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Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disorder caused by an mRNA splicing defect that folds the r(CCUG)gene into an extended hairpin with periodically repeating internal loops. A compound has now been designed that improves DM2-associated defects using information about RNA–small molecule interactions. Structural analysis of the three internal loops reveals that the loop in L1 is stabilized by one hydrogen bond and a water-mediated hydrogen bond, while L2 and L3 loops are stabilized by two hydrogen bonds. Molecular dynamics reveal that the CU pairs are stabilized by Na+ and water molecules.

Most Detailed Picture Yet of Muscular Dystrophy

Scientists from the Florida campus of The Scripps Research Institute have created an atomic-level view of a genetic defect causing a form of muscular dystrophy, md type 2. Such a discovery may lead to the design of drugs with the potential to counter and/or reverse the disease.

“This the first time the structure of the RNA defect that causes this disease has been determined,” said TSRI Associate Professor Matthew Disney, who led the study. “Based on these results, we designed compounds that, even in small amounts, significantly improve disease-associated defects in treated cells.”

Myotonic dystrophy type 2 is a relatively rare form of muscular dystrophy that is somewhat milder than myotonic dystrophy type 1, the most common adult-onset form of the disease.

Both types of myotonic dystrophy are inherited disorders that involve progressive muscle wasting and weakness, and both are caused by a type of genetic defect known as a “RNA repeat expansion,” a series of nucleotides repeated more times than normal in an individual’s genetic code. The repeat binds to the protein MBNL1, rendering it inactive and resulting in RNA splicing abnormalities—which lead to the disease.

Many other researchers had tried to find the atomic-level structure of the myotonic dystrophy 2 repeat, but had run into technical difficulties. In a technique called X-ray crystallography, which is used to find detailed structural information, scientists manipulate a molecule so that a crystal forms. This crystal is then placed in a beam of X-rays, which diffract when they strike the atoms in the crystal. Based on the pattern of diffraction, scientists can then reconstruct the shape of the original molecule.

Prior to the new research, which was published in an advance, online issue of the journal ACS Chemical Biology, scientists had not been able to crystallize the problematic RNA.

The Scripps Florida team spent several years on the problem and succeeded in engineering the RNA to have crystal contacts in different positions. This allowed the RNA to be crystallized—and its structure to be revealed.

Using information about the RNA’s structure and movement, the scientists were able to design molecules to improve RNA function.

The new findings were confirmed using sophisticated computational models that show precisely how the small molecules interact with and alter the RNA structure over time. Those predictive models matched what the scientists found in the study—that these new compounds bind to the repeat structure in a predictable and easily reproducible way, attacking the cause of the disease.

“We used a bottom-up approach, by first understanding how the small components of the RNA structure interact with small molecules,” said Jessica Childs-Disney of TSRI, who was first author of the paper with Ilyas Yildirim of Northwestern University. “The fact that our compounds improve the defects shows that our unconventional approach works.”

The study, “Myotonic Dystrophy Type 2 RNA: Structural Studies and Designed Small Molecules that Modulate RNA Function,” is published in Chemical Biology, a publication of the American Chemical Society (ACS).

Abstract
Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disorder caused by a r(CCUG) expansion (r(CCUG)exp) that folds into an extended hairpin with periodically repeating 2×2 nucleotide internal loops (5′CCUG/3′GUCC). We designed multivalent compounds that improve DM2-associated defects using information about RNA–small molecule interactions. We also report the first crystal structure of r(CCUG) repeats refined to 2.35 Å. Structural analysis of the three 5′CCUG/3′GUCC repeat internal loops (L) reveals that the CU pairs in L1 are each stabilized by one hydrogen bond and a water-mediated hydrogen bond, while CU pairs in L2 and L3 are stabilized by two hydrogen bonds. Molecular dynamics (MD) simulations reveal that the CU pairs are dynamic and stabilized by Na+ and water molecules. MD simulations of the binding of the small molecule to r(CCUG) repeats reveal that the lowest free energy binding mode occurs via the major groove, in which one C residue is unstacked and the cross-strand nucleotides are displaced. Moreover, we modeled the binding of our dimeric compound to two 5′CCUG/3′GUCC motifs, which shows that the scaffold on which the RNA-binding modules are displayed provides an optimal distance to span two adjacent loops.

Authors
Jessica L. Childs-Disney †, Ilyas Yildirim ‡, HaJeung Park †§, Jeremy R. Lohman †, Lirui Guan †, Tuan Tran †, Partha Sarkar , George C. Schatz ‡, and Matthew D. Disney *†

†Department of Chemistry and §Translational Research Institute, The Scripps Research Institute, 130 Scripps Way, Jupiter, Florida 33458, United States

‡ Department of Chemistry, Northwestern University, 2145 Sheridan Road, Evanston, Illinois 60208-3113, United States

The study was supported by the National Institutes of Health (grant R01 GM079235), the Muscular Dystrophy Association (grant 254929), TSRI and the PS-OC Center of the NIH (grant 1U54CA143869-01).

About The Scripps Research Institute
The Scripps Research Institute (TSRI) is one of the world's largest independent, not-for-profit organizations focusing on research in the biomedical sciences. TSRI is internationally recognized for its contributions to science and health, including its role in laying the foundation for new treatments for cancer, rheumatoid arthritis, hemophilia, and other diseases. An institution that evolved from the Scripps Metabolic Clinic founded by philanthropist Ellen Browning Scripps in 1924, the institute now employs about 3,000 people on its campuses in La Jolla, CA, and Jupiter, FL, where its renowned scientists—including three Nobel laureates—work toward their next discoveries. The institute's graduate program, which awards PhD degrees in biology and chemistry, ranks among the top ten of its kind in the nation. For more information, see www.scripps.edu.