Research projects of NBP
Physics DNA and RNA : single molecule studies
In the past decade, local force measurement techniques such as atomic force microscopy and optical tweezers were used to study elastic properties and mechanically induced structural transitions of nucleic acids at the single molecule level. Single molecule manipulation was also increasingly used for investigations of DNA-dependent enzymatic processes, related to unfolding and modifying DNA, protein-DNA interactions, replication and transcription. Compared to classical techniques of molecular biology, single molecule measurements avoid the averaging over a large number of events and can thus potentially provide detailed and complementary information.
The nucleic acids DNA and RNA play a central role in biology and the understanding of nucleic acid related molecular processes is steadily growing. X-ray scattering, electron microscopy and nuclear magnetic resonance techniques are very important in this field, as they provide high resolution structures of RNA and of protein-nucleic acid complexes. This structural information has to be complemented by dynamic studies to reveal the conformations, energy landscapes, length and timescales of molecular rearrangements involved in the function of the macromolecule.
Single molecule manipulation of nucleic acids allows to apply an external mechanical constraint (force and/or torque) in a well defined way and to measure the time-dependent response in length, force, angle or torque. These measurements are usually done in solution and can provide access to the folding dynamics and the elastic properties of nucleic acids. Forces that are generated in the course of biochemical reactions can be measured and the reaction can be investigated as a function of an external mechanical load. We use optical tweezers to perform single molecule force measurements on DNA and RNA and two investigate DNA and RNA-dependent enzymatic processes.:
Transistor based detection of bio molecules
The international effort to sequence the human genome is well advanced. The information obtained in the form of a four letter alphabet represents an extremely precious basis for future investigations. Important forthcoming topics relate to the global analysis of the messenger RNA, the proteins, and the associated mechanisms of regulation; as well as the molecular characterisation of diseases. DNA and protein microarrays, which allow investigating many different molecular interactions in parallel, are two techniques which are particularly well suited for such global analysis
Hybridization, the specific recognition of DNA and RNA sequences by complementary base pairing, is central to fundamental biological processes like replication, transcription and translation and represents the basic principle of important techniques, like PCR and DNA microarrays. Our research aims DNA and protein microarrays with pure electronic detection, to avoid labelling, improve reliability and simplify read-out by integration with silicon based microelectronics. We already achieved detection of label-free DNA bound to the surface of integrated silicon field effect transistor arrays, detection of single base pair mutations by combining allele-specific PCR and transistor based measurements and detection of hybridization between DNA oligonucleotides.
Manipulation of DNA and RNA single molecules using alpha hemolysin nanopores
Nanopores allow us to manipulate untethered single molecules. We use alpha-hemolysin nanopores to sequentially open helical structues in DNA or RNA in order to understand the principles of the unzipping processes in confined geometry or shearing modes.
We detect the cotranscriptional folding of RNA structures as they emerge from the pore lumen. The RNA structure unzips on one side and rezips on the other side of the nanopore.