Prebiotic DNA replication and analyzing a genetic disorder with thermophoresis

The work presented in this Thesis is divided into three independent parts. Parts I and III are connected by the topic of the Origin of Life. Parts II and III are based on the same biophysical analysis method of microscale thermophoresis (MST). Part I focuses on prebiotic replication of DNA. Different approaches to replicate RNA and DNA under prebiotic conditions are discussed in the introduction. A nonenzymatic DNA replication system is presented as an approaches to a prebiotic replicator. Two DNA strands of 24 bases cross catalyze each other's synthesis from four 12 bases long substrate strands, two of which are in situ activated by a condensing agent 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide (EDC). DNA amplified (2.2~\pm~0.5)-fold in 6 hours. Stronger amplification is suppressed by EDC-dependent modification of substrate strands. Detailed kinetic modeling was realized with the system of ordinary differential equations and allowed to understand critical parameters of the reaction. Further research should be focused on finding ways to recycle substrate or to reduce its modification. This could lead to DNA replication and elongation experiments starting from short random DNA strands. Such experiments could be analyzed with next-generation sequencing due to the formation of the natural phosphodiester bonds at the ligation sites. In part II, MST is used to develop a diagnostic assay for α1-antitriypsin (AAT) deficiency disorder working directly in blood plasma. In AAT deficiency, individuals have reduced plasma concentration of AAT, which makes them susceptible to lung emphysema among other symptoms. Surprisingly, concentration of AAT does not correlate with the severity of the symptoms manifestation. In a standard MST approach, the amplitude of the obtained binding curve is not important for the affinity analysis. In an MST-based competition assay introduced in this work, the amplitude of the obtained binding curve is sensitive to both the affinity of AAT binding to its target and the concentration of AAT in plasma. In a proof-of-principle experiment, blood plasma of individuals with AAT deficiency was used to quantify the amplitude. The amplitude showed a better correlation to the severity of the disease status than the concentration of AAT alone, suggesting that AAT affinity to its target is different in different individuals. Direct measurement of AAT affinity to its target in plasma of individuals with strong vs weak manifestation of symptoms showed that affinity was higher in individuals who were only mildly affected. This suggests that plasma composition has an effect on AAT affinity. In part III, experimental systems are developed in order to test the stereochemical theory of the origin of the genetic code. The genetic code defines the assignment of 20 amino acids to their codons. The hypotheses to what determined the assignment have been widely discussed in literature but the question remains one of the experimentally most challenging in the origin of life field. The stereochemical theory states that amino acids have an intrinsic binding affinity towards their cognate codons. This can be tested by directly measuring the interaction between the activated amino acids and RNA structures containing codons. RNA structures were developed based on literature research, and the affinity was measured with the MST method. Proof-of-principle experiments for three RNA structures were performed, and the outline for future work is detailed in the end.