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. 2025 Aug 29;20(9):2243–2254. doi: 10.1021/acschembio.5c00372

Streamlined Fragment-Based Discovery Platform for Targeting Structured RNAs

Yilin Jia †,‡, Amirhossein Taghavi †, Patrick R A Zanon †, Matthew D Disney †,‡,*
PMCID: PMC12455570  PMID: 40879339

Abstract

Fragment-based drug discovery typically relies on specialized spectrometric methods to identify low-affinity compounds that bind to biomolecules. Here, we report a proof-of-concept study on the development of a streamlined fragment-based screening platform for small molecules targeting RNA. This method employs low molecular weight fragments appended with a diazirine reactive moiety and an alkyne tag. Upon photolysis and click chemistry with an azide-containing fluorophore, these compounds can be visualized for binding to the r­(CUG) repeat expansion [r­(CUG)exp] implicated in myotonic dystrophy type 1 (DM1). Fragments were found to bind the 1 × 1 nucleotide U/U internal loops formed when r­(CUG)exp folds, guiding the design of homodimeric compounds capable of interacting with adjacent internal loops in a single molecule. One dimeric compound exhibited enhanced affinity and was converted into a proximity-induced covalent binder for prolonged target occupancy. This work establishes a versatile platform for targeting structured RNAs with potential applications across a variety of disease-relevant RNA targets.

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Introduction

Most of the human genome, although not translated into proteins, is actively transcribed into RNA, which is essential in a wide range of cellular processes through both coding and noncoding functions. RNA dysfunction and aberrant expression are often associated with the onset and progression of disease. RNA functionality is intrinsically tied to its structure, which affects its stability, localization, and interactions with proteins and other nucleic acids. , Therefore, targeting RNA structure with small molecules offers a promising approach to modulate gene expression associated with diseases and potentially expand the scope of druggable targets.

Fragment-based drug discovery (FBDD) has emerged as an effective alternative to conventional high-throughput screening, which typically involves large libraries of high molecular weight molecules. These molecules frequently present optimization challenges due to their size, structural complexity, and limited ligand efficiency. In contrast, FBDD focuses on the discovery of simple hit compounds that allow for efficient optimization into potent ligands and has been successfully applied to challenging biological targets. − However, applying FBDD to RNA targets introduces unique challenges due to the dynamic and flexible nature of RNA molecules as well as typically weak affinities and short residence times of the initial fragment hits. , Multiple biophysical − and chemical biology − methods have been used to identify fragment binders for RNA targets.

Various studies have demonstrated the feasibility of covalent small-molecule modification of RNA targets. − In this work, we utilized fully functionalized fragments (FFFs) to overcome the key limitations of FBDD. , These FFFs are equipped with diazirine photoaffinity labels that enable the capture of weak to moderate interactions through Ultraviolet (UV)-induced covalent modification of bound RNA targets. Further, an embedded alkyne handle facilitates the visualization and/or enrichment of the target molecule via click chemistry (Figure A). Here, by harnessing the advantages of photoaffinity modification and signal amplification through the generation of a fluorescence signal, we developed a platform for identifying fragments that target specific RNA structures with low to moderate binding affinity. These identified low-affinity RNA-binding fragments were then rationally optimized into potent ligands targeting RNA structures.

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Screening of FFFs for binding a r­(CUG) repeat by TAMRA labeling and gel electrophoresis. (A) Schematics of FFF probe architecture and general label-free or Cy5-labeled in-gel fluorescence screening workflow. Chemical structure of the control diazirine probe, 1 is also provided. (B) Left: secondary structure of r­(CUG)12, used for the first-round screening of each FFF at a concentration of 100 μM. Right: results of first-round screening (n = 1), affording 46 hits with labeling score >1, which were advanced to secondary validation at a concentration of 25 μM. Red data points: compound 1. (C) Left, secondary structures of the Cy5-labeled RNAs used in a counter-screen to explore fragment specificity. Right, results of the counter-screen (n = 1), affording 14 hits selected for validation of binding by NMR spectrometry. All TAMRA-labeling screening experiments were performed in 20 mM HEPES, pH 7.4.

For proof of concept, the pathogenic RNA element operating in myotonic dystrophy type 1 (DM1), r­(CUG)exp, was employed. This expanded triplet repeat forms stable structures within the 3′ untranslated region (UTR) of the dystrophia myotonic protein kinase (DMPK) mRNA, in particular a periodic array of 1 × 1 nucleotide U/U internal loops. These loops bind and sequester muscleblind-like 1 (MBNL1), a key protein that regulates alternative pre-mRNA splicing, leading to toxic gain-of-function effects and disruption of normal splicing processes. By using this RNA as a model system, an FBDD platform was developed to identify small molecules that bind r­(CUG)exp. These strategies are broadly applicable to a variety of RNA targets for which the identification and exploitation of druggable pockets is challenging.

Results

Development of a TAMRA-Labeling Screening Platform for RNA and Identification of 14 Hits That Selectively Label r­(CUG)

Previously, the screening of FFFs against RNA targets involved enriching radioactively labeled RNA using a method dubbed Chemical Cross-Linking and Isolation by Pull-Down (Chem-CLIP), , a process that was also relatively labor intensive. To address these limitations, a streamlined, RNA label-free screening method was developed. In the general workflow, each FFF was incubated with the target RNA, followed by UV irradiation and a subsequent click reaction to attach the fluorescent dye tetramethylrhodamine (TAMRA) azide to the RNA-fragment adduct. In order to remove interfering excess, unreacted TAMRA azide, the samples were cleaned up by solid phase-enhanced precipitation prior to analysis by gel electrophoresis. Fluorescence was quantified, followed by poststaining with SYBR Gold to normalize for total RNA loaded (Figure A). The diazirine probe with a propanamide group instead of a fragment ligand (1, Figure A) was used as a control for nonspecific labeling. A labeling score was calculated for each fragment by dividing the TAMRA signal intensity by the SYBR Gold signal intensity, where the ratio for control probe 1 was set to equal 1 (Figure A). Thus, a labeling score >1 indicates increased labeling due to the RNA-binding element. The score also serves as a quantitative metric to compare binding of the fragments to the RNA target across individual gels.

Enhancing the precision of RNA loading quantification, Cyanine 5 (Cy5) fluorophore-labeled RNA was utilized to circumvent the need for SYBR Gold staining, which can be less effective when FFFs exhibit strong covalent labeling. This phenomenon, observed as a weakened SYBR Gold signal in extensively TAMRA-labeled RNA, likely results from interference with SYBR Gold dye intercalation, as noted in our related studies. Consequently, we further shortened the workflow by eliminating the poststaining step (Figure A). Dual-color imaging for gel electrophoresis was implemented to simultaneously quantify fragment labeling and RNA loading, thereby optimizing both the accuracy and efficiency of our analysis.

A library of 187 FFFs tailored to interact with RNA structures was selected (Figure A). The potential RNA-binding elements of the fragments predominantly consist of heteroaromatic ring systems (Figure S1A), which can stack or hydrogen bond with RNA nucleobases. , In addition to these planar systems, the incorporation of aliphatic moieties contributes to the spatial configuration of the molecules and overall hydrophobic character. The chemical properties of the RNA-binding elements, excluding the diazirine and alkyne moieties, were also assessed to confirm the suitability for fragment-based drug design. Most compounds have a molecular weight below 300 Da (average M W = 266 ± 27 Da) and cLogP value <3 (average cLogP = 1.06 ± 1.06), with all compounds containing fewer than three hydrogen-bond donors (HBD; average HBD = 1.19 ± 0.62) (Figure S1B). These characteristics align with the fragment screening “Rule of Three”, which is further tuned to RNA recognition by including a greater number of hydrogen-bond acceptors (HBA; average HBA = 3.97 ± 1.13) (Figure S1B).

As aforementioned, the RNA target selected for these proof-of-concept studies was the triplet repeat expansion r­(CUG)exp that causes DM1, for which therapeutic strategies are largely limited to managing symptoms rather than addressing the underlying molecular cause. , Here, a validated model of the repeat expansion, r­(CUG)12, harbors five 1 × 1 nucleotide U/U internal loops that are bound by MBNL1 (Figure B, left). At a concentration of 100 μM, 46 compounds exhibited a labeling score >1 (Figure B, right), and secondary validation at a concentration of 25 μM of FFF afforded 25 compounds showing ≥1.8-fold increase in labeling score (Figure S2A). Here, both the first-round screen and secondary validation were performed using agarose gel electrophoresis to enable higher-throughput sample processing and SYBR gold staining.

The specificity of the binding fragments was subsequently evaluated by studying binding to a control base-paired RNA construct where 1 × 1 nucleotide U/U internal loops were replaced with A/U base pairs (Figure C, left). In this counter-screen, polyacrylamide gel electrophoresis (PAGE) and Cy5-labeled RNAs were used to improve resolution as well as provide direct and accurate quantification, affording 14 hit fragments with >2.5-fold higher labeling scores to r­(CUG)12 and a selectivity ratio (normalized TAMRA to Cy5 signal for CUG relative to the base-paired control) > 2 (Figures C, right, S2B, and C). These 14 hits (hit rate ∼7.5%) were selected for further validation of direct interaction with the r­(CUG) repeat using nuclear magnetic resonance (NMR) spectrometry.

Physicochemical analyses were completed to compare fragments that bind r­(CUG)12 at a concentration of 100 μM vs. nonbinding fragments and fragments that bind r­(CUG)12 at a concentration of 25 μM vs. nonbinding fragments at the same concentration (Table S1 and Figures S3A). Only the binding elements were considered in these analyses; that is, the diazirine and alkyne were excluded. When comparing fragments that bind r­(CUG)12 at both the 100 and 25 μM doses, four parameters distinguish them from nonbinders: the number of aromatic atoms (p = 0.0015 and p = 0.0002, respectively); number of aromatic rings (p = 0.0002 and p < 0.0001); fraction of sp3 hybridized carbons (p = 0.0072 and p = 0.0152); and polar surface area (p = 0.039 and p = 0.0041). The hits contain a significantly lower fraction of sp3 hybridized carbons and greater numbers of aromatic atoms and rings, suggesting a preference for planar structures and the importance of stacking interactions in RNA binding. An analysis of the different monocyclic heterocycle frequencies in 25 hits and 162 nonhits revealed potential preferred functional binding elements that interact with r­(CUG)exp, such as imidazole and pyridazine (Figure S3B). In general, the structural features enriched among these compounds align with characteristics reported for RNA-binding small molecules in larger, previously published data sets. Moreover, these findings provide guidelines for the design of RNA-focused fragment libraries.

NMR Studies Identified 2 as a Modest r­(CUG) Repeat Binder

Ligand-observed 1H NMR experiments were performed to study the noncovalent interactions of the fragments and the RNA target. , For each compound, 1D 1H NMR spectra were recorded in the absence and presence of r­(CUG)12 (compound/RNA = 20:1) without UV irradiation. The following techniques were employed to study binding: (1) chemical shift perturbations (CSPs) and line broadening (LB), which reflect changes of the ligand proton chemical environment and exchange dynamics upon RNA binding; (2) Carr–Purcell–Meiboom–Gill (CPMG), − which distinguishes the differences in transverse relaxation time (T2) of unbound free ligands (long T2, sharp NMR signal) and bound ligands (short T2, broad NMR signal); and (3) Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY), which differentiates Nuclear Overhauser Effect (NOE) magnetization transfer of unbound ligands (positive NOE from water) and bound ligands (negative NOE from water via RNA), leading to oppositely phased NMR signals (Figure A).

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Study of FFF-r­(CUG) repeat interactions via NMR spectrometry. (A) Scheme of ligand-observed NMR experiments to study the interactions between RNA and small molecules. (B) Chemical structure of the four hit compounds exhibiting interactions with r­(CUG)12 by ligand-observed NMR experiments. (C) 1D 1H NMR spectra of 2 (150 μM) in the presence and absence of r­(CUG)12 (7.5 μM), which demonstrated the strongest interactions of the four fragments (Buffer: 10 mM sodium phosphate, pH 6.0 and 0.1 mM EDTA).

Initial NMR experiments were performed in a low salt buffer (10 mM sodium phosphate, pH 6.0, and 0.1 mM EDTA), as these low molecular-weight fragments are expected to exhibit weak binding affinity that could be masked by electrostatic screening under higher ionic strength conditions. , Notably, four of the 14 specific fragments (2-5) demonstrated interactions with r­(CUG)12, as defined as evidence of binding in at least two of the three NMR-based experiments (Figures B, S4, and S5). Among the four compounds, fragment 2 met the most stringent criteria, exhibiting marked CSP (>6 Hz) and LB, disappearance of aromatic signals in T2-CPMG (>20%), and signal inversion in WaterLOGSY studies (Figure C). Therefore, compound 2 was selected for further investigation.

Consequently, the RNA-binding elements within FFF 2, that is, without diazirine and alkyne functionality, were synthesized, affording 6, a propanamide form of the fragment monomer (Figure A). To study the interaction of 6 with the r­(CUG) repeat, an RNA-observed 1D 1H NMR experiment by monitoring the imino protons of uracil and guanine was utilized (Figure B). It has previously been shown that the imino protons within the 1 × 1 nucleotide U/U internal loop are dynamic and sensitive to perturbation upon small molecule binding, thereby inducing chemical shift perturbations or reductions in peak intensity (exchange broadening).

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Evaluation of the binding of fragment 6 to the 1 × 1 nucleotide U/U internal loops present in r­(CUG)exp and optimization of 6 into PEG homodimer. (A) Chemical structure of 6, the RNA-binding element derived from FFF 2. (B) Scheme of the RNA-observed imino proton NMR experiments to study target engagement of the small molecule. (C) Imino proton spectra of r­(CUG)4 as a function of 6 concentration, indicating modest but specific interactions with the 1 × 1 nucleotide U/U loops. (D) Strategy of fragment dimerization to obtain a ligand with increased potency and selectivity. (E) Chemical structure of the PEG5 dimer 7. (F) Imino proton spectra of r­(CUG)4 as a function of 7 concentration, where enhanced (as compared to 6) and specific interactions with the 1 × 1 nucleotide U/U internal loops were observed (Buffer: 10 mM sodium phosphate, pH 6.0 and 0.1 mM EDTA).

To enable clear peak assignments and assess binding specificity, a simplified r­(CUG)4 RNA with two adjacent U/U loops and a GAAA tetraloop was utilized (Figure C), also serving as a minimal model for evaluating dimeric ligands in the subsequent optimization strategy. The proton spectra of r­(CUG)4 were recorded in the absence and presence of 6. Addition of 0.5 and 1 equiv of 6 relative to the number of 1 × 1 nucleotide U/U binding sites resulted in modest engagement of the RNA, as evidenced from a reduction of peak intensity of two sets of peak at ∼10 ppm (U8/22 and U5/19) that correspond to the two 1 × 1 U/U internal loops (Figure C). Compound 6 also showed peak reduction and chemical shift perturbations of the adjacent guanine bases, which form the loops’ closing base pairs (G6/20, G9/23). It is noteworthy that 6 showed minimal effect on imino protons of other uracils and guanines outside the two 1 × 1 nucleotide U/U internal loop motifs (U11, U25, and G10/24). These data suggest that 6 is a modest but specific binder of the 1 × 1 nucleotide U/U internal loop, encouraging further structural optimization of 6 to enhance its binding affinity to r­(CUG)exp. Overall, the chemical shift perturbation and line broadening of adjacent guanines and cytosines indicate changes in the local chemical environment of the adjacent bases, possibly by formation of stacking interactions. Taken together with the planar and aromatic ring-rich features of the FFF library, we hypothesize that the preferable binding mode for these fragments targeting r­(CUG) might be insertion into the site where the 1 × 1 nucleotide U/U internal loop and stacking with the uridines and adjacent bases.

PEG-Linked Dimerization Improved Spatial Engagement with r­(CUG)exp

Fragment linking is a core strategy in FBDD to obtain lead compounds with enhanced affinity and selectivity. This strategy involves linking weak-affinity fragments that bind to adjacent sites on a target molecule, minimizing the system’s free energy and improving binding characteristics. Strategic formation of heterodimers engaging adjacent and distinct binding sites of the target RNA has been shown to significantly enhance both potency and bioactivity. Furthermore, considerable progress has been made to address toxic repeat RNAs with small-molecule homodimers incorporating appropriate linkers (Figure D). ,− Thus, to develop a more potent ligand for r­(CUG)exp based on 6, a dimerization strategy was employed, hypothesizing that two adjacent 1 × 1 nucleotide U/U internal loops within the r­(CUG) repeat could be bound simultaneously.

For the rational design of such a dimeric binder, structures of r­(CUG) repeats, elucidated by NMR spectrometry (PDB: 5VH8 ) or X-ray crystallography (PDB: 1ZEV ) were analyzed and indicated that two consecutive U/U loops are spanned by an approximate linear distance of 12–15 Å. Taking into account the flexibility and curvature of the RNA groove, we estimated that a linker approximately 1.5-fold of this length would be ideal. Incorporating a tether with five polyethylene glycol (PEG) units, which affords ∼23 Å, homodimer 7 was synthesized (Figure E). Imino proton NMR spectra confirmed the enhanced binding of 7 compared to the monomeric ligand (Figure F). At equivalent concentrations of the binding motifs compared to 6, 7 exhibited a greater extent of 1 × 1 nucleotide U/U internal loop U8/22 and U5/19 peak intensity decrease (80 vs. 30% reduction), as well as a more prominent decrease for G6/20, indicating an interaction of the linker with the groove between the two 1 × 1 nucleotide U/U internal loops (80 vs. 25% reduction), while maintaining the minimal interactions with bases outside of the 1 × 1 nucleotide U/U internal loop region (Figure F).

Upon addition of 50 mM NaCl to the NMR buffer, the interactions of both 6 and 7 with r­(CUG)4 were significantly reduced (Figure S6), indicating their overall modest binding affinity and underscoring the need for further optimization. For example, at 1:1 equivalence of binding elements relative to the number of U/U loops, 6 and 7 resulted in 5 and 15% reductions in U8/22 and U5/19 peak intensity, respectively. Nonetheless, these results suggest that dimerization of 6 with a PEG5 linker enables the engagement of adjacent 1 × 1 nucleotide U/U internal loops, considerably improving the binding interactions of the initial hit fragment.

Polyamine-Linked Dimerization Substantially Enhanced Selective Ligand Binding to r­(CUG)exp

Given that the PEG5 linker enabled favorable positioning of the binding modules, we sought to further enhance binding affinity by exploiting potential interactions of the linker with the RNA. Polyamines are basic amine-containing molecules biosynthesized in living cells that naturally bind to nucleic acids and modulate RNA structure. , Potentially leveraging the electrostatic interactions between the cationic polyamines and the RNA phosphate backbone at physiological pH, two polyamine dimers linked by putrescine and alkyl chains of two different lengths were synthesized (8 and 9), aiming to harness these interactions for enhanced RNA binding (Figure A).

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Structural optimization of polyamine dimers and biochemical evaluation of 8. (A) Chemical structures of two polyamine dimers, 8 and 9. (B) Imino proton spectra of r­(CUG)4 RNA as a function of 8 concentration, where potent and specific interactions with the 1 × 1 nucleotide U/U internal loops were observed. (C) Imino proton spectra of BP4 RNA with and without the addition of 50 μM of 8. The buffer in panels B and C was 10 mM sodium phosphate, pH 6.0, 50 mM NaCl, and 0.1 mM EDTA. (D) Thermal melting experiment of r­(CUG)10 alone and as a function of the concentration of 6, 7, or 8. Data points shown are three technical replicates from one independent experiment. (E) BLI interferogram to measure the binding of 8 binding to biotinylated r­(CUG)10, affording a K d of 12 ± 4 μM with a hill slope of 1.4 ± 0.3 to biotinylated r­(CUG)10. The curve shown is the average of three independent experiments. (Mean ± SD; buffer: 1.47 mM KH2PO4, 8.09 mM NaH2PO4, pH 7.3, 137.9 mM NaCl, 2.67 mM KCl, and 0.02% (v/v) Tween-20).

In imino proton NMR spectra using the higher ionic strength buffer (10 mM sodium phosphate, pH 6.0, 50 mM NaCl, and 0.1 mM EDTA), 8 demonstrated significant enhancement in 1 × 1 nucleotide U/U internal loop target engagement, effectively flattening the imino proton peaks at U8/22 and U5/19 at 1:1 equivalence and showing substantial line broadening and chemical shift perturbations near adjacent guanine bases (G6/20, G9, and G23), indicative of interactions of the linker within the RNA groove (Figure B). The weak changes in imino protons outside the 1 × 1 nucleotide U/U internal loop motifs (U11, U25, and G10/24) also demonstrated 8’s targeted interaction. In contrast, 9 showed less effective engagement of the 1 × 1 nucleotide U/U internal loops by showing less reduction in peak intensity and significantly increased nonspecific interactions as evidenced by severe line broadening and chemical shift perturbations of U11, U25, and G10/24 (Figure S7). The different behaviors of the two polyamine-linked dimers align well with our linker length hypothesis and rational design. Homodimer 8, with a longer linker (∼25 Å), comparable to that of compound 7, provides superior binding characteristics. Conversely, 9 with a short linker (∼18 Å) might not be able to span the two 1 × 1 nucleotide U/U internal loops. Collectively, these results emphasize that the optimal linker length is critical for the specificity and efficacy in targeting juxtaposed RNA structures.

To further evaluate the specificity of 8, an imino 1H NMR experiment with a base-paired RNA construct (BP4) was carried out. Only 15% of line broadening for uracils of the AU base pairs and neighboring G6 and G20 was observed (Figure C). These data demonstrated that dimerization using a polyamine linker of appropriate length significantly enhanced the binding affinity of the ligand to the r­(CUG) repeat while retaining specificity for the 1 × 1 nucleotide U/U internal loop.

As an orthogonal method to compare the interactions of the monomer 6 and homodimer 7 and 8 with the r­(CUG)exp repeat structure, thermal melting experiments were performed using a 5′-carboxyfluorescein (FAM) and 3′-Black Hole Quencher (BHQ) dually labeled r­(CUG)10 construct (five 1 × 1 nucleotide U/U internal loops; Figure S8A). In the folded state, FAM fluorescence is quenched due to the proximity to BHQ. As the RNA unfolds with increasing temperature, the distance between FAM and BHQ increases, resulting in an increase in fluorescence signal. The melting temperature (T m) of the RNA, which reflects the transition from the folded to unfolded state, can be determined by analyzing the derivative of the change in fluorescence signal.

Monomer 6 showed no effect on the T m of r­(CUG)10 at the concentration of 50 μM, while the PEG-linked dimer 7 modestly increased T m by 1.3 ± 0.5 °C at 50 μM. In contrast, the polyamine-linked dimer 8 exhibited a dose-dependent stabilization effect at low concentrations, effectively increasing T m by 3.1 ± 0.7 °C at a concentration as low as 5 μM (Figures D and S8B). These results are consistent with the NMR studies, confirming that rational dimerization provides substantial improvements in binding affinity and thermal stabilization over the monomeric compound, with the polyamine-tethered dimer exhibiting greater interactions with the RNA target than the PEG-linked analogue.

The dissociation constant (K d) of the optimized polyamine dimer 8 was measured by biolayer interferometry (BLI) using 5′-biotinylated r­(CUG)10 immobilized onto a streptavidin sensor tip; the corresponding base-paired control RNA lacking U/U internal loops was immobilized on the reference sensor to subtract nonspecific interactions (Figure S9A), as previously applied to other targets. Steady state analysis of the binding response of 8 in 1× Dulbecco’s phosphate-buffered saline (DPBS) supplemented with 0.02% (v/v) Tween 20 yielded a K d of 12 ± 4 μM (Figures G and S9B). In contrast, 6 showed no detectable binding signal by BLI under these conditions (Figure S9C), highlighting the advantage of the TAMRA-labeling screening platform in capturing modest-affinity fragment hits that would have been missed by direct biophysical screening alone. These data suggest that the optimization of the initial fragment binder into a dimeric ligand conferred a substantial increase in binding affinity that is retained under buffer conditions with physiological ionic strength.

Conversion of 8 to Covalent Modifier 12 Selectively Modified r­(CUG) Repeats In Vitro

Covalent RNA small-molecule binders represent a promising therapeutic strategy due to their ability to form irreversible or highly stable bonds with the target, which prolongs target engagement and increases target occupancy. , We aimed to convert the r­(CUG) repeat binders into covalent modifiers to prolong target occupancy of the RNA and further demonstrate a modular approach to the targeted covalent modification of RNA. N-Chloroethyl anilines are derived from chlorambucil and can form aziridinium species that predominantly react with N7-guanine, making it suitable for reacting with r­(CUG) repeats. An alkyne handle was further appended for functionalization, enabling, e.g., TAMRA-labeling assays as described above. In addition to a control probe lacking the RNA-binding ligand (10), reactive derivatives of monomer 6 and polyamine dimer 8 were designed and synthesized (11 and 12, respectively; Figure A).

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Design of N-chloroethyl aniline covalent binders and their evaluation for binding the r­(CUG) repeat in vitro. (A) Chemical structures of N-chloroethyl aniline probes, including a control probe, and r­(CUG) repeat probes based on monomer 6 and dimer 8. (B) TAMRA labeling of Cy5-r­(CUG)12 or fully base-paired control BP12 by 10, 11, or 12 (2 μM RNA, 37 °C, 18 h incubation; buffer: 20 mM HEPES, pH 7.4, and 50 mM NaCl). The extent of labeling was calculated by TAMRA/Cy5 ratio with normalization to 25 μM of 10. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001, as determined by a one-way Analysis of Variance (ANOVA) with multiple comparisons. (C) Representative MALDI-TOF mass spectra to analyze the reaction of probe 12 with r­(CUG)12 (left) or BP12 (right). (Reaction conditions: 10 μM RNA, 37 °C, 18 h incubation in a buffer containing 20 mM HEPES, pH 7.4, and 50 mM NaCl.)

The ability of 11 and 12 to modify r­(CUG) repeats and the specificity of the modification were assessed by the TAMRA-labeling assay. At concentrations of 25 μM and 50 μM, 11 showed labeling scores of ∼2.5 (relative to 10 at respective concentrations), while 12 exhibited significantly higher cross-linking to the r­(CUG)12 with labeling scores of 6.8 and 9.0, and little modification to the base-paired RNA (Figures B and S10A), in agreement with the binding affinity and selectivity of the noncovalent binders. This specific modification was further confirmed by MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-of-Flight) mass spectrometry. With an 18 h incubation at 37 °C, r­(CUG)12 was modified by 12 in a dose-dependent manner (Figure S10B). Over 60% of r­(CUG)12 (10 μM) was modified when incubated with 100 μM of 12 (2 equiv as compared to the concentration of internal loops), while the corresponding base-paired construct BP12 remained unmodified (Figure C). [Note: r­(CUG)12 contains five 1 × 1 nucleotide U/U internal loops, that is, two binding sites for the dimer. Thus, the mass corresponding to two adducts is expected, although cross-linking of one dimer may impede binding of the second sterically or by induction of structural changes in the RNA. Such an effect would be expected to be less pronounced in longer r­(CUG) repeats in disease contexts where more independent binding sites are available.] Significant, but not complete, modification of the RNA was observed. The extent of covalent modification depends not only on noncovalent binding affinity, but also on multiple factors, including the reactivity and kinetics of the covalent warhead. This includes the rate-limiting formation of the aziridinium intermediate from the N-chloroethyl group, the competing hydrolysis reactions, and the availability of suitable nucleophiles such as N7-guanine residues near the binding site, which can be affected by RNA structural dynamics.

To validate that selective modification of r­(CUG)12 by 12 is driven by specific molecular recognition rather than the intrinsic reactivity preference of the N-chloroethyl warhead, a competition experiment, monitored by MALDI-TOF mass spectrometry, was performed. Compound 12 (20 μM) was incubated simultaneously with r­(CUG)12 (10 μM) and increasing concentrations of BP12 (1, 2, and 4×). Notably, the presence of BP12 did not affect 12’s modification of r­(CUG)12, and no modification product of BP12 was detected under any condition (Figure S10C). These results demonstrated that covalent modification by 12 is indeed driven by structural recognition of the r­(CUG) repeat. Appending an electrophilic warhead to the optimized dimer therefore enabled selective and efficient modification to r­(CUG), leveraging the inherent selectivity of the noncovalent RNA-binding element. This strategy holds promise for developing irreversible RNA-targeted therapeutics with a prolonged target occupancy.

Discussion

In this study, we report a streamlined, nonradioactive platform for identifying small molecules that selectively engage structured RNA through noncovalent interactions as well as covalent modification. This TAMRA-labeling method allows for rapid screening of fragment-based libraries, offering a cost-effective, scalable alternative to traditional radioactive labeling techniques. , Our approach integrates gel-based quantification, dual-color imaging, and downstream NMR validation, enabling the efficient identification of selective RNA-binding fragments.

Among the 187 FFFs screened, 14 fragments that strongly and specifically labeled (labeling score >2.5, selectivity ratio >2) r­(CUG)12 were identified. Structurally, these hits were enriched for small aromatic scaffolds with heterocyclic functionalities, suggesting that hydrogen bonding and stacking interactions with the 1 × 1 nucleotide U/U internal loops are important for binding. Detailed analysis of molecular descriptors for these compounds will inform future design of more focused screening libraries optimized for RNA targeting. As more data are obtained using this platform, further elucidation of structural and chemical features that enhance RNA binding will be possible, which will in turn guide effective medicinal chemistry optimization to obtain more potent RNA-binding ligands.

Compound 2 emerged as a bona fide binder by NMR techniques, and NMR experiments confirmed that its binding is centralized to the 1 × 1 nucleotide U/U internal loops and adjacent guanineskey structural features of r­(CUG) repeat expansions. By stripping the diazirine and alkyne handles, a minimal recognition motif (6) that retained 1 × 1 nucleotide U/U internal loop selectivity was defined. Although compound 6 exhibited modest affinity, its targeted binding pattern served as a strong foundation for further optimization. Using fragment-linking strategies, we substantially improved the binding affinity and selectivity via homodimerization. A PEG5-linked dimer (7) improved engagement with the 1 × 1 nucleotide U/U internal loops, and a polyamine-linked dimer (8) showed even more pronounced interactions due to electrostatic stabilization of the RNA-ligand complex. Notably, 8 demonstrated target selectivity, enhanced thermal stabilization of r­(CUG) repeats, and micromolar binding affinity (K d = ∼10 μM).

To extend the pharmacological potential of these ligands, we designed covalent modifiers using an N-chloroethyl aniline warhead. − The reactive dimer (12) selectively alkylated r­(CUG)12 but not the base-paired RNA, while no cross-linking was observed for the electrophile lacking the RNA binder. More broadly, the synergistic platform can enable identification of binders to RNA that can be optimized through dimerization as well as covalency.

This platform is broadly applicable to other structured RNAs that cause disease and may help to uncover druggable pockets in these targets. RNA repeat expansions such as r­(CAG), r­(G4C2), and highly structured viral and noncoding RNAs are the logical next applications. While the fragments in the library are relatively simple and the properties analyzed represent broad physicochemical trends, we hypothesize that these features may reflect general principles of RNA recognition, which can be explored by studying a wide variety of RNA targets. Compared to widely used NMR- or mass spectrometry-based fragment screening methods that rely on specialized instrumentation, our platform significantly reduces equipment requirements, lowers cost, and improves throughput. Furthermore, in contrast to conventional fluorescence-based methods, our covalent modification strategy enables the capture of modest or transient interactions, improving sensitivity. Admittedly, as a gel-based assay, our method still requires manual sample loading. While we were able to screen hundreds of compounds within 2–3 days, larger libraries would require additional automation to maintain efficiency. Overall, this platform offers a generalized and practical screening method for early-stage RNA-targeted fragment discovery and is complementary to existing fragment-based screening methods.

Because our approach starts with small, fragment-like compounds, it opens the door to multiple optimization strategiesincluding modular assembly through hetero- or homodimerization, the latter as shown here for targeting RNA repeat expansions. Moreover, the use of covalent probes enables a streamlined transition to binding site mapping strategies via covalent adduct formation. Such structural information can be leveraged to rationally design hetero- or homodimers that link fragments targeting proximal but distinct sites within the RNA. In cases where a single dominant site is identified, fragment-to-lead optimization via file mining of related analogues or structure-based design guided by docking of RNA structural models could be effective next steps. The flexibility of this strategy is a major strength, allowing follow-up experiments to be tailored to the target’s structural features and tractability.

Importantly, this type of in vitro screening complements cell-based RNA-targeting efforts, such as recent studies using live-cell chemical probes to detect small-molecule engagement of structured RNAs in situ. , While cell-based platforms can capture specificity, cellular permeability, and functional relevance, they may miss lower-affinity binders or early-stage fragments that lack selectivity but could be useful in lead optimization campaigns. Our in vitro system enables direct detection of binding to defined RNA motifseven when selectivity is incompleteand is especially well suited to rigid, highly structured RNAs that maintain stable folds outside the cellular environment.

Together, in vitro and cellular screening strategies provide a synergistic path forward: in vitro assays help define and characterize ligandable pockets and guide early hit optimization, while cell-based approaches validate biological relevance and downstream activity. This dual-track approach is likely to be critical for the successful development of selective and potent RNA-targeted therapeutics. By validating both modular and covalent strategies for RNA engagement, this study provides a conceptual and technical roadmap for discovering first-in-class therapeutics targeting structured, disease-causing RNAs.

In conclusion, a streamlined platform for identifying low molecular weight fragments that specifically bind to RNA targets in a radioactivity-free, cost-effective, and time-efficient manner was developed. This approach not only simplifies the initial screening process but also integrates advanced validation methods such as the use of NMR techniques to study direct interactions. A key aspect was the optimization of an initially modest binder into a significantly more potent ligand through dimerization, illustrating a strategy that can be readily extended to other RNA repeat disorders. The dimerization approach also allows for the development of heterodimers that target distinct and proximal binding sites, which can be mapped by mutational analysis. Overall, our workflow is highly adaptable and suitable for a broad range of RNA targets. By covering both the discovery and optimization phases, this study provides a model for future research and applications in RNA-targeted small-molecule development. As a result, we envision that it will enable the ready identification of RNA binders and therefore foster the development of small molecules for the investigation and modulation of RNA structure and biology.

Methods

General Methods

All RNAs were purchased from Dharmacon (Horizon Discovery Bioscience Limited) with HPLC purification and deprotection from the manufacturer. RNA concentration was quantified by UV/vis spectrometry using its absorbance at 260 nm at 85 °C and the extinction coefficient provided by the manufacturer. The sequences of all RNA constructs used in this study and their corresponding applications are listed in Supporting Table S2. The fully functionalized fragment library was purchased with customization from Enamine.

TAMRA Labeling Assay

RNA was folded in 20 mM HEPES, pH 7.4 or 20 mM HEPES, pH 7.4 supplement with 50 mM NaCl by heating to 95 °C for 2 min, followed by cooling on ice for 15 min. For diazirine-containing compounds, 13.5 μL of 2.2 μM RNA was incubated with 1.5 μL of 1 or FFF at the desired concentration for 30 min at RT. The mixtures were then irradiated with UV light (Stratalinker 2400 Bulb, 365 nm) using a UVP cross-linker (UV Stratalinker 2400) for 20 min. For N-chloroethyl aniline-containing compounds, 13.5 μL of 2.2 μM RNA was incubated with 1.5 μL of compound at the desired concentration for 24 h at RT.

For click reaction to attach the fluorophore TAMRA, a 2.67 μL aliquot of a click reaction mixture containing TAMRA azide (4 equiv to alkyne-containing probes, same below, Vector Laboratories, catalog #CCT-AZ109), CuSO4 (10 equiv), THPTA (50 equiv), and sodium ascorbate (250 equiv) was added. The click reaction was incubated at 37 °C for 90 min. The RNA was cleaned up using the RNAClean XP beads (Beckman Coulter, catalog #A63987) according to the manufacturer’s protocol with the following adjustment: initial incubation of the RNA mixture with 1.8 volumes of beads was supplemented with 2.7 volumes of isopropanol with mixing by pipetting. All incubations, click reactions, and cleanup processes were completed in 384-well plate format (Greiner, catalog #781185) with muti-channel pipets. Following cleanup, the RNA was resolved on a 3% (w/v) agarose gel or a denaturing 15% (w/v) polyacrylamide gel using 1× Tris-boric acid-EDTA (TBE) buffer.

For assay completed with SYBR Gold staining, TAMRA fluorescence was first measured by imaging the gel using a Molecular Dynamics Typhoon 9000 instrument (Ex: 546 nm; Em: 579 nm). The gel was then stained with SYBR Gold (dilution factor 1:10000, Invitrogen, catalog #S11494) in 1× TBE for 10 min with gentle shaking and imaged to visualize the amount of RNA loaded (Ex: 496 nm; Em: 539 nm). For Cy5-labeled RNA constructs, the gel was imaged in the TAMRA and Cy5 channels (Ex: 651 nm; Em: 670 nm) using a ChemiDoc MP System. Quantification was performed by Image Lab Volume Tools Rectangle, with local subtraction.

NMR Spectrometry

Ligand-Observed 1H NMR Experiments

Ligand-observed 1H NMR experiment spectra were acquired on a Bruker Advance III 600 MHz spectrometer equipped with a cryoprobe at 298 K with 1024 scans for each experiment. The following pulse sequences and parameters were used:

  • 1.

    1D 1H NMR with water suppression (used to monitor CSP/LB): zgesgp, relaxation delay = 1.0 s.

  • 2.

    CPMG: cpmg_espg2d, time delay = 300 ms, relaxation delay = 2.0 s.

  • 3.

    WaterLOGSY: ephogsygpno.2, mixing time = 1.5 s, relaxation delay = 2.0 s. Spectra were processed without rephasing to preserve NOE-derived signal polarity.

The compound was dissolved in 5 μL of DMSO-d 6 and then diluted into 485 μL of 10 mM sodium phosphate, pH 6.0, and 0.1 mM EDTA containing 5% (v/v) D2O to afford a final concentration of 150 μM. After acquiring spectra for the compound alone, the solution was removed and then added to the RNA of interest, folded as follows. A 10 μL aliquot of 375 μM r­(CUG)12 was folded in 10 mM sodium phosphate, pH 6.0, and 0.1 mM EDTA by heating to 95 °C for 2 min, followed by cooling on ice for 15 min. After briefly centrifuging to collect the RNA, the compound solution was added to the RNA and gently mixed by pipetting. The sample was then transferred to an NMR tube for data collection (ratio compound: RNA = 20:1).

RNA-Observed Imino 1H NMR Experiments

RNA-observed 1H NMR spectra were acquired on a Bruker Advance III 700 MHz spectrometer equipped with a cryoprobe at 283 K by using the zgesgp pulse sequence with 512 scans and a relaxation delay of 1.5 s for each experiment. The RNA of interest (380 μL of 52.63 μM), r­(CUG)4 or the corresponding fully paired control RNA, was folded by heating to 95 °C for 2 min, followed by cooling on ice for 15 min in 1.05× of the indicated buffer (10 mM sodium phosphate, pH 6.0, and 0.1 mM EDTA with or without 50 mM NaCl). After folding, 20 μL of D2O was added (final 5% (v/v) D2O). The RNA was then transferred to an NMR tube for data acquisition. After collecting the imino 1H spectrum of the RNA alone, the compound of interest was added. Each compound was dissolved in DMSO-d 6, and then 2–4 μL aliquots were added to the RNA. After mixing the samples, imino 1H spectra were acquired, repeating the process if compound titration was performed.

Supplementary Material

cb5c00372_si_001.pdf (6.1MB, pdf)

Acknowledgments

This work was supported by the U.S. National Institutes of Health (R35 NS116846 to M.D.D.), the Muscular Dystrophy Association (MDA 1069959 to M.D.D. and Development Grant ID #963835 to A.T.), and the German Research Foundation (DFG) through a Walter Benjamin fellowship (#515396515 to P.R.A.Z). Purchase of the Bruker Avance III 600 MHz NMR instrument used in these studies was supported in part by the National Institutes of Health (S10 OD021550). We thank J. Childs-Disney for advice and critical review of the manuscript. The following graphical illustrations were created with Biorender (Components of Figure A: https://BioRender.com/3tdj2jj; Figure A: https://BioRender.com/ddgvg6o; Figure B: https://BioRender.com/knmzozc).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.5c00372.

  • Tables and figures; additional experimental details; oligonucleotides sequences; synthetic procedures, and characterizations (1H NMR, 13C NMR, HRMS, and HPLC spectra) of the new compounds (PDF)

The authors declare the following competing financial interest(s): M.D.D is a founder of Ribonaut Therapeutics.

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