Biological solution conditions and flanking sequence modulate LLPS of RNA G-quadruplex structures

Visualization of LLPS and aggregation for 5′-flanking GQs by confocal microscopy. Shown are 5′-mixed (column 1), 5′-A₉ (column 2), 5′-C₉ (column 3), and 5′-U₉ (column 4). Each row indicates a different condition: no polyamines (row 1), biological 0.4 mM spermidine and 0.3 mM spermine (row 2), and 0.3 mM spermine-only (row 3). All samples were in the background of 10 mM HEPES (pH 7.5) and K₁₅₀N₁₀M_0.5. Multiple 80 × 80 µm² fields were visualized, and representative images are shown. No LLPS was found in the absence of polyamines fields (row 1) or 5′-C₉ fields (column 3).