Controlling DNA compaction and the interaction with model biomembranes

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

DNA condensation using cationic dendrimers-morphology and supramolecular structure of formed aggregates

The control of DNA condensation, i.e. packaging or compaction, is essential for the living cell, but also important in many applications. One example is gene therapy that often utilises vehicles with the ability to condense DNA and thereby protect DNA against degradation, transport DNA across membranes (which act as barriers towards gene delivery), and regulate gene expression. This review discusses the ability of poly(amido amine) dendrimers to condense DNA molecules via attractive electrostatic interactions, which in turn leads to self-assembled structures with a rich variety of morphologies. The process of condensation is cooperative and kinetically controlled, and the structure of the aggregates strongly depends on the size and charge of the dendrimer, and the salt concentration of the aqueous solution. While globular aggregates are formed by large dendrimers, rods and toroids are formed by smaller sized dendrimers with lower total charge per molecule. The globular aggregates appear to be disordered, but the smaller dendrimers give rise to high-ordered packing of the DNA in ordered arrays according to a square or hexagonal unit cell. The high-ordered packing also indicates that the dendrimers deform while inducing the DNA to condense

Analytical Model Study of Dendrimer/DNA Complexes.

The interaction between positively charged poly(amido amine) (PAMAM) dendrimers of generation 4 and DNA has been investigated for two DNA lengths; 2000 basepairs (bp; L = 680 nm) and 4331 bp (L = 1472.5 nm) using a theoretical model by Schiessel for a semiflexible polyelectrolyte and hard spheres. The model was modified to take into account that the dendrimers are to be regarded as soft spheres, that is, the radius is not constant when the DNA interact with the dendrimer. For the shorter and longer DNA, the estimated optimal wrapping length, l(opt) is approximately 15.69 and approximately 12.25 nm, respectively, for dendrimers that retain their original size (R(o) = 2.25 nm) upon DNA interaction. However, the values of l(opt) for the dendrimers that were considered to have a radius of (R = 0.4R(o)) 0.9 nm were 9.3 and 9.4 nm for the short and long DNA, respectively, and the effect due to the DNA length is no longer observed. For l(opt) = 10.88 nm, which is the length needed to neutralize the 64 positive charges of the G4 dendrimer, the maximum number of dendrimers per DNA (N(max)) was approximately 76 for the shorter DNA, which is larger than the corresponding experimental value of 35 for 2000 bp DNA. For the longer DNA, N(max) approximately 160, which is close to the experimental value of 140 for the 4331 bp DNA. Charge inversion of the dendrimer is only observed when they retain their size or only slightly contract upon DNA interaction

Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy

Interactions between DNA and poly(amido amine) dendrimers on Silica surfaces

This study increases the understanding at a molecular level of the interactions between DNA and poly(amido amine) (PAMAM) dendrimers on solid surfaces, which is a subject of potential interest in applications such as gene therapy. We have used in situ null ellipsometry and neutron reflectometry to study the structure of multilayer arrangements formed by PAMAM dendrimers of generation 2 (G2), 4 (G4), and 6 (G6) and DNA on silica surfaces. Specifically, we adsorbed cationic dendrimer layers, then we condensed DNA to form dendrimer-DNA bilayers, and last we exposed further dendrimer molecules to the interface to encapsulate DNA in dendrimer-DNA-dendrimer trilayers. The dendrimer monolayers formed initially result in the deformation of the cationic adsorbates as a result of their strong electrostatic attraction to the hydrophilic silica surface. The highest surface excess and most pronounced deformation occurs for the G6 molecules due to their relatively large size and high surface charge density. G6-functionalized surfaces give rise to the highest surface excess of DNA during the bilayer formation process. This result is explained in terms of the high number of charged binding sites in the G6 monolayer and the low electrostatic repulsion between DNA and exposed patches of silica surface due to the relatively thick G6 monolayer. The binding strengths of the silica-dendrimer and dendrimer-DNA interactions are demonstrated by the high stability of the interfacial bilayers during rinsing. For the formation of trilayers of dendrimers, DNA, and dendrimers, G2 adsorbs as a smooth layer while G4 and G6 induce the formation of less well-defined structures due to more complex DNA layer morphologies

Condensation of DNA using poly(amido amine) dendrimers: effect of salt concentration on aggregate morphology

The condensation of DNA and poly(amido amine) dendrimers of generation 1, 2, 4, 6, and 8 has been studied as a function of salt concentration in order to reveal the forces that control the aggregate size and morphology. For the lower generation dendrimers (1, 2, and 4) a dramatic increase in aggregate size occurs as a result of an increase in salt concentration. Toroidal aggregates having an outer diameter of up to several hundreds of nm are observed. For the higher generation 6 dendrimers, the size of the condensed DNA aggregates does not change, however, an alteration in morphology is seen at high salt concentration, as more rod-like aggregates are observed. The size and morphology of generation 8 dendrimers are seemingly insensitive to salt concentration. It is believed that the effective neutralisation of the dendrimer and DNA charge in the aggregate is the reason for the observed effects. It is further shown that the 2D hexagonal lattice spacing observed in toroids is close to constant irrespective of the size of the cation responsible for the DNA condensation

Characterization of cereal β-glucan extracts from oat and barley and quantification of proteinaceous matter.

An extraction method for mixed-linkage β-glucan from oat and barley was developed in order to minimize the effect of extraction on the β-glucan structure. β-Glucan were characterized in terms of molecular size and molar mass distributions using asymmetric flow field-flow fractionation (AF4) coupled to multiangle light scattering (MALS), differential refractive index (dRI) and fluorescence (FL) detection. The carbohydrate composition of the extracts was analysed using polysaccharide analysis by carbohydrate gel electrophoresis (PACE) and high-performance anion-exchange chromatography (HPAEC). Whether there were any proteinaceous moieties linked to β-glucan was also examined. Purified extracts contained 65% and 53% β-glucan for oats and barley, respectively. The main impurities were degradation products of starch. The extracts contained high molecular weight β-glucan (105-108 g/mol) and large sizes (root-mean-square radii from 20 to 140 nm). No proteins covalently bound to β-glucan were detected; therefore, any suggested functionality of proteins regarding the health benefits of β-glucan can be discounted