PhD defence Ivo Severins (CJ Lab)

The sequence-dependence of biomolecular interactions involving nucleic acids and proteins is essential for numerous processes inside the cell. Insights into the underlying molecular mechanisms have been obtained using various biochemical and biophysical methods on two different levels — bulk and single-molecule. These have complemented each other as single-molecule studies excel in observing multi-state dynamic interactions, but perform only at low throughput; while bulk studies can probe many different sequences simultaneously, but providing limited kinetic information. To unite the strengths of both levels, we developed high-throughput Single-molecule Parallel Analysis for Rapid eXploration of Sequence space (SPARXS), that allows the study of molecular structure, kinetics and interactions for thousands of different sequences simultaneously at the single-molecule level. We, for the first time, combine single-molecule fluorescence with next-generation Illumina sequencing. As a proof of principle we apply SPARXS to study the sequence-dependent kinetics of the four-way DNA Holliday junction, occurring among others during homologous recombination and double strand break repair. Using SPARXS we observe the dynamic behavior of 120,000 Holliday junction molecules covering 3750 distinct core sequences, a result unattainable with previous techniques. We found that the purine-pyrimidine distribution and the GC content at the junction core influence the total transition rates through their stacking forces. Overall, the mechanistic insights obtained using SPARXS will give an entirely new view on the relationship between sequence, structure and function.

Promotors:
Chirlmin Joo and John van Noort (Universiteit Leiden)