Final Report Summary - DNAMETRY (DNA based nanometry: Exploring chromatin structure and molecular motors)
Recent results of the ERC starting grant funded project include (i) the development of new single-molecule detection methods based on the combination of magnetic force spectroscopy and optical methods together with on purpose-tailored DNA nanostructures and (ii) application of these methods to unravel the mechanisms of enzymes involved in DNA metabolism. The project is centered at the interface between nanotechnology and molecular biology: Nanotechnological tools shall be used to understand design principles of biomolecules, biological nanostructures and biological motors, which in turn shall be used to develop smarter nanotools and functional elements.
Regarding the development of new single-molecule technology the following achievements were made: 1) A new setup that combines single-molecule fluorescence imaging and simultaneous operation and detection of a magnetic tweezers has been brought to operation. 2) This setup allows to use a DNA nanogear to move fluorescent particles with nanometer precision in a given direction, which was applied to calibrate fluorescent illumination fields. 3) A simple method for direct torque measurements based on magnetic tweezers was established that allows now so-called torque spectroscopy. 4) Mechanical measurements on single three-dimensional nanostructures could be established. These revealed an extra-ordinary mechanical stability of such structures, which paves the way for their use as mechanical sensors such as in ultra-fast torque spectroscopy. 5) Three-dimensional DNA structures were used as sensors to study the behavior of large objects on lipid membranes.
These technical developments as well as the expertise of the research group in single molecule experiments were employed to understand the behavior of single cellular proteins: 1) RecQ helicases fulfill essential and very specialized functions in DNA damage repair. It could now be shown that such closely related enzymes can exhibit antagonistic activities. While AtRecQ2 was found to unwind DNA hairpins a second helicase AtRecQ3 was preferentially closing DNA hairpin structures. This behavior may be the basis for the specialized functions of these enzymes. 2) Type III restriction enzymes employ helicase motor domains in order to destroy viral DNA. By direct observation of fluorescently labeled enzymes on DNA it could be revealed that the motor domains do not drive a directed motion but rather act as a switch in order to trigger a fast long-range diffusion of these enzymes on DNA that is a prerequisite for the later DNA destruction. 3) The eviction forces of nucleosomes (protein complexes that compact DNA in the cell nucleus) from DNA were studied and differences between different nucleosome variants were found. 4) The mechanism by which CRISPR-Cas enzymes recognize their targets could be revealed. During this recognition the RNA of these complexes hybridizes with target DNAs in a zipper-like fashion. Understanding the nature of the zipper mechanism will help to improve the targeting of these enzyme systems in genome engineering applications.
The long-term goal of the project is to develop better technology in order to obtain a more and more detailed understanding of molecular machines that full-full important functions within the cell. The project made already considerable progress in this direction and will be continued further in this sense. The future aim of the research group to apply the improved technologies directly within cells but also to learn from the mechanisms of single cellular machines in order to develop smart nanoobjects and tools.
Regarding the development of new single-molecule technology the following achievements were made: 1) A new setup that combines single-molecule fluorescence imaging and simultaneous operation and detection of a magnetic tweezers has been brought to operation. 2) This setup allows to use a DNA nanogear to move fluorescent particles with nanometer precision in a given direction, which was applied to calibrate fluorescent illumination fields. 3) A simple method for direct torque measurements based on magnetic tweezers was established that allows now so-called torque spectroscopy. 4) Mechanical measurements on single three-dimensional nanostructures could be established. These revealed an extra-ordinary mechanical stability of such structures, which paves the way for their use as mechanical sensors such as in ultra-fast torque spectroscopy. 5) Three-dimensional DNA structures were used as sensors to study the behavior of large objects on lipid membranes.
These technical developments as well as the expertise of the research group in single molecule experiments were employed to understand the behavior of single cellular proteins: 1) RecQ helicases fulfill essential and very specialized functions in DNA damage repair. It could now be shown that such closely related enzymes can exhibit antagonistic activities. While AtRecQ2 was found to unwind DNA hairpins a second helicase AtRecQ3 was preferentially closing DNA hairpin structures. This behavior may be the basis for the specialized functions of these enzymes. 2) Type III restriction enzymes employ helicase motor domains in order to destroy viral DNA. By direct observation of fluorescently labeled enzymes on DNA it could be revealed that the motor domains do not drive a directed motion but rather act as a switch in order to trigger a fast long-range diffusion of these enzymes on DNA that is a prerequisite for the later DNA destruction. 3) The eviction forces of nucleosomes (protein complexes that compact DNA in the cell nucleus) from DNA were studied and differences between different nucleosome variants were found. 4) The mechanism by which CRISPR-Cas enzymes recognize their targets could be revealed. During this recognition the RNA of these complexes hybridizes with target DNAs in a zipper-like fashion. Understanding the nature of the zipper mechanism will help to improve the targeting of these enzyme systems in genome engineering applications.
The long-term goal of the project is to develop better technology in order to obtain a more and more detailed understanding of molecular machines that full-full important functions within the cell. The project made already considerable progress in this direction and will be continued further in this sense. The future aim of the research group to apply the improved technologies directly within cells but also to learn from the mechanisms of single cellular machines in order to develop smart nanoobjects and tools.