Abstract
Nanotechnology and synthetic biology currently constitute one of the most innovative, interdisciplinary fields of research, poised to radically transform society in the 21st century. This paper concerns the synthetic design of ribonucleic acid molecules, using our recent algorithm, RNAiFold, which can determine all RNA sequences whose minimum free energy secondary structure is a user-specified target structure. Using RNAiFold, we design ten cis-cleaving hammerhead ribozymes, all of which are shown to be functional by a cleavage assay. We additionally use RNAiFold to design a functional cis-cleaving hammerhead as a modular unit of a synthetic larger RNA. Analysis of kinetics on this small set of hammerheads suggests that cleavage rate of computationally designed ribozymes may be correlated with positional entropy, ensemble defect, structural flexibility/rigidity and related measures. Artificial ribozymes have been designed in the past either manually or by SELEX (Systematic Evolution of Ligands by Exponential Enrichment); however, this appears to be the first purely computational design and experimental validation of novel functional ribozymes. RNAiFold is available at http://bioinformatics.bc.edu/clotelab/RNAiFold/.
Highlights
Ribonucleic acid enzymes (a.k.a. ribozymes) are catalytic RNAs with enzymatic capabilities that, similar to their protein counterparts, can catalyze and accelerate the rate of biochemical reactions while maintaining a great specificity with respect to the substrate they act upon
Given the target Rfam consensus structure S of Peach Latent Mosaic Viroid (PLMVd) AJ005312.1/282-335, which is identical with the minimum free energy (MFE) secondary structure using RNAfold 1.8.5, 16 highly conserved positions nucleotides were taken as constraints in the generation of over one million sequences solving the inverse folding problem, as determined by RNAiFold 1.8.5
Time series for cleavage fraction and kinetics curves for a typical designed hammerhead ribozyme (HH1) and the fastest designed ribozyme (HH7) are shown in Figure 5, while similar figures for the remaining designed hammerheads appear in Supplementary Information (SI) Figure 2
Summary
Ribonucleic acid enzymes (a.k.a. ribozymes) are catalytic RNAs with enzymatic capabilities that, similar to their protein counterparts, can catalyze and accelerate the rate of biochemical reactions while maintaining a great specificity with respect to the substrate they act upon. Several laboratories have successfully produced synthetic RNA sequences capable of self-cleaving, sensing small molecules in vivo or in vitro, as well as regulating gene expression [8, 9]. Many of these efforts have focused on the creation of allosteric ribozymes, or gene regulatory elements that can be used for further application
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
