Using the above knowledge we attempted to create a supershifting pseudoknot (Figure 2 ). To this end we substituted G for C20, removed C24 and restored the G2-C18 and C10-G32 base pairs, as present in the SRV-1 pseudoknot. To our surprise this mutant was 3-fold less active in frameshifting than the parent SF206 pseudoknot. Close inspection of the sequence indicated a possible alternative structure containing two hairpins competing with pseudoknot formation (Figure 2 ). To prevent this alternative structure, we mutated C10-G32 to U-A (SF223) or G-C (SF224). In these constructs the U-A pair is predicted to destabilize the 4-and 7-bp hairpins and the G-C base pair is predicted to completely disrupt the 4-bp hairpin and destabilize the 7-bp hairpin. Frameshift levels of SF224 increased approximately 2-fold compared to SF222, while that of SF223 increased more than 3-fold suggesting that the role of alternative structure formation should not be underestimated.