Complete RNA inverse folding: computational design of functional hammerhead ribozymes

TL;DR

This paper presents a computational method, RNAiFold, for designing functional hammerhead ribozymes, which are experimentally validated, marking a novel achievement in purely computational RNA design.

Contribution

The study introduces RNAiFold, a new algorithm capable of designing all RNA sequences with a specified structure, and demonstrates its effectiveness by creating functional hammerhead ribozymes.

Findings

Designed ten functional hammerhead ribozymes computationally

Validated ribozymes through cleavage assays confirming functionality

Identified potential correlations between cleavage rate and structural measures

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/.