Structural Polymorphism of (Cag)N Repeat RNA Elucidated using Single Molecule Nanomanipulation

Abstract

Abnormal expansions of CAG trinucleotide repeats are responsible for 9 hereditary human disorders including Huntington's disease and a variety of Spinocerebellar ataxias. Disease symptoms typically manifest when the number of repeats exceeds a given threshold (typically 35+ repeats). It has been hypothesized that genetic instability and RNA toxicity arise due to the structurally polymorphic nature of expanded repeats. However, it is technically difficult to study the structure and folding of a heterogeneous population of RNAs. To overcome this problem, we applied a single-molecule approach to monitor folding of individual RNAs with up to 100 CAG repeats using optical tweezers. Compared to RNAs with non-repetitive sequences, a (CAG)n RNA folds slowly and non-cooperatively in multiple back-and-forth small steps, suggesting that the molecule undergoes an extensive conformational search. Most surprisingly, as a (CAG)100 RNA is extended by >100 nm, tension on the molecule remains largely unchanged at ∼13.5±0.4 pN, which is not expected for an elastic polymer. This unusual viscoelasticity implies that force is unevenly distributed in the molecular structures of the (CAG)n RNAs which possibly take the form of multi-branched, similarly-sized hairpins. The relatively constant unfolding force indicates that the folding energy landscape is almost flat at the force, and the small force fluctuation suggests that the energetic barriers between different conformations are very low. When a (CAG)n RNA is nanomanipulated to be partially unfolded, the molecule is trapped in a subset of conformations, as evident by the constant value of the mean extension. This observation leads to a hypothesis that the folding of (CAG)n RNAs is dominated by topological constraints, and the existing conformation is affected by preceding structures. Our findings support the structural polymorphism hypothesis for the (CAG)n RNAs and provide evidence for sequential folding of repeated sequences.