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Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1 riboswitches in E.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.

SUBMITTER: Neuner E

PROVIDER: S-EPMC6284575 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1 riboswitches in E.coli.

Neuner Eva E Frener Marina M Lusser Alexandra A Micura Ronald R

RNA biology 20181026 10

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 riboswi ...[more]

PMID: 30332908

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Project description:We report here the behavior of naturally occurring and rationally engineered preQ1 riboswitches and their application to inducible gene regulation in mycobacteria. Because mycobacteria lack preQ1 biosynthetic genes, we hypothesized that preQ1 could be used as an exogenous nonmetabolite ligand to control riboswitches in mycobacteria. Selected naturally occurring preQ1 riboswitches were assayed and successfully drove preQ1-dependent repression of a green fluorescent protein reporter in Mycobacterium smegmatis Using structure-based design, we engineered three preQ1 riboswitches from Thermoanaerobacter tencongensis, Bacillus subtilis, and Lactobacillus rhamnosus toward achieving higher response ratios and increased repression. Assuming a steady-state model, variants of the T. tencongensis riboswitch most closely followed the predicted trends. Unexpectedly, the preQ1 dose response was best described by a model with a second, independent preQ1 binding site. This behavior was general to the preQ1 riboswitch family, since the wild type and rationally designed mutants of riboswitches from all three bacteria behaved analogously. Across all variants, the response ratios, which describe expression in the absence versus the presence of preQ1, ranged from <2 to ∼10, but repression in all cases was incomplete up to 1 mM preQ1. By reducing the transcript expression level, we obtained a preQ1 riboswitch variant appropriate for inducible knockdown applications. We further showed that the preQ1 response is reversible, is titratable, and can be used to control protein expression in mycobacteria within infected macrophages. By engineering naturally occurring preQ1 riboswitches, we have not only extended the tools available for inducible gene regulation in mycobacteria but also uncovered new behavior of these riboswitches.IMPORTANCE Riboswitches are elements found in noncoding regions of mRNA that regulate gene expression, typically in response to an endogenous metabolite. Riboswitches have emerged as important tools for inducible gene expression in diverse organisms. We noted that mycobacteria lack the biosynthesis genes for preQ1, a ligand for riboswitches from diverse bacteria. Predicting that preQ1 is not present in mycobacteria, we showed that it controls optimized riboswitches appropriate for gene knockdown applications. Further, the riboswitch response is subject to a second independent preQ1 binding event that has not been previously documented. By engineering naturally occurring riboswitches, we have uncovered a new behavior, with implications for riboswitch function in its native context, and extended the tools available for inducible gene regulation in mycobacteria.

Project description:Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4-L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2) and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC₅₀ value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T(m) of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (K(d)≈10⁻⁷ M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC₅₀ values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are useful for improving and building potential pro-apoptotic ligands.

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Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ1 riboswitches in E.coli.

Publications

Superior cellular activities of azido- over amino-functionalized ligands for engineered preQ<sub>1</sub> riboswitches in E.coli.

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