Hairpin ribozyme mediated RNA recombination

Abstract

We have engineered a hairpin ribozyme that in a two-step reaction supports RNA recombination by catalysing the cleavage of two non-functional RNA precursors (first step) followed by the recombination of two of the cleavage fragments into a functional RNA (second step). The recombination product (yield: 76%) is a fully functional hammerhead ribozyme.

Engineering of hairpin ribozyme variants for RNA recombination and splicing

“…On the contrary, if fragments are less stably bound to the ribozyme, such that upon cleavage dissociation is fast, cleavage will be the major reaction outcome. This allows tuning the ribozyme for either cleavage or ligation by conformational control . The minimal motif would be the preferred structure if cleavage of an RNA substrate is desired, whereas stabilization of the ribozyme–substrate complex at the secondary and tertiary structure level would assist fragment ligation.…”

“…). Upon cleavage and product dissociation, fragments are rebound and ligated in a recombined fashion, thus delivering a fully functional hammerhead ribozyme with yields up to 76% . So far, reactions have been carried out with a ratio of ribozyme to the two initial substrates of 4:8:1, thus not really demonstrating multiple turnover conditions.…”

“…Upon cleavage and product dissociation, fragments are rebound and ligated in a recombined fashion, thus delivering a fully functional hammerhead ribozyme with yields up to 76% . So far, reactions have been carried out with a ratio of ribozyme to the two initial substrates of 4:8:1, thus not really demonstrating multiple turnover conditions. One may assume that the first part of the reaction (cleavage of substrates) would proceed with multiple turnover, whereas the second part of the reaction (ligation of recombined fragments) would produce again a rather stable ribozyme–product complex and thus encounter the same problem of product inhibition as discussed above for the twin ribozyme.…”

“…Apart from replication, other functionalities that contribute to higher genetic complexity and extended functional space are of equally high relevance to RNA world scenarios. Along this line, we have engineered a number of hairpin ribozyme variants that mediate RNA processing reactions in diverse scenarios, including recombination, splicing, circularization (back‐splicing), and oligomerization . The hairpin ribozyme is a rather small RNA that can make itself from even shorter fragments, which cooperate to form a functional complex .…”

“…For the example of the hairpin ribozyme, we have demonstrated by conformational control that the inherent cleavage doi: 10.1111/nyas.14052 and ligation activity can be tuned to support diverse RNA processing pathways, [7][8][9][10][11][12]20 which in the RNA world may have contributed to (1) the emergence of RNAs with catalytic activity, (2) the extension of genetic space by oligomerization and/or recombination, and (3) the control of activity by splicing or allosteric regulation. Here, we review our latest efforts to engineer hairpin ribozymes into variants for RNA recombination, 9 regular RNA splicing, and back-splicing. 10,12 The hairpin ribozyme…”