To examine if the mutants with disrupted base-triple interactions still adopt the pseudoknot conformation, non-denatured gel electrophoresis and enzymatic structure probing experiments were performed on CCC/GU RNA, and the results were compared with those of DU177 pseudoknot. Analysis of non-denatured gel confirmed that both RNA constructs adopt a major conformation ( Figure 3A ). However, distinct gel mobility of these two RNAs suggested that they might have different conformations. The enzymatic probing data are shown and summarized in Figure 3B -E. We used DU177 RNA as the standard for comparison because its NMR structure is well defined (33) . For both DU177 and CCC/GU RNAs, the distributions of cleavage patterns by ribonuclease T2, the probe for single-stranded region, are in agreement with the formation of the two loop regions. In addition, the distributions of cleavage patterns by RNase V1, the probe for duplex and stacked conformations, can be localized to the two predicted stem regions for both RNAs. Specifically, both RNAs possess signature V1 cleavage for the quasi-continuous helical region corresponding to the 3 0 -portion of stem 1 and the 5 0 -portion of stem 2 ( Figure 3B,D) . Therefore, these probing data indicate that CCC/GU RNA also adopts a pseudoknot conformation and suggest that the other mutants containing more base triples should also adopt the pseudoknot conformations. In the related UV-melting and singlemolecule mechanical unfolding experiments for DU177 and CCC/GU RNAs, transitions corresponding to the melting or mechanical unfolding of stems 1 and 2 were also observed and are consistent with the probing data (33, 38) . In addition, an extra transition corresponding to the disruption of tertiary interaction network was also reported in single-molecule unfolding studies and can only be observed in DU177 RNA (38) . Taken together with the mutagenesis studies above, the results of these experiments suggest that DU177 RNA can be converted into a À1 PRF-incompetent pseudoknot by disruption of the triplex structure spanning the helical junction.