Engineering a family of synthetic splicing ribozymes

Abstract

Controlling RNA splicing opens up possibilities for the synthetic biologist. The Tetrahymena ribozyme is a model group I self-splicing ribozyme that has been shown to be useful in synthetic circuits. To create additional splicing ribozymes that can function in synthetic circuits, we generated synthetic ribozyme variants by rationally mutating the Tetrahymena ribozyme. We present an alignment visualization for the ribozyme termed as structure information diagram that is similar to a sequence logo but with alignment data mapped on to secondary structure information. Using the alignment data and known biochemical information about the Tetrahymena ribozyme, we designed synthetic ribozymes with different primary sequences without altering the secondary structure. One synthetic ribozyme with 110 nt mutated retained 12% splicing efficiency in vivo. The results indicate that our biochemical understanding of the ribozyme is accurate enough to engineer a family of active splicing ribozymes with similar secondary structure but different primary sequences.

Figure 1.

The secondary structure of the Tetrahymena ribozyme consists of a series of paired helical regions. The canonical numbering is based on the wild-type ribozyme.

Figure 2.

The structure information diagram from the group IC1 ribozyme alignment is mapped to the Tetrahymena secondary structure. At each position, the most common base (consensus) is shown. See text for details.

Figure 3.

The sequence of a designed ribozyme is shown. Red bases indicate ‘harmless’ positions and blue bases indicate ‘likely mutable’ positions (as defined in text). Lowercase letters indicate bases that were swapped from the wild-type ribozyme sequence in Figure 1.

Figure 4.

For the non-starred synthetic ribozymes in Table 1, the number of nucleotides changed is plotted versus splicing efficiency. The splicing efficiency dropped linearly as more nucleotides were changed.