High-Throughput Mutational Analysis of a Twister Ribozyme

Abstract

Recent discoveries of new classes of self-cleaving ribozymes in diverse organisms have triggered renewed interest in the chemistry and biology of ribozymes. Functional analysis and engineering of ribozymes often involve performing biochemical assays on multiple ribozyme mutants. However, because each ribozyme mutant must be individually prepared and assayed, the number and variety of mutants that can be studied are severely limited. All of the single and double mutants of a twister ribozyme (a total of 10 296 mutants) were generated and assayed for their self-cleaving activity by exploiting deep sequencing to count the numbers of cleaved and uncleaved sequences for every mutant. Interestingly, we found that the ribozyme is highly robust against mutations such that 71 % and 30 % of all single and double mutants, respectively, retain detectable activity under the assay conditions. It was also observed that the structural elements that comprise the ribozyme exhibit distinct sensitivity to mutations.

Keywords: RNA; deep sequencing; high-throughput assays; ribozymes.

Figure 1

A) Structure of the Osa‐1‐4 ribozyme based on the crystal structure described by Liu et al.6 The nucleotides are numbered according to Ref. 6. The arrowhead indicates the cleavage site. The bases shown in red are more than 97 % conserved among twister ribozymes.5 B) llustration of the library preparation procedure.

Figure 2

Visualizations of the activity of the ribozyme mutants. A) Relative activity (RA) of 144 single mutants of the Osa‐1‐4 ribozyme. B) Two‐dimensional RA matrix of all single and double mutants of the Osa‐1‐4 ribozyme. Single mutants are represented on the diagonal side. Several characteristic clusters of double mutants are shown in the boxes and are discussed in the main text. The full RA matrix with numerical RA data and mutations is provided in the Supporting Information.