To test whether the C΄/D΄ motifs of snR13, snR48, U18 and U24 alone are capable of directing multi-site modification we inserted the C΄/D΄ motifs from these snoRNAs into an artificial snoRNA construct, based on the human U24 snoRNA, that we have previously described (24). In the control construct for these experiments, the C΄/D΄ motif from the human U24 snoRNA (which unlike its yeast counterpart follows the consensus standard) was placed adjacent to a guide sequence that targets nt S1316, an unmodified region of the yeast 18S rRNA (Figure 1D; nt with black background indicates the predicted target site). The C΄/D΄ motifs of snR13, U18 and U24 were cloned into the artificial snoRNA construct to target site S1315 based on the established 5-base-pair rule for 2΄-O-methylation (Figure 1D). If the C΄/D΄ motifs are capable of multiple modifications then site S1316 should also be modified. Since snR48 does not contain a recognisable D΄ motif, we cloned the C΄/D΄ motif such that it should, based on snR48 modification of its natural target site, target nucleotides S1316 and S1314. The constructs expressing the artificial snoRNAs were transformed into yeast cells. Northern blotting was used to demonstrate that the snoRNAs were all expressed to the expected levels (Supplementary Figure S2A). The modified nucleotide in the target region of the 18S rRNA was then determined by primer extension under reduced nucleotide concentrations where 2΄-O-methylated nucleotides cause the reverse transcriptase to terminate at a higher frequency.