January 28, 2025
Using Depixus’ magnetic force spectroscopy platform to explore small molecules targeting RNA riboswitches
We’ve been working with Dr Jay Schneekloth, a leading expert in RNA-targeted drug discovery, to use Depixus’ magnetic force spectroscopy (MFS) platform to probe the interactions between the PreQ1 bacterial riboswitch and its natural ligand or a synthetic small molecule.
In a paper published in Nature Communications,1 we showed how our magnetic force spectroscopy technology provided novel insights into the distinct mechanisms of action of different ligands upon the target RNA at single molecule resolution.
What are riboswitches, and why are they important in drug development?
Riboswitches are structured sequences of RNA that are commonly found in the 5′ untranslated regions of bacterial mRNAs and help to control metabolic pathways. They work through direct binding of a ligand such as a metabolite to the riboswitch, which induces a conformation change to their secondary structure and alters gene expression.
The PreQ1 riboswitch 2 regulates downstream gene expression for the biosynthesis of queuosine – an important non-canonical nucleoside found in many bacterial and eukaryotic species – in response to a metabolite called PreQ1 (7-aminomethyl-7-deazaguanine). Upon binding of PreQ1 the RNA changes into a shape known as a pseudoknot, 3 which alters gene expression at either the transcriptional or translational level.
Figure 1: Schematic of the Bsu PreQ1 riboswitch. (a) Riboswitch sequence in mRNA. (b–f) Proposed secondary structures of the aptamer domain (b, hairpin; c, prefolded form; d, H-type pseudoknot) and the expression platform (e, antiterminator loop; f, terminator loop). Nucleotides colored in blue, yellow, and magenta form the base pairs that change among these secondary structures. Taken from Sarkar, Ishii & Tahara (2021).
The ability of riboswitches to modulate bacterial gene expression in response to small molecules makes them an attractive group of novel antibacterial drug targets.4
Using magnetic force spectroscopy to explore RNA-ligand interactions
We used our magnetic force spectroscopy (MFS) technology to probe the dynamic interactions between the PreQ1 riboswitch RNA from Bacillus subtilis (Bsu) and its natural ligand, PreQ1, or a synthetic ligand named Compound 4.
With our technology, we precisely measured the effects of applying constant or gradually changing (ramped) forces to hundreds of immobilized riboswitch RNAs simultaneously (Figure 2A). This enabled us to study the response of the RNA structures to the applied forces in the absence and presence of the ligands, and to visualize changes in conformation as the structures unfolded and refolded.
We found that both PreQ1 and Compound 4 stabilized the riboswitch RNA structure, requiring more force to unfold it. We were also able to explore the concentration dependency of their effects (Figure 2B).
While both PreQ1 and Compound 4 bound to the riboswitch RNA and stabilized it, analysis of the data streams from single molecules generated by our instrument revealed that the two ligands had different modes of action (Figure 2C). Unlike the natural ligand, Compound 4 does not induce the pseudoknot structure in the RNA – a distinction that would be missed by bulk, averaged measurement techniques.
Figure 2: (A) Overview of Depixus’ scalable magnetic force spectroscopy platform for exploring the interactions of small molecule ligands with target RNA structures. (B) Dose-response curve for RNA unfolding with Compound 4 and PreQ1(C) Raw traces from single molecule constant force experiments with control, Compound 4 and PreQ1 with density histograms shown to the right. Taken from Parmar et al. (2024).1
In addition, Schneekloth and colleagues explored other structural aspects of the interaction between the riboswitch and various ligands, and their impact on gene expression in live bacterial cells. They were also able to show that Compound 4 could bind in a similar way to PreQ1 riboswitches from other species of bacteria, building up a fuller picture of the properties of this novel molecule.
Magnetic force spectroscopy delivers deeper insights for RNA-targeted drug development
Overall, these findings demonstrate that even though different ligands may appear to be the same in terms of RNA binding site and their effect on gene expression, other factors – including selectivity, mode of recognition, and impacts on both conformational kinetics and thermodynamics – can all play a role in the ability of a compound to modulate biological function. Such subtle yet important distinctions may not be apparent using analytical methods that rely on surrogate outputs or bulk measurements.
Our unique single molecule approach enabled the Schneekloth team to visualize the binding of a novel ligand to its target in real time and gather insights into the distinct mechanisms of action, which would not have been possible using other methods. These results demonstrate the capabilities of Depixus magnetic force spectroscopy (MFS) technology to deliver valuable data about individual dynamic molecular interactions at scale.
Depixus MAGNA One is the first laboratory instrument that makes the power of magnetic force spectroscopy accessible and scalable. With Depixus MAGNA One you can now identify compounds that bind to RNA, and directly reveal how they impact its structure and function at the level of individual molecules, supporting lead selection and lead validation in the development of novel RNA-targeted therapeutics. You can also use it to generate detailed information about the kinetics and concentration dependence of ligand binding.
- Check out our in-depth app note to learn more about how Depixus’ magnetic force spectroscopy technology can be used to analyze riboswitch-ligand interactions.
- Get in touch to find out how you can explore RNA-targeted therapeutics with Depixus MAGNA One.
References:
1. Parmar, S., Bume, D.D., Connelly, C.M. et al. (2024) Mechanistic analysis of Riboswitch Ligand interactions provides insights into pharmacological control over gene expression. Nat Commun 15, 8173 (2024).doi:10.1038/s41467-024-52235-3
2. McCown PJ, Liang JJ, Weinberg Z, Breaker RR. (2014) Structural, functional, and taxonomic diversity of three preQ1 riboswitch classes. Chem Biol. Jul 17;21(7):880-889. doi:10.1016/j.chembiol.2014.05.015
3. Sarkar B, Ishii K, Tahara T (2021). Microsecond Folding of preQ1 Riboswitch and Its Biological Significance Revealed by Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy. J Am Chem Soc. 2021 Jun 2;143(21):7968-7978.doi:10.1021/jacs.1c01077
4. Howe JA, Wang H, Fischmann TO, et al. (2015) Selective small-molecule inhibition of an RNA structural element. Nature. Oct 29;526(7575):672-7.doi: 10.1038/nature15542