We therefore next tested the importance of the last nucleotide of the guide and first nucleotide of the D΄ box in the human U24 C΄/D΄ motif, which modifies a single site when expressed in the context of our artificial snoRNA targeting S1316. Insertion of a U between the C and U of the D΄ box, changing the last nucleotide of the guide to C and the first nucleotide of the D΄ box to U (Figure 4C; D΄1 Uins) should, based on the five nucleotide rule, result in modification of S1315. However, this mutation resulted in the modification of position S1316 (Figure 4D). Importantly, when a U was inserted between the guide and the D΄ box site (Figure 4C; D΄-1 Uins) nucleotide S1315 was modified. This therefore supports our proposal that the last nucleotide of the guide can influence the site to be modified.