Figure 7.

VAI-hhRz chimeric expression construct. (A) The VAI-hhRz chimera RNA is expressed from a gene with an intragenic Pol-III promoter (A and B boxes) and terminates after a polyuridine tetramer. The loop sequence into which the hhRz cDNA is cloned between the SalI and PstI sites is stabilized by a long (19 bp) GC rich stem. An adapter sequence inserted between and separating the SalI and PstI sites in the loop makes hhRz cDNA cloning much more efficient. Other than this adapter element, the overall sequence of the pGVAL vector was not altered from the vector which has been used successfully in prior studies (Lieber and Strauss, 1995). (B) The hhRz was cloned into an expected loop region engineered into the adenoviral VAI RNA (Lieber and Strauss, 1995). This VAI RNA has stable apical and basal stems and its central domain (functional) (box) was obviated by the engineered stem loop structure to hold the hhRz and create a flexible steric environment where it could function (box). (C). Stabilized hhRz design. A 7 nt/7 nt symmetrical antisense flank design was used centered on the intended NUH↓ cleavage motif. The catalytic core is shown using the established numbering scheme (Hertel et al., 1992). The nt in the core enzyme that is mutated to form a catalytically inactivated hhRz is also shown (G5C). Stem II was extended by 2 bp to add six hydrogen bonds to the helical stack in order to stabilize folding. This extended stem was capped with a single stranded 5' UUCG 3' loop that also promotes stabilization of the overlying stem structure. Stable folding of Stem II protects the catalytic domain from misfolding. Stem II is engineered with a rare 7 bp RsrII site that allows straightforward identification of successful hhRz cDNA ligation into pGVAL-ad.