivatives in vitro and in vivo. We found that synthetic preQ1 ligands with amino-modified side chains that protrude from the ligand-encapsulating binding pocket, and thereby potentially interact with the phosphate backbone in their protonated form, retain or even increase binding affinity for the riboswitches in vitro. They, however, led to significantly lower riboswitch activities in a reporter system in vivo in E. coli. Importantly, when we substituted the amino- by azido-modified side chains, the cellular activities of the ligands were restored for the class-I conditional gene expression system and even improved for the class-II counterpart. Kinetic analysis of ligand binding in vitro revealed enhanced on-rates for amino-modified derivatives while they were attenuated for azido-modified variants. This shows that neither high affinities nor fast on-rates are necessarily translated into efficient cellular activities. Taken together, our comprehensive study interconnects in vitro kinetics and in vitro thermodynamics of RNA-ligand binding with the ligands’ in vivo performance and thereby encourages azido- rather than amino-functionalized design for enhanced cellular activity.

Unraveling the structure and biological functions of RNA triple helices

“…Finally, it should be noted that riboswitches using a triple helix as a ligand‐binding site are not coupled to a single mechanism for controlling gene expression. The ligand‐bound forms of the c‐di‐GMP‐II (Lee et al, 2010) and guanidine‐III (Sherlock & Breaker, 2017) riboswitches turn ON translation while PreQ 1 ‐II (Dutta, Belashov, & Wedekind, 2018; Meyer et al, 2008; Neuner, Frener, Lusser, & Micura, 2018; Van Vlack, Topp, & Seeliger, 2017), PreQ 1 ‐III (Liberman et al, 2015; Van Vlack et al, 2017), metX SAM‐II (Gilbert et al, 2008), and SAM‐V riboswitches (L. Huang & Lilley, 2018; Poiata et al, 2009) turn OFF translation. Thus, there are instances where ligand binding leads to sequestration of the ribosome‐binding site into a triple helix to turn OFF translation and other instances where the ribosome‐binding site is released upon ligand binding, thereby turning ON translation.…”

Synthesis of O6-alkylated preQ1 derivatives

“…Finally, Carell reported a cycloaddition route relying on α-brominated 3-phthalimidopropanal and diaminopyrimidin-4-one [ 24 – 25 ]. We further optimized this path for the synthesis of 15 N-labeled prequeuosine nucleobase derivatives [ 26 ] required for advanced NMR spectroscopic applications [ 27 ], and for the syntheses of azido- or amino-functionalized preQ 1 derivatives needed for cellular applications with engineered riboswitches [ 28 ]. Finally, we point out that only a single synthetic route has been published to a potential O 6 -methylated precursor of m 6 preQ 1 , namely N 9-trimethysilylethyl protected m 6 preQ 0 [ 20 ].…”

“…vanced NMR spectroscopic applications [27], and for the syntheses of azido-or amino-functionalized preQ 1 derivatives needed for cellular applications with engineered riboswitches [28]. Finally, we point out that only a single synthetic route has been published to a potential O 6 -methylated precursor of m 6 preQ 1 , namely N9-trimethysilylethyl protected m 6 preQ 0 [20].…”

Changed reactivity of secondary hydroxy groups in C8-modified adenosine – lessons learned from silylation

“…Oligoribonucleotides carrying site-specific modifications are highly required as models for structure and function studies, driven by the ongoing discovery of new RNAs and their investigation [1][2][3][4][5][6]. This has put demand also on synthetic chemistry to provide suitable compounds at monomeric and oligomeric level.…”