Affinity-Based Profiling of the Flavin Mononucleotide Riboswitch

Abstract

Riboswitches are structural RNA elements that control gene expression. These naturally occurring RNA sensors are of continued interest as antibiotic targets, molecular sensors, and functional elements of synthetic circuits. Here, we describe affinity-based profiling of the flavin mononucleotide (FMN) riboswitch to characterize ligand binding and structural folding. We designed and synthesized photoreactive ligands and used them for photoaffinity labeling. We showed selective labeling of the FMN riboswitch and used this covalent interaction to quantitatively measure ligand binding, which we demonstrate with the naturally occurring antibiotic roseoflavin. We measured conditional riboswitch folding as a function of temperature and cation concentration. Furthermore, combining photoaffinity labeling with reverse transcription revealed ligand binding sites within the aptamer domain with single-nucleotide resolution. The photoaffinity probe was applied to cellular extracts of Bacillus subtilis to demonstrate conditional folding of the endogenous low-abundant ribD FMN riboswitch in biologically derived samples using quantitative PCR. Lastly, binding of the riboswitch-targeting antibiotic roseoflavin to the FMN riboswitch was measured in live bacteria using the photoaffinity probe.

A Riboswitch-Driven Era of New Antibacterials

“…Its FMN ligand is directed in the center of the junctional region and one of its four oxygens is used for hydrogen bonding, whereas phosphate oxygens interact with several conserved guanines, such as G33 [ 73 ]. On the other hand, riboflavin which lacks the phosphate exhibits 1000-fold lower binding affinity to the FMN aptamer of F. nucleatum and B. subtilis [ 74 , 75 , 76 ].…”

Toehold-Exchange-Based Activation of Aptamer Switches Enables High Thermal Robustness and Programmability

“…Molecular switches that undergo conformational changes upon target binding are naturally evolved machinery to regulate diverse biological functions . For example, riboswitches are widely adopted by living organisms to control gene expressions in response to environmental stimuli. , Inspired by nature, chemists and synthetic biologists have developed intense research interests to create artificial chemically activated switches that can be used to mimic processes in living systems and further expand their functionality for uses in biosensing, imaging, , therapeutics, , and beyond. , In this regard, aptamers are ideal functional molecules to develop artificial molecular switches because they not only are synthetic binders with high affinity and selectivity but can also be readily engineered via Watson–Crick base pairing. To be a molecular switch, an aptamer must be selected or engineered so that it shifts reversibly and controllably between an inactive “off” and a binding-competent “on” state, which is often achieved by caging an aptamer in a metastable inter- or intramolecular duplex motif (Figure a) …”

“…1 For example, riboswitches are widely adopted by living organisms to control gene expressions in response to environmental stimuli. 2,3 Inspired by nature, chemists and synthetic biologists have developed intense research interests to create artificial chemically activated switches that can be used to mimic processes in living systems and further expand their functionality for uses in biosensing, 4 imaging, 5,6 therapeutics, 7,8 and beyond. 9,10 In this regard, aptamers are ideal functional molecules to develop artificial molecular switches because they not only are synthetic binders with high affinity and selectivity but can also be readily engineered via Watson−Crick base pairing.…”

Chemical RNA Cross-Linking: Mechanisms, Computational Analysis, and Biological Applications

“…A different azido derivative bears the azide group at the end of a triethylene glycol linker attached to the primary amine of AMT and forms the basis of Cross-linking Of Matched RNA And Deep Sequencing (COMRADES) . Using this psoralen derivative, cross-linked RNA can be selectively captured and enriched using a biotin ligation and streptavidin pulldown. − The presence of the azide group did not affect the cross-link efficiency and using this method the researchers determined the architecture of Zika virus inside cells …”