The most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) is an expanded G 4 C 2 RNA repeat [r(G 4 C 2 ) exp ] in chromosome 9 open reading frame 72 (C9orf72), which elicits pathology through several mechanisms. Here, we developed and characterized a small molecule for targeted degradation of r(G 4 C 2 ) exp . The compound was able to selectively bind r(G 4 C 2 ) exp 's structure and to assemble an endogenous nuclease onto the target, provoking removal of the transcript by native RNA quality control mechanisms. In c9ALS patientderived spinal neurons, the compound selectively degraded the mutant C9orf72 allele with limited off-targets and reduced quantities of toxic dipeptide repeat proteins (DPRs) translated from r(G 4 C 2 ) exp . In vivo work in a rodent model showed that abundance of both the mutant allele harboring the repeat expansion and DPRs were selectively reduced by this compound. These results demonstrate that targeted small-molecule degradation of r(G 4 C 2 ) exp is a strategy for mitigating c9ALS/FTD-associated pathologies and studying disease-associated pathways in preclinical models.

Genetically defined amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), collectively named c9ALS/FTD, are triggered by hexanucleotide GGGGCC repeat expansions [r­(G4C2)exp] within the C9orf72 gene. In these diseases, neuronal loss occurs through an interplay of deleterious phenotypes, including r­(G4C2)exp RNA gain-of-function mechanisms. Herein, we identified a benzimidazole derivative, CB096, that specifically binds to a repeating 1 × 1 GG internal loop structure, 5′CGG/3′GGC, that is formed when r­(G4C2)exp folds. Structure–activity relationship (SAR) studies and molecular dynamics (MD) simulations were used to define the molecular interactions formed between CB096 and r­(G4C2)exp that results in the rescue of disease-associated pathways. Overall, this study reveals a unique structural feature within r­(G4C2)exp that can be exploited for the development of lead medicines and chemical probes.

A solid-phase DNA-encoded library (DEL) was studied for binding the RNA repeat expansion r(CUG)exp, the causative agent of the most common form of adult-onset muscular dystrophy, myotonic dystrophy type 1 (DM1). A variety of uncharged and novel RNA binders were identified to selectively bind r(CUG)exp by using a two-color flow cytometry screen. The cellular activity of one binder was augmented by attaching it with a module that directly cleaves r(CUG)exp. In DM1 patient-derived muscle cells, the compound specifically bound r(CUG)exp and allele-specifically eliminated r(CUG)exp, improving disease-associated defects. The approaches herein can be used to identify and optimize ligands and bind RNA that can be further augmented for functionality including degradation.

The hexanucleotide repeat expansion GGGGCC [r­(G4C2)exp] within intron 1 of C9orf72 causes genetically defined amyotrophic lateral sclerosis and frontotemporal dementia, collectively named c9ALS/FTD. , the repeat expansion causes neurodegeneration via deleterious phenotypes stemming from r­(G4C2)exp RNA gain- and loss-of-function mechanisms. The r­(G4C2)exp RNA folds into both a hairpin structure with repeating 1 × 1 nucleotide GG internal loops and a G-quadruplex structure. Here, we report the identification of a small molecule (CB253) that selectively binds the hairpin form of r­(G4C2)exp. Interestingly, the small molecule binds to a previously unobserved conformation in which the RNA forms 2 × 2 nucleotide GG internal loops, as revealed by a series of binding and structural studies. NMR and molecular dynamics simulations suggest that the r­(G4C2)exp hairpin interconverts between 1 × 1 and 2 × 2 internal loops through the process of strand slippage. We provide experimental evidence that CB253 binding indeed shifts the equilibrium toward the 2 × 2 GG internal loop conformation, inhibiting mechanisms that drive c9ALS/FTD pathobiology, such as repeat-associated non-ATG translation formation of stress granules and defective nucleocytoplasmic transport in various cellular models of c9ALS/FTD.

Since conventional computers are straining to handle the increased size and sophistication of non-numeric processing (data management, information retrieval, artificial intelligence), a new class of non-numeric architectures is evolving. The segment sequential architecture is one of these. Further development of this architecture requires new techniques for multiple cell operation and intercell communication to handle control and search operations. This paper describes such techniques for instruction fetching, operand recall, string, set and tree context searching, and pointer transfer. It is expected that combinations of these techniques will appear in future architectures that are needed for non-numeric processing.