Sequence and ionic requirements of pUG fold quadruplexes

Poly(UG) repeats or pUG RNAs can fold into a left-handed parallel quadruplex, the pUG fold. The pUG fold directs the epigenetic amplification of RNAi in <i>Caenorhabditis</i> elegans, and pUG sequences are abundant in eukaryotic transcriptomes. Here, we report the sequence and ionic requirements for pUG folding. The pUG fold preferentially incorporates 12 guanosines but has an otherwise flexible sequence requirement. The uridines can be substituted with other nucleotides, with some sequence variants folding better than pUG RNA. The GA repeat sequence (GA)₁₂ also forms a pUG-like fold, albeit with lower thermodynamic stability than (GU)₁₂. The pUG fold can tolerate multiple deoxyribose substitutions but does not fold when the backbone is entirely deoxyribose. It has a high affinity and specificity for potassium ions (<i>K</i>_1/2 = 6 mM) and does not fold in sodium or ammonium. Addition of 2 mM Mg²⁺ does not further stabilize the pUG fold, and the polyamines spermine and spermidine do not affect its stability. Finally, the pUG fold is sensitive to surrounding sequence context and complementary flanking sequences can stabilize pUG folds, while other sequences can interfere with folding. These data provide an improved ability to understand and predict pUG folds.