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.

Structure-Specific RNA Targeting by Small Molecules: A Dual-Assay Approach Using SPR and Fluorescence Displacement

RNA dysregulation (typically overexpression of mRNA and miRNA) has been implicated in myriad human diseases, thus making it a suitable therapeutic target, particularly in case of undruggable proteins. Certain inhibitors of transcription, such as Rifampicin and Actinomycin D has been explored as potential chemotherapeutic agents. However, since they are general blockers of transcription machinery and lack target specificity, these are associated with toxicity and are not preferred. Alternatively, therapeutic strategies aimed at specific RNA molecules relies on sequence-based targeting of its unstructured regions through the use of complementary oligonucleotides, which mediate Ribonuclease-dependent RNA cleavage. This approach however, is associated with challenges of effective delivery to the cells and optimal dosing.

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 tuned 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 systems (Cytiva Biacore T200 and the upcoming Biacore 1S+) provide 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 combination of computation and in vitro methods. 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. We observed dose-dependent sensorgrams for both WT as well as mutant RNA across a range of compound concentrations as indicated. The data was fit in a 1:1 binding kinetic model (Figure 1B). Comparison of the reference corrected data revealed that the C-Jun binder exhibits ~1.2-fold selectivity for binding to the WT RNA over mutant RNA. The same binding data was analyzed by affinity fit model and indicated a similar trend i.e. the C-Jun binder exhibits ~1.5-fold selectivity for binding to the WT RNA as compared to the mutant RNA (Figure 1C).

To further analyze the specific interaction of C-Jun binder with WT RNA as compared to the mutant RNA, we subtracted the binding response values of interaction of C-Jun binder with mutant RNA from the WT RNA. The data was analyzed by 1:1 kinetic fit model and we observed a moderate affinity of C-Jun binder for the WT RNA over the mutant RNA (Figure 1D).

Figure 1: of small molecule (C-Jun binder) and RNA interaction by surface plasmon resonance (SPR).

A. Differential folding of wild type (WT) and mutant C-Jun mRNA as analyzed by the RNA-fold server. The WT RNA possess a unique kink (encircled in red), which is absent in the mutant RNA.

B. Dose-dependent sensorgrams for the interaction of the small molecule C-Jun binder with mutant and wild type mRNA. The extent of interaction is indicated as Response units and is reference corrected (i.e. the response of C-Jun binder binding to the biotin blocked surface is subtracted from the active flow cell i.e. wild type or mutant RNA). The C-Jun binder is used at a maximum concentration of 200 µM maximum concentration with a two-fold serial dilution. The data is fit using the 1:1 kinetic model. 

C. The same binding data is fit in an affinity model and exhibits binding saturation at higher concentrations i.e. 100 and 200 µM.

D. The binding affinity and specificity of the C-Jun binder with WT and mutant RNA was represented as binding response of mutant RNA subtracted from WT RNA. The data was fit as 1:1 kinetic binding model.

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. 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.

The biological outcome of such RNA-binding small molecules can be tuned by appending to them a functional group that can recruit and locally activate a Ribonuclease. Such bi-functional molecules, named RiboTACs, can selectively target and degrade specific RNA molecules inside cells. The effects of such a molecule can be seen at both the protein and mRNA level of Jun.

Using RT-qPCR, we can see that the Jun RiboTAC can robustly reduces the levels of Jun mRNA present in the cells, while the Jun binder lacking the ribonuclease recruiter has no effect on mRNA levels.

Figure 3: Jun mRNA RT-qPCR results from DMSO (control), Jun binder at 5uM and Jun ribotac at 5uM. Normalised to DMSO using GAPDH as housekeeping gene.

This reduction is also observed at the protein level. Using the JESS automated western blotting system, we see that Jun protein levels are depleted upon treatment with the Jun RiboTAC – an effect not observed with the Jun-binding small molecule

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.