Ligation of random pools and autonomous ribozymatic RNA replication in thermal non-equilibria

Salditt, Anna (2023): Ligation of random pools and autonomous ribozymatic RNA replication in thermal non-equilibria. Dissertation, LMU München: Fakultät für Physik

Abstract

A central quality of life is its ability to store, replicate, and translate (sequence) information. Naturally, this provokes the question of how replication emerged on early Earth. Due to the ability of RNA to store sequence information as well as to fold into catalytically active complexes (ribozymes), its replication and the search for ribozymes receive abiding interest. Efficient RNA replication is a prerequisite for open-ended evolution and RNA world scenarios, where ribozymes catalyze multiple chemical reactions. However, they face the same obstacle - the product-template inhibition after a successful copying step. How can thermal non-equilibria help overcome this obstacle? This thesis aims to show that thermal non-equilibrium systems not only help certain replication processes but even make them possible in the first place. In the first part, the templated ligation of random or semirandom pools of 12 nt long DNA oligomers by a DNA Taq polymerase is used as a model system for early non-enzymatic replication systems. Thermal cycling, a most simple nonequilibrium, enables templated ligation of these random pools to elongate to longer product strands with a reduced sequence space. The employed cycling conditions critically influence the outcome and can either hinder or promote the extension of random pools depending on the size of their sequence space. Further, spiking the system with subsets of sequences, each capable of forming its own replication network, alters the elongation behavior significantly. This demonstrates how biases in the starting pool are amplified and lead to different long sequences. In the second part of this work, two different geophysical scenarios of the early Earth are experimentally mimicked to accommodate replication with ribozymes. In the first setting, a cylindrical water-filled chamber with a point-like temperature source induces temperature cycles by convectively shuttling molecules between the hot temperature spot and the cold outer region. In this chamber, multiple rounds of PCR-like replication can be driven using 24-3 ribozyme polymerase. At the same time, the longer RNA is selectively accumulated away from higher temperatures, protecting the polymerase from high temperatures and associated hydrolysis. In the second setting, an air-water interface in a temperature gradient adds another level of complexity to the system. Within the same reaction compartment, molecules can now reside in different phases, salt concentrations, and temperatures, allowing different reactions, such as replication and separation of RNA strands without human intervention. Even complete replication cycles of RNA sequences complementary to the replicating sunY ribozyme are thus possible, indicating a pathway to autocatalytic selfreplication. Moreover, this setting allows for one-pot reactions that enable replication of an active hammerhead ribozyme, subsequent separation of template and copy, and secondary cleavage reaction. This is a critical step in an RNA world scenario where ribozymes must perform multiple catalytic functions in parallel.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: origin of life, RNA, replication, ligation, network, non-equilibrium, reaction compartments
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 530 Physik
• Fakultäten: Fakultät für Physik
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 14. Dezember 2023
• 1. Bericht­erstatter:in: Braun, Dieter