Objective: To determine whether CAG trinucleotide repeat number intrinsically governs liquid-liquid phase separation (LLPS) of single-stranded DNA (ssDNA).

Background: LLPS has emerged as a fundamental mechanism underlying biomolecular condensate formation. Expansions of trinucleotide repeats are associated with multiple inherited neurodegenerative disorders, and characteristically manifest only when repeat counts exceed a critical threshold. Whether repeat number alone serves as an intrinsic determinant of ssDNA phase behavior, independent of protein cofactors, remains poorly understood.

Method: Fluorescently labeled ssDNAs containing 10 or 33 CAG triplet repeats (10×CAG and 33×CAG) were synthesized. Control ssDNAs of equivalent length (~250 bases), with 50% GC content and similar base composition to 33×CAG, were used for comparison. CAG repeats were cloned via sequential repeat-directed elongation in a modified vector. Molecular crowding agents (spermine, spermidine, poly-L-lysine) were employed to promote condensate formation. Droplet formation was assessed by fluorescence and differential interference contrast microscopy; material properties were characterized by time-lapse imaging of fusion events, Ostwald ripening, and fluorescence recovery after photobleaching (FRAP).

Results: Escalating CAG repeat number conferred an elevated capacity for droplet nucleation and growth relative to sequence-matched controls, establishing a repeat-length-dependent enhancement of LLPS. 33×CAG ssDNA constructs formed spherical droplets exhibiting inter-droplet coalescence and Ostwald ripening, confirming liquid-phase thermodynamic properties. FRAP analysis demonstrated rapid fluorescence recovery with an estimated half-life of 3.75 seconds, indicating high internal molecular mobility consistent with a liquid condensate state.

Conclusion: Our findings propose that repetitive ssDNA sequences possibly drive condensate assembly in a repeat-length-sensitive manner, and that the resulting liquid droplets are competent to undergo further liquid-to-solid transition. Repeat expansions appear to provide templates for multivalent base-pairing, driving phase separation at pathologically relevant repeat numbers. These findings suggest that sequence-specific phase transitions of DNA may represent a contributing molecular mechanism in TRDs, offering a nucleic acid-centric perspective on condensate-driven neurodegeneration.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/triplet-repeat-length-determines-the-liquid-liquid-phase-separation-propensity-of-single-dtranded-dna/