A Rational Approach to Targeting Unstable RNA Repeats

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PROJECT SUMMARY A number of genetic disorders, including myotonic dystrophy (DM1 and DM2), fragile X tremor/ataxia, Huntingtin's disease, Machado-Joseph disease, spinocerebella ataxia, and more recently, amyotrophic lateral sclerosis (ALS, also known in the U.S. as Lou Gehrig's disease), occur as the result of unstable repeat expansion. For many of these repeat sequences, once transcribed they adopt a peculiar hairpin structure that closely resembles the binding site of an RNA splicing factor muscleblind-like 1 (MBNL1) protein. Sequestration of MBNL1 not results in the loss of its function, but also traps the gene transcript from being exported to the cytoplasm and from being translated into a functional protein. It has been demonstrated by several labs that disruption of the RNAexp-MBNL1 complex leads to reversion in the disease phenotype, in particular DM1. While MBNL1 has long been recognized as a bona fide therapeutic target of DM1, it remains a challenge to design molecules that can recognize and bind CUGexp, the cause of DM1, with high affinity, sequence-specificity, and selectivity, and displace MBNL1. The proposed research aims at developing a novel molecular platform for targeting CUGexp, as a proof-of-concept for treating neurodegenerative diseases associated with repeat expansion. Aim 1. Synthesis of chemical building blocks and the corresponding ligands. In the preliminary study we have performed MD simulations and carried out chemical reactions demonstrating the validity of the design concept and the feasibility of the synthetic routes. In the proposed study, we will scale up the monomer production and prepare the corresponding ligands for binding study. Aim 2. Determination of the binding properties of ligands. We will employ an array of biophysical methods, including UV-vis, CD, ITC, SPR, and electrophoretic mobility-shift assays to determine the binding properties of the newly designed ligands. Gaining a full understanding of the binding kinetics and thermodynamics is an important first step toward developing molecular therapies for treating the aforementioned genetic diseases. The proposed molecular design concept is general, applicable not only to targeting CUGexp, but also a slew of other repeated expansion sequences. If successfully developed, the proposed research will have far-reaching implication for the treatment of neurodegenerative diseases associated with repeat expansion.