Programmed Chemistry in Lipid-Nanocompartments

For many years, liposomes have been used as a model system to study membrane fusion ofliving cells. The advances in artificial liposome fusion platforms have increased the efficiencyand content mixing of the fusion to a point, at which reactions can be performed inside liposomes. In the group of Assoc. Prof. S. Vogel, lipidated nucleic acids (LiNAs) have been designed to induce programmed liposome fusion with high content mixing.The aim of this PhD thesis is to utilize and expand the LiNA fusion platform by establishing aconcept for programmed synthesis using liposome fusion, demonstrated by enzymatic oligosaccharide synthesis. In the work presented, an 8-Aminonaphthalene-1,3,6-trisulfonic acid(ANTS), p-Xylene-bis(N-pyridinium bromide) (DPX) fluorescence quenching assay was established, providing the ability to determine the number of fused liposomes and monitor thefusion during synthesis. The fusion conditions were optimized and a 1:3 ratio between the twoliposome populations resulted in higher number of fused liposomes from the starting population. A fluorescent starting substrate was synthesized by attaching ANTS to N-acetylglucosamine, and the enzymatic reactions using glycosyltransferases B4GALT1 and B3GNT2 wereoptimized for multi-step synthesis of poly-N-acetyllactosamine (poly-LacNAc). Due to thelimited freeze-thaw stability of B4GALT1, resulting in encapsulation problems, the bacterialequivalent LgtB from Neisseria meningitidis was expressed in E. coli. LgtB demonstratedgreatly improved freeze-thaw stability and was successfully encapsulated in liposomes, resulting in the first enzymatic synthesis of LacNAc by liposome fusion (Figure 1). Although multistep synthesis was not achieved in this thesis, a promising system was developed, and variousconditions were optimized. The properties of bacterial glycosyltransferases from Neisseriameningitidis encourages further development of the system