Computational design and experimental verification of pseudoknotted ribozymes

Abstract

The design of new RNA sequences that retain the function of a model RNA structure is a challenge in bioinformatics because of the structural complexity of these molecules. RNA can fold into secondary and tertiary structure by forming stem loops and pseudoknots. A pseudoknot is a set of base pairs between a region within a stem-loop and nucleotides outside of this stem-loop; this motif is very important for numerous functional structures. It is important for any computational design algorithm to take into account these interactions to give a reliable result for any structures that include pseudoknots. In our study, we validated synthetic ribozymes designed by Enzymer which implements algorithms allowing for the design of pseudoknots. Ribozymes are catalytic RNAs that have activities similar to those of enzymes. Ribozymes like the hammerhead and the glmS have a self-cleaving activity that allows them to liberate the new RNA genome copy during rolling-circle replication or to control the expression of the downstream genes, respectively. We demonstrated the efficiency of Enzymer by showing that the pseudoknotted hammerhead and glmS ribozymes it designed were extensively modified compared to wild-type sequences and were still active.