10. S UMMARY
In summary, the potent combination of microfluidics with small-angle X-ray microdiffraction and confocal Raman microscopy allows for detailed insights into the evolution of dendrimer and linker-histone induced DNA compaction. In particular, the laminar flow conditions inside microchannels provide the possibility to investigate interaction processes in a time-resolved manner. Diffusive mixing in microchannels creates tunable reaction conditions with defined changes in local concentrations.
Multiple experimental data are therefore aquired at any desired complex composition on a single device, while consuming extremely small amounts of material and without any concerns for radiation damage. The fact that it is possible to improve the correlation length of biomaterials, by superimposing stress, is a clear advantage of using flow to assemble such materials, which normally form liquid-crystalline phases.
The major obstacle for performing X-ray diffraction measurements directly on a microfluidic chip was the lack of cheap, tunable, and robust devices with thin, low-absorbing and low-scattering windows. Therefore, the successful development of such X-ray compatible microfluidic devices is a central aspect of the present work. The newly developed microflow foils now place researchers in an advantageous position to approach important biophysical questions regarding biomatter self-assembly and interactions in a variety of environments. It is anticipated that microflow foils will provide a robust and affordable approach not only to continuous flow X-ray microdiffraction, but also in micro total analysis systems (“µ-TAS”) and bioassays.
Here, two different classes of compaction agents have been studied: dendrimers of varying generation and linker-histones H1.
10. Summary
Dendrimers, which are a unique class of precisely engineered, highly branched macromolecules, can be viewed as compact, spherical objects with a smeared surface region. In order to precisely control and understand dendrimer induced DNA compaction, it is of crucial importance to first characterize the compaction agent.
Therefore, dendrimer properties have been analyzed in detail. In particular, the phenomenon of charge-induced dendrimer swelling has been experimentally quantified for PPI and PAMAM dendrimers and over a wide range of generations. The results clearly show highly predictable, charge-induced changes in dendrimer conformation.
Therefore, the discrepancy between theory and experiments that existed in literature up to now is eliminated. Moreover, the observed response of dendrimers to superimposed electrostatics can be generalized to all types of dendrimers.
Through the variation of dendrimer size and charge, the entire spectrum of naturally occurring condensation agents is bridged, ranging from small cations, such as spermine/spermidine encountered in viruses, to the much larger histone proteins, in eukaryotic cells. Therefore, dendrimers are perfectly suited to study DNA compaction and to mimic the influence of electrostatic interactions on DNA compaction in vivo. In particular, the dynamic assembly of DNA condensates by cationic dendrimers with sizes and charges situated between that of small multivalent organic cations and larger histone-like proteins is analyzed. Performing measurements on the 2D columnar mesophase formation in flow for the first time enables access to very low N/P ratios in a controlled manner. Most surprising, our results indicate that in an undercharged regime it is possible that DNA condensation occurs locally without direct contact to cationic compaction agents. The consistency of results obtained from DNA complexes with three different types of dendrimers suggests that the observed DNA compaction mechanism at low N/P ratios is a general phenomenon and may also exist in biological cells.
PAMAM dendrimers generation 6 have dimensions and charges comparable to those of the histone core found in chromosomal DNA packing. The observed interactions with this dendrimer of increased size and charge are in strong contrast to those of DNA and smaller cations. Just as in the case of protein folding, studying PAMAM 6/DNA complex formation in microflow reveals a DNA condensation process that exhibits multistage dynamics. Consistently, interaction kinetics at high pH conditions (pH = 8.5) are found to be particularly slow and the organization of PAMAM 6/DNA in a highly ordered 3D hexagonal lattice takes place on remarkably long time scales of years.
The principle organization of the DNA chain on the PAMAM 6 surface includes local wrapping of DNA around the dendrimers and is primarily determined by the pH dependent dendrimer valency. Increasing dendrimer charge density from 1.46e+/nm2
10. Summary
(pH = 8.5) to about 1.7e+/nm2 (pH = 5.5) results in a transition from a state, where only a finite length of DNA is adsorbed on the dendrimer surface in approximately half of a turn to a full wrapping of DNA in approximately two turns. The cylindrically shaped, fully wrapped conformation of PAMAM 6/DNA entities at low pH is strikingly similar in shape and size to the structure of nucleosome core particles (NCPs). In this sense, PAMAM 6/DNA entities are excellent biomimetics of NCPs. Consequently, one may conclude that at least for a simple replication of the beads on a string structure without paying attention to DNA functionality, a specific arrangement of charged patches on the histone octamer is not required. The uniform spherical charge distribution of the
“artificial protein” results in a structurally analogous packing of DNA. An important point is that for PAMAM 6/DNA entities, connecting (linker-)DNA strands are still present in the structure. Therefore, the PAMAM 6/DNA system represents an ideal starting point towards an experimental realization of the 30nm fiber (e.g. by adding linker-histones).
The importance of investigations of DNA condensates is not only relevant from a fundamental point of view. These structures are also extensively used for the delivery of therapeutic genes to living cells. Owing to the increased fraction of DNA, which is tightly bound, the wrapping scenario provides a plausible rationale for the increased stability of DNA complexes with higher generation dendrimers against nuclease digestion. This is a predominant issue reported in literature. Following this line of argumentation, low pH conditions are better suited for the purpose of DNA delivery inside cells since a larger fraction of DNA is wrapped around the dendrimer. In this scenario, the DNA is also protected. Therefore, results presented in this thesis are believed to contribute in the field of biotechnology towards the construction of new vectors for DNA gene delivery.
In addition to dendrimers, linker-histone H1 proteins are also used to compact DNA.
Exploiting the powerfull combination of X-ray microdiffraction and microfluidics allows for the first time to gain access to H1/DNA structure formation dynamics.
Observed SAXS patterns clearly show that the interaction of H1 with DNA is a two step process: an initial unspecific binding of H1 proteins to DNA is followed by a rearrangement of molecules in the complexes. Results suggest that the conformational transition of the linker-histone tails from their rather extended conformation in aqueous solution to their fully folded state upon interaction with DNA is responsible for the conformational phase transition of H1/DNA complexes. This kind of rearrangement is most likely necessary to determine the curvature and the path of initial parts of linker-DNA at the entry and exit points of the nucleosome.
The systems studied in this thesis are inherently significant to the fields of biology and biotechnology. The results reported here are expected to have a direct bearing on the
10. Summary
understanding of chromatin fiber folding into higher order structures. The underlying concepts and techniques may be generalized and used to experimentally access additional relevant biophysical problems and to realize new biomimetic systems.