Analyzing and Tuning Ribozyme Activity by Deep Sequencing To Modulate Gene Expression Level in Mammalian Cells

Self-cleaving
ribozymes, in combination with aptamers and various
classes of RNAs, have been heavily engineered to create RNA devices
to control gene expression. Although understanding of sequence–function
relationships of ribozymes is critical for such efforts, our current
knowledge of self-cleaving ribozymes is mostly limited to the results
from small scale mutational studies performed under different conditions,
or qualitative results of mutate-and-select experiments that may contain
experimental biases. Here, we applied our strategy based on deep sequencing
to comprehensively assay a large number of mutants to systematically
examine the effect of the P4 stem sequence on the activity of an HDV-like
ribozyme. We discovered that the ribozyme activity is highly sensitive
to the sequence and the apparent stability of the varied positions.
Furthermore, we demonstrated that the collection of the ribozyme variants
with different activities can be used as a convenient device to fine-tune
the level of gene expression in mammalian cells.