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Practical applications for Martini coarse-grained DNA

Bruininks, B.M.H. (2015) Practical applications for Martini coarse-grained DNA. Master's Thesis / Essay, Biology.

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In this project the applicability of the newly developed coarse-grained (CG) Martini double stranded DNA (dsDNA) model was explored in two ways. One being the investigation of the applicability of the usage of the CG dsDNA model for base flipping research. The other being the usage of the model to build and analyze lipoplexes (dsDNA encapsulated cationic). Base flipping is involved in both expression and maintenance of the genome. It plays an important role for the recognition of damaged DNA and is often needed for modification of the nucleotides – e.g. methylation of cytosine. The exact mechanisms by which these protein-DNA interactions occur is unclear and CG molecular dynamics simulations could provide new insights. By locally uncoupling the elastic network – which is present in the Martini dsDNA model – base(s)/nucleotide(s) should have enough freedom to allow base flipping to occur. Different degrees of uncoupling were tested and visually analyzed. Uncoupling of the elastic network did allow base flips to occur with the uncoupling of a single nucleotide or nucleotide-pair giving the best results. Uncoupling a single base did not lead to base flips and uncoupling more than one (pair) caused loss of the double helix character. However, uncoupled bases interacted too strong with their complementary bases independent of their directionality. Including directionality in the non-boned potential could improve the behavior of the flipped out bases and the behavior of the model as a whole, though this would increase computational load. For now, one should be extremely careful when using the current CG dsDNA model for base flipping related research. Complexes of DNA and cationic lipids (lipoplexs) can be used as an alternative for viral-vector mediated gene transfer. They do not cause an immune response and are easier to make. The downside of non-viral vectors is that they often have a low transfection efficiency due to the low escape of genetic material from the endosome. A CG model could allow the study of lipoplex-membrane interactions at an otherwise inaccessible resolution – both in time and size. The development of the lipoplex model was performed in three steps: first a periodic lipoplex was built containing lipids in the inverted hexagonal phase (HII) with the strands of DNA in the hydrated channels. Second, the complex was solvated in water with additional lipids to create the coat around the HII phase inner core of the lipoplex. Third, the fusion of such lipoplexes with an endosomal resembling membrane was explored. Three levels of saturation for the cationic lipid (DOTAP) were tested to study their effect on the fusion speed of the lipoplex with the membrane. All three steps of building the lipoplex succeeded. The internal DNA distances were close to the values previously observed by small-angle-X-ray diffraction and CG periodic lipoplex models. The solvated lipoplex appeared stable in the microsecond time range. Remarkably, if the channels of the lipoplex were closed from the bulk, exchange of water between channels and the bulk occurred quite readily. No difference in fusion speed between saturated and single unsaturated tails of cationic lipids was found. Double-unsaturated tails in the cationic lipid did seem to have an effect on fusion speed though more simulations are needed to make the trend significant. The solvated lipoplex model has been built and appears to be a new asset for cationic lipid mediated gene transfer research but much more can be analyzed, like the effects of anionic lipids in the membrane on lipoplex-membrane fusion. Overall the Martini dsDNA force field showed to be accurate if the internal structure was rigid – as was the case for the lipoplex simulations. If internal movement of the dsDNA is mandatory – e.g. base flipping – the model showed to be of less value, though this could be overcome by future improvements.

Item Type: Thesis (Master's Thesis / Essay)
Degree programme: Biology
Thesis type: Master's Thesis / Essay
Language: English
Date Deposited: 15 Feb 2018 08:09
Last Modified: 15 Feb 2018 08:09

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