Surprisingly, we saw modest GFP expression compared to a control with an inactive intron. G2Is tend to silence host genes by cleaving the mRNA, so lowered expression is not unexpected. These results indicate that the protein-free intron is active in E. coli cells. (8/n)

According to conventional wisdom, G2Is require protein cofactors to aid in self-splicing under intracellular conditions, hindering their application as protein-free genome editors.

We designed an assay for splicing-dependent fluorescent protein expression — a GFP gene interrupted by Arq.I2 (7/n)

Unlike typical de novo designed ribozymes, Arq.I2 outperforms most wild type variants characterized thus far! It reacts with fast kinetics, having a rate of splicing comparable to those of the fastest known natural G2Is! (6/n)

All three candidates were active in vitro, some even outperforming a variant of a natural intron.
Arq.I2 is a very unusual intron — it requires extreme measures to denature it for PAGE, and remains catalytically active in the absence of monovalent cations. (5/n)

In our latest paper we combined the inverse folding algorithm aRNAque and rational design to generate unique synthetic G2Is.
aRNAque's evolutionary algorithm was able to finetune intron folding, resulting in unusually stable structures. (4/n)

Perhaps the most unexpected finding was that the ribozyme was capable of reacting with stable dsDNA (incl. plasmids)! This activity was not thought possible in the absence of protein cofactors, as proteins have been deemed necessary to unwind stably duplexed nucleic acids. (5/7)

It turned out that the ribozyme readily hydrolyzed its DNA substrate, which explained the depletion of RSP at long timepoints. G2Is are the only ribozymes known to be capable of DNA hydrolysis. (4/7)

We found that a well-studied G2I was actually capable of complete reverse-splicing into ssDNA in vitro, in the absence of any proteins. Surprisingly, the time course of this reaction had a peak-shaped curve, hinting at the existence of a competing irreversible reaction. (3/7)

The ribozyme component catalyzes its integration (reverse splicing) into DNA genomes, but its reliance on protein cofactors to perform this activity has contributed to the view that G2Is evolved after the emergence of both translation and the transition to DNA genomes. (2/7)