Stem-loop 1 structure is required to maintain repeat A silencing ability. Silencing ability of 2-mer repeat A construct is retained when forced to form stem-loop 1 structure, but abrogated when pairing between the monomers is enforced. (A) Sequence of the canonical repeat A 2-mer and four mutant constructs that either enforce formation of stem-loop 1 (A1, A2) or an alternative folding (B1, B2) of repeat A sequences, as indicated by schematics. Dashes indicate no change in sequence. (B) Mean Enhanced Green Fluorescent Protein gene (EGFP) expression following five days of transgene induction, measured by flow cytometry, relative to d0 (two-tailed paired t-test). Error bars indicate ± 1 S.D. of two independent single-cell clones and a total of seven biological replicates.