Sequence-controlled RNA self-processing: computational design, biochemical analysis, and visualization by AFM

Abstract

Reversible chemistry allowing for assembly and disassembly of molecular entities is important for biological self-organization. Thus, ribozymes that support both cleavage and formation of phosphodiester bonds may have contributed to the emergence of functional diversity and increasing complexity of regulatory RNAs in early life. We have previously engineered a variant of the hairpin ribozyme that shows how ribozymes may have circularized or extended their own length by forming concatemers. Using the Vienna RNA package, we now optimized this hairpin ribozyme variant and selected four different RNA sequences that were expected to circularize more efficiently or form longer concatemers upon transcription. (Two-dimensional) PAGE analysis confirms that (i) all four selected ribozymes are catalytically active and (ii) high yields of cyclic species are obtained. AFM imaging in combination with RNA structure prediction enabled us to calculate the distributions of monomers and selfconcatenated dimers and trimers. Our results show that computationally optimized molecules do form reasonable amounts of trimers, which has not been observed for the original system so far, and we demonstrate that the combination of theoretical prediction, biochemical and physical analysis is a promising approach toward accurate prediction of ribozyme behavior and design of ribozymes with predefined functions.

Predicting RNA 3D structure using a coarse-grain helix-centered model

“…We suggest that such a model can also be useful for the elucidation and identification of different RNA species in atomic-force microscopy images where the positions of the individual atoms are largely indistinguishable (Petkovic et al 2015). Given fluorescence resonance energy transfer (FRET) data, the structures generated by our model can provide the experimentalist with an overview of the global structure of the RNA molecule without the overwhelming precision (and uncertainty) of an all-atom model.…”

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.…”

“…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 .…”

In vitro circularization of RNA

“…Previously, we have engineered hairpin ribozyme variants that fold into 2 alternative cleavage active conformations, thus cutting off their 5 0 -and 3 0 -terminus, and thereby producing a 5 0 -OH group and a 3 0 -terminal 2 0 ,3 0 -cyclic phosphate. 29,75,76 These characteristic ends are required for ligation, which then can take place in an intramolecular manner as depicted in Fig. 3.…”

“…We have demonstrated hairpin ribozyme supported RNA circularization for the generation of 83mers. 29,75,76 However, as shown in Fig. 3, the hairpin ribozyme structure may be embedded in RNA sequences of variable length (gray lines), thus paving the way toward preparation of large RNA circles.…”