Group II introns

Szokoli, Deni (2026): Group II introns: computational de novo design and protein-free DNA hydrolysis and transesterifications. Dissertation, LMU München: Faculty of Biology

Abstract

Group II introns are ancient self-splicing ribozymes and retroelements. They are among the first and most complex ribozymes ever discovered, and are thought to have had an outsized effect in shaping Eukaryote evolution. Although they have long been speculated to be remnants of the RNA World, their reliance on intron-encoded proteins for self-splicing and retrotransposition has cast doubt on this hypothesis. Despite the fact that some introns are known to self-splice in the absence of proteins, there has thus far been no evidence that they are capable of fully reverse splicing into DNA targets without the aid of protein cofactors. In the first part of my dissertation, I demonstrate that a group II intron can perform all intron-catalyzed reactions completely independently of protein cofactors. Not only is the ribozyme capable of fully reverse splicing into single-stranded DNA in vitro, but it was also found that it was capable of hydrolyzing DNA substrates, and remarkably, of unwinding and reacting with stable dsDNA duplexes. I argue that by expanding their known catalytic repertoire beyond what would be required to survive the transition from RNA to DNA genomes, these results support the possibility of an RNA World origin of group II introns. This newfound capacity of group II introns as protein-free DNA processing enzymes has sparked renewed interest in their development as genome editors. I anticipate that the ability to design custom group II introns from scratch will be important to fully realize their biotechnological potential. However, computational approaches to design RNA catalysts have only ever been successful in generating small ribozymes. Methods for engineering large ribozymes, such as group II introns, remain underdeveloped, as their complexity and size have thus far discouraged any design efforts. In the second part of my dissertation, I used an RNA inverse folding algorithm to design group II introns de novo, resulting in three novel self-splicing ribozymes with exceptionally stable structures. One of these introns, Arq.I2, was revealed to be a highly active ribozyme in vitro, with self-splicing rates that are comparable to the fastest naturally occurring group II introns. Remarkably, even though most group II introns are believed to require a maturase protein under intracellular conditions, Arq.I2 was shown to self-splice in Escherichia coli cells. These results show that existing inverse folding algorithms are capable of generating novel, highly active variants of large and complex ribozymes with relative ease. This paves the way for the design of bespoke group II intron-derived ribozymes for their potential application in biotechnology and therapeutics. In light of these results, I review and discuss the evolution, biological function, and possible ancient origins of group II introns, as well as their potential for biotechnological application.

• Item Type: Theses (Dissertation, LMU Munich)
• Keywords: Group II introns, computational design, RNA, genome editors, ribozymes
• Subjects: 500 Natural sciences and mathematics; 500 Natural sciences and mathematics > 570 Life sciences
• Faculties: Faculty of Biology
• Language: English
• Date of oral examination: 9. April 2026
• 1. Referee: Griesbeck, Oliver
• Signature of the printed copy: 0001/UMC 32013