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We’ve collaborated with Professor Matthew Disney at the Wertheim UF Scripps Institute for Biomedical Innovation & Technology in Florida, US, to use our scalable magnetic force spectroscopy (MFS) single molecule interactomics platform to explore compounds targeting an RNA repeat expansion in neurological disease.

The new study, published as a preprint on bioRxiv and submitted to a high impact peer reviewed journal, demonstrates how our unique technology provides valuable insights into biomolecular interactions and the effects of small molecules on these dynamics to accelerate the development of novel therapeutics.

What are repeat expansion disorders?

Repeat expansion disorders are a group of more than 40 hereditary neurological and neuromuscular diseases, including Huntington’s disease, myotonic dystrophy type 1, and spinocerebellar ataxias (SCA).

These disorders are caused by the presence of unusually long stretches of repeated three base sequences in particular genes. Repeat sequence RNAs (e.g., CUG, CAG) resulting from these expansions can cause toxicity by translating into poly-amino acid repeats or folding into stable three-dimensional structures that interfere with gene expression and cellular functions.

These devastating incurable conditions urgently require novel therapeutic approaches. One promising strategy involves targeting these aberrant RNA structures or the proteins that bind them with small molecules in order to mitigate their impact.

Exploring novel therapeutic approaches for CAG expansion diseases

Huntington’s disease and several spinocerebellar ataxias are characterized by expanded CAG triplets, resulting in aberrant RNA structures.

These structures are bound by essential cellular proteins, such as muscleblind-like splicing regulator 1 (MBNL1), a regulator of alternative RNA splicing. The entrapment of MBNL1 leads to splicing defects, contributing to disease pathogenesis.

In this study, Disney and colleagues set out to dissect the binding and mechanisms of action of three small molecules that target the CAG expansion.

Exploring biomolecular interactions with scalable MFS

NMR experiments were carried out to determine how each of the three compounds binds to a single CAG repeat RNA at a detailed atomic level.

We then used our large-scale single molecule MFS technology to study larger 21 CAG repeats (r(CAG)21) as well as CUG repeats (r(CUG)21) which also sequester MBNL1, causing the disease myotonic dystrophy type 1. We then used MFS to probe the biomolecular interactions between these RNAs and MBNL1, assess the effect of binding of the three compounds to r(CAG)21, and carry out three-way binding experiments to explore the impact of each compound on MBNL1 binding to r(CAG)21.

Force ramp experiments, which apply a gradually increasing force to the folded RNA strands, revealed that r(CUG)21 RNA unfolds at higher forces than r(CAG)21 which is consistent with known thermodynamics (Figure 1A). Force ramp experiments also showed that MBNL1 binding decreases the force required to unfold and refold both r(CAG)21 and r(CUG)21 structures, demonstrating that MBNL1 destabilizes these RNAs (Figure 1B). We also showed that MBNL1 preferentially binds to single-stranded CAG and CUG repeats, with a higher affinity for r(CUG)21 than r(CAG)21.

Next, we analyzed the effect of each of the three small molecules on folding and unfolding of the r(CAG)21 structure. Results revealed that none of the three compounds significantly altered the structural stability of the RNA, although deeper analysis indicated that compound 3 and possibly compound 2 may interfere with RNA refolding.

We then investigated the effect of each of the compounds on the interaction between r(CAG)21 and MBNL1 at two different concentrations (10 and 100 µM).

• Compound 1: At both concentrations, no discernible effect on MBNL1’s destabilizing impact on r(CAG)21 unfolding was observed. However at 100 µM, compound 1 made it easier for the RNA to refold in the presence of MBNL1. Probability analysis of unfolding and refolding suggested that compound 1 may inhibit MBNL1 binding by reducing the availability of CAG repeats.
• Compound 2: At 100 µM, compound 2 enhanced MBNL1’s effect on unfolding, possibly by increasing MBNL1’s binding rate. However, its impact on refolding was variable.
• Compound 3: While compound 3 facilitated MBNL1 binding during unfolding, it had inconsistent effects on refolding. This suggests it may stabilize the RNA-protein complex and/or hinder RNA refolding, allowing more MBNL1 binding.

These MFS observations confirm that compound 1 prevents MBNL1 protein from binding to r(CAG)21 by either competing with MBNL1, or inhibiting binding. The effects of compound 2 were more variable and difficult to interpret, as the hypothesized mechanism of action (binding to and stabilizing the internal amino acid loop) was not clearly confirmed by the MFS experiments. Compound 3, however, was shown by MFS to enhance MBNL1 protein binding to r(CAG)21, most likely during the unfolding stage.