HDV Family of Self-Cleaving Ribozymes - Progress in Molecular Biology and Translational Science - page 132

of the molecule before and after the reaction. 77,86,87 When the ribozyme is
labeled at the top of P2 and bottom of P4 helices, an inhibited precursor
ribozyme showed compaction upon Mg 2 þ binding, whereas the product
form showed extension of the molecule (approximately 50 and approxi-
mately 66 ˚ , respectively). These results suggest that the presence of the
leader sequence in the precursor ribozyme causes bending in the structure
such that the distal ends of P2 and P4 are closer before the reaction and
further apart in the product form. Similar data were obtained for a trans -
cleaving version of the ribozyme. 88 Interestingly, mutation of the active-site
cytosine affects the overall shape of the molecule. Mg 2 þ -dependent con-
formational changes differed for the C75 and C75U variants, where the
wild-type ribozyme showed shorter P2-P4 end-to-end distance in both
precursor and product states. 87
5. RIBOZYMES OUTSIDE OF THE HEPATITIS DELTA VIRUS
5.1. The CPEB3 ribozyme
The first HDV-like ribozyme isolated outside of the virus was found embed-
ded within the second intron of the mammalian gene encoding cytoplasmic
polyadenylation element binding protein 3 ( CPEB3 ). 2 Although the mam-
malian CPEB3 ribozymes are identical to the HDV molecule in terms of
secondary structure, they are quite divergent at the sequence level. Identi-
fication of the CPEB3 ribozyme was thus not possible with sequence-based
approaches. Instead, a novel in vitro selection technique was employed to
isolate self-cleaving molecules from their genomic sources.
5.2. Selection scheme for CPEB3 ribozyme identification
In a typical in vitro selection, a large pool of either synthetic or genomic
sequences containing known regions flanking a tract of random nucleotides
is evolved in vitro to contain a very small subset of the starting sequences that
exhibit a desired trait. 89-91 This trait can be any selectable molecular pheno-
type, fromligandbinding to enzymatic catalysis, so long as there exists amech-
anismto distinguish betweenmolecules with andwithout the chosen activity.
For selections of ligand-binding RNA or DNA sequences, discrimina-
tion between active and inactive sequences can be achieved simply by
flowing the pool through a column containing the target ligand. Sequences
unable to bind the target are washed through, whereas those capable of bind-
ing can be competitively eluted from the column. 92 This activity-enriched
pool is then either directly polymerase chain reaction (PCR) amplified via