Ribozyme cDNA constructs designed against NUH↓ cleavage sites in accessible and inaccessible regions (see Table 1) were directionally ligated into the Sal I/Pst I sites in pNEB-VAI-hhRz-1 or pNEB-VAI-hhRz-2 vectors. pNEB-VAI-hhRz-1 and pNEB-VAI-hhRz-2 vectors were generated by cloning the gene for VAI as a BssHII-XbaI fragment from pAdVAntage (E1711; Promega) into pNEB193-T7 (modified by us from pNEB193 from New England Biolabs to have a T7 promoter immediately upstream of the multiple cloning site) and then making extensive further modifications.10 (link),35 (link) Modified stem-loop structures (designed using secondary structure analysis) were added as a series of adapters. Details on the construction of the pNEB-VAI-hhRz-1 plasmid, also known as pUC-VAL, was previously described.17 (link) Details on the construction of the pNEB-VAI-hhRz-2 construct scaffold (pPrislei) are presented (Supplementary Materials). All constructs were confirmed by DNA sequencing. Because there is a strong intragenic RNA-Pol-III promoter (A, B boxes) in the VAI sequence, both in vitro transcription (with upstream T7 promoter) and in cellula transcription can occur from the same plasmid for either type of construct. Expected RNA structures of native VAI RNA and those of the two engineered VAI hhRz scaffold constructs are shown (Fig. 1).