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.
Keywords: RNA engineering; deep sequencing; ribozyme.