Developing RNA binding small molecule therapeutics requires robust and reliable methods to study molecular interactions, as these therapies are gaining significant attention for their potential to target previously “undruggable” diseases. Surface Plasmon Resonance (SPR) and Fluorescent Dye Displacement assays are two powerful techniques that offer unique insights into RNA-ligand binding dynamics. In this blog, we explore how these methods work, and their growing role in advancing the rapidly expanding field of RNA-targeted drug discovery.

Potential applicability of SPR and Fluorescent Dye Displacement assay in the discovery of RNA binding small molecule therapeutics

RNA dysregulation has been implicated in myriad human diseases, thus making it a suitable therapeutic target. Classically, therapeutic strategies aiming at RNA have been limited to sequence-based targeting of its unstructured regions through the use of complementary oligonucleotides, which mediate Ribonuclease-dependent RNA cleavage. However, RNA is remarkably structured and intricately folded as hairpin loops through its intramolecular base pairing, forming a series of unique secondary structures. These RNA folds, similar to motifs or domains in proteins, may offer a unique opportunity for targeting by small molecules through specific binding. The biological outcome of such RNA binding small molecules can be further enhanced by appending a functional group to it, which can recruit a Ribonuclease, activate it and cleave the target RNA. However, studying these RNA small molecule interactions has been largely hindered by the limited availability of robust methods with high throughput. Surface Plasmon Resonance (SPR), a label-free technique for studying biomolecular interactions, has been widely employed for screening and identification of small molecule binders of proteins. We explored the potential applicability of this technique for studying the specific interactions between RNA and small molecules.

Capabilities in RNA small molecule interaction studies by SPR assay at o2h

Leveraging our expertise in small molecule discovery using SPR (Surface Plasmon Resonance) platform for target proteins at o2h discovery, we have developed an SPR-based assay for studying the specific interaction between RNA and small molecules. Our SPR system (Cytiva Biacore T200) provides a highly sensitive approach for analysing the specific interaction between RNA and small molecules. These assays provide a robust platform for medium throughput screening of a library of small molecules binding to a unique RNA fold of a specific RNA molecule.

Case study

Our case study is based on a seminal research paper by Tong et al. (2023). The article demonstrates specific interaction of small molecules with unique RNA folds through the use of Fluorescent dye displacement assay. Collectively, Tong et al. demonstrated the structure-activity relationships (SAR) between small molecules and their preferred RNA 3D folds.

We adopted the above study with reference to c-Jun mRNA and synthesized the compounds at o2h accordingly. To study the specific interaction between RNA and small molecules, we adapted our highly sensitive Biacore T200 SPR system. In this assay, we captured the wild type and mutant (as a control) form of c-Jun mRNA (the Ligand) on two individual flow cells of a sensor chip. The wild type RNA consisted of a unique kink/bulge which was absent in the mutant RNA (Figure 1A). We analysed their interaction with reported specific small molecule – Jun binder (Tool compound/Positive control) by SPR. Binding data is a correction which involves subtraction of the binding Response values of mutant RNA from the wild type RNA, revealed its binding specificity and high affinity towards wild type c-Jun RNA but showed poor binding to the mutant counterpart. We also observed dose-dependent sensorgrams across a range of compound concentrations (Figure 1B). To further test the binding specificity and robustness of the SPR binding assay, we evaluated an unrelated RNA binding compound, Myc-binder (Negative control) known to bind c-Myc mRNA for its binding affinity towards the c-Jun RNAs (WT and mutant forms). As expected, this compound did not show any preference for binding towards c-Jun RNA, and with the reference correction, revealed no binding or negative sensograms (Figure 1C).      

Our SPR assay thus enabled us to characterize the specificity of a small molecule binding to a unique secondary structure in an RNA molecule demonstrating specificity towards the WT c-Jun RNA. This approach will be instrumental in screening a library of compounds capable of binding with specificity and high affinity to a unique RNA fold.

Such RNA binding small molecules can be conjugated to a second small molecule that recruits and locally activates RNase L to constitute a RiboTAC (RNA Targeting Chimera) and given o2h significant experience with PROTACs we can adapt our learnings in building improved, novel bioactive small molecule RNA degraders, similar to a PROTAC approach.

Figure 1: SPR-enabled Screening of RNA-Fold-Specific Small Molecule Binders.

A. Secondary structures of c-Jun RNA (wild-type [WT] and mutant forms). The “kink” present in the WT RNA, which serves as the binding site for the small molecule, is highlighted in blue and shown in the inset. The corresponding region where the kink is absent in the mutant RNA is also indicated.

B. The tool compound, c-Jun binder, was evaluated for binding to both WT and mutant c-Jun RNA across a range of concentrations. The sensorgrams (binding curves) are displayed as response units (RU) and are reference subtracted (binding to WT c-Jun RNA minus binding to mutant c-Jun RNA). Kinetic parameters are shown below: Ka (association constant), Kd (dissociation constant), KD (equilibrium dissociation constant), and Rmax (maximum response).

C. An unrelated compound, c-Myc binder, was similarly tested for binding to both WT and mutant c-Jun RNA. Sensorgrams show that this compound does not bind to c-Jun RNA, with reference correction leading to negative sensograms for the mutant c-Jun RNA.Fluorescent dye displacement assay for analysing RNA binding small molecules

We have also developed fluorescent dye displacement assays to complement our SPR studies for analysing RNA-small molecule interactions. This assay utilises nucleic acid-binding dyes that preferentially bind to specific structural features within RNA secondary structures, such as loops and bulges. These dyes can be displaced by potent small molecule RNA binders that interact with the same regions, leading to a reduction in fluorescence, which is detected in a plate reader format.

In the example below, we use ToPro dye, which preferentially binds to bulge and loop regions in RNA and exhibits fluorescence only when bound to nucleic acids. As demonstrated in the SPR assay, we tested the interaction of c-Jun mRNA with a known c-Jun RNA binder (tool compound/positive control). A strong, concentration-dependent reduction in ToPro fluorescence was observed, indicating the displacement of the dye by the small molecule. This fluorescence decrease was not seen when the mutant mRNA, lacking the specific bulge/kink binding site, was tested. From these experiments, we were able to determine the EC50 of the small molecule binding to c-Jun mRNA.

Figure 2: Fluorescent Displacement Assay for RNA-Small Molecule Interactions

Top: Schematic of the fluorescent displacement workflow. A nucleic acid-binding dye, which fluoresces only when bound to RNA, is incubated with a folded RNA molecule. Upon the addition of a small molecule binder, the dye is displaced, leading to a reduction in fluorescence.

Bottom: Fluorescence readout showing displacement of the ToPro RNA binder by the c-Jun binder. As the concentration of the c-Jun binder increases, fluorescence decreases. No displacement is observed with the mutant c-Jun mRNA, which lacks the bulge/kink site targeted by the small molecule binder, as fluorescence remains unchanged at all concentrations. EC50 = 1.5 µM ± 0.2.

This assay provides a versatile approach for screening RNA binders that target specific secondary structure elements. By combining nucleic acid binders that preferentially bind to particular structural features and with mutant RNA fragments that lack the targeted element, we can identify small molecules that bind with high specificity. The assay has been optimized in a 384-well format for high-throughput screening, enabling the rapid testing of large compound libraries and accelerating structure-activity relationship (SAR) studies.

In addition, we can support follow-up in vitro (cell-based) studies to further evaluate promising compounds identified through these screens. For more information or to explore collaboration opportunities, reach out to us at discovery@o2h.com.