Assembling a single-molecule view on nucleosome dynamics

The main focus of this thesis is a better understanding of the basic compaction mechanism of our DNA using multiple single-molecule techniques. The stretched-out length of our DNA is enormous compared with the dimensions of a cell. To make DNA fit within a cell it is systematically wrapped around proteins (histone octamers), forming nucleosomes....

Single-molecule studies of the twisted, knotted, and broken genome

This thesis describes a series of single-molecule experiments aimed at understanding the physical properties of DNA itself and the proteins that interact with it. We developed and applied sensitive techniques that allowed us to directly probe the conformation and interactions of individual DNA molecules and proteins. Magnetic and optical...

Seek and Destroy: Single-molecule perspective on the target search and recognition by the Cas9 endonuclease

The most commonly known relationship between the macromolecules of life – DNA, RNA and protein – is the central dogma which states that information flows from DNA to RNA through transcription and from RNA to protein through translation. The discovery of non-coding RNAs has altered and expanded this picture by revealing many more functions of RNA...

A Random Walk Towards the Golden Fleece: Single-molecule Investigations of Argonaute Target Search

In this thesis we used single-molecule FRET to investigate the kinetic properties of a protein called Argonaute. While traditionally one uses bulk methods to investigate the molecular properties of proteins, bulk methods do not confer information that is transient, since that is inherently averaged out. Single-molecule methods, as their name...

Intercalating Electron Dyes for TEM Visualization of DNA at the Single-Molecule Level

Staining compounds containing heavy elements (electron dyes) can facilitate the visualization of DNA and related biomolecules by using TEM. However, research into the synthesis and utilization of alternative electron dyes has been limited. Here, we report the synthesis of a novel DNA intercalator molecule, bis-acridine uranyl (BAU). NMR...