Controlling DNA compaction and the interaction with model biomembranes

Sammanfattning: The studies described in this thesis forms part of a larger collaborative project, Neonuclei, with the objective to design a module for packaging the negatively charged DNA into a ”neonucleus” with transcription competence. There are many applications that require control of DNA compaction, i.e. packaging or condensation. One example is the systemic delivery of DNA for gene therapy. In this work, DNA compaction was induced by using positively charged binding agents, e.g. poly(amido amine) (PAMAM) dendrimers. DNA undergoes a transition from a semi-flexible coil to a more compact globule due to the electrostatic attractive interaction between DNA and the dendrimer. The process of compaction is cooperative and kinetically controlled, and the structure of the well-defined aggregates strongly depends on the size and charge of the dendrimer and the ionic strength of the aqueous solution. This is demonstrated by dynamic light scattering, fluorescence spectroscopy and cryogenic transmission electron microscopy. The smaller sized dendrimers, which have a lower total charge per molecule, allow the formation of well-structured rods and toroids. In contrast, globular and less defined aggregates, which are less stable against precipitation, are formed with larger dendrimers. The implication is that the conformation of DNA is closely related to the biological function, i.e. that compacted DNA is not transcription competent and also protected against degradation. This was shown by using a number of biochemical assays in addition to fluorescence spectroscopy. The use of a DNA compacting agent serves furthermore as a vehicle to aid in the transport of DNA across membranes which act as barriers towards gene delivery. PAMAM dendrimers of varying size and charge traverse model biomembranes composed of supported phospholipid bilayers and lower PAMAM dendrimers are concluded to transport across bilayers without affecting the bilayer integrity. This is shown by in situ null ellipsometry and neutron reflectometry and is verified by coarse grained simulations. The ability of PAMAM dendrimer/DNA aggregates to penetrate model biomembranes is reduced compared to dendrimers alone. We also show, using ellipsometry, QCM-D, neutron reflectometry and scattering, that DNA adsorb to zwitterionic bilayers even without the presence of multivalent ions, i.e. in the absence of strong electrostatic attractive interaction. The results obtained are important in relation to the design of efficient transfection mediators with membrane permeating ability.

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