Programmed Chemistry in Lipid-Nanocompartments

Nikolaj Risgaard

doi:10.21996/b8bs-w860

Programmed Chemistry in Lipid-Nanocompartments

Nikolaj Risgaard

Publikation: Afhandling › Ph.d.-afhandling

116 Downloads (Pure)

Abstract

Liposomer har i mange år været brugt som modeller til at undersøge membran fusion i levendeceller. Fremskridt i de kunstige liposom fusions metoder har medført, at fusionseffektivitetener blevet forbedret til et stadie, hvor man kan udføre reaktioner inde i liposomerne. Assoc.Prof. S. Vogels forskningsgruppe har designet lipiderede nukleinsyrer (LiNAs) til at udføreprogrammeret liposom fusion, hvor en stor mængde af indholdsstofferne bliver blandet.Formålet med denne ph.d.-afhandling er at benytte LiNA fusionsplatformen til at udvikle etkoncept for at udføre syntese med liposom fusion, demonstreret med enzymatisk syntese afoligosakkarider. I dette projekt bliver en metode til at dæmpe fluorescensen af 8-Aminonaphthalene-1,3,6-trisulfonic acid (ANTS) med p-Xylene-bis(N-pyridinium bromide) (DPX) etableret for at bestemme antallet af fusionerede liposomer og følge fusionen undervejs i syntesen.Fusionsbetingelserne blev optimeret, og en 1:3 ratio mellem de to liposom populationer resulterede i et større antal af fusionerede liposomer fra startpopulationen. Et fluorescerende udgangsstof blev syntetiseret ved at sammensætte ANTS og N-acetylglukosamin, og brugt til atoptimere betingelserne i enzymatiske reaktioner med glycosyltransferaserne B4GALT1 ogB3GNT2 til syntesen af poly-N-acetyllactosamine (poly-LacNAc). Da B4GALT1 udviste dårlig stabilitet efter at være blevet frosset og optøet, hvilket resulterede i indkapslingsproblemer,blev den tilsvarende bakterielle glycosyltransferase LgtB fra Neisseria meningitidis udtrykt iE. coli. LgtB udviste bedre stabilitet efter at være blevet frosset og optøet, hvorfor den succesfuldt blev indkapslet i liposomer, som resulterede i den første syntese af LacNAc ved hjælp afliposom fusion (Figur 1). På trods af, at det ikke var muligt at udføre en syntese i flere trin, såblev et lovende system udviklet og en lang række betingelser optimeret. Egenskaberne af glycosyltransferaserne fra Neisseria meningitidis opfordrer til fortsat at forbedre systemet.

Originalsprog	Engelsk
Bevilgende institution	Syddansk Universitet
Vejledere/rådgivere	Vogel, Stefan, Hovedvejleder
Dato for forsvar	1. jun. 2022
Udgiver	Syddansk Universitet. Det Naturvidenskabelige Fakultet
DOI	https://doi.org/10.21996/b8bs-w860
Status	Udgivet - 9. sep. 2022

Dokumenter og links

10.21996/b8bs-w860

Open Access VersionForlagets udgivne version, 40,5 MB

SDU link

Tjek adgangen til materialet i Syddansk Universitetsbiblioteks søgemaskine

2 Tidsskriftartikel

Label-free observation of DNA-encoded liposome fusion by surface plasmon resonance
Löffler, P. M. G., Risgaard, N. A., Svendsen, B. L., Jepsen, K. A., Rabe, A. & Vogel, S., 2023, I: Chemical Communications. 59, 70, s. 10548-10551
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review
Single-particle combinatorial multiplexed liposome fusion mediated by DNA
Malle, M. G., Löffler, P. M. G., Bohr, S. S. R., Sletfjerding, M. B., Risgaard, N. A., Jensen, S. B., Zhang, M., Hedegård, P., Vogel, S. & Hatzakis, N. S., maj 2022, I: Nature Chemistry. 14, 5, s. 558-565
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

Citationsformater

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abstract = "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",

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AB - 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

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M3 - Ph.D. thesis

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Programmed Chemistry in Lipid-Nanocompartments

Abstract

Dokumenter og links

SDU link

Fingeraftryk

Relaterede publikationer

Label-free observation of DNA-encoded liposome fusion by surface plasmon resonance

Single-particle combinatorial multiplexed liposome fusion mediated by DNA

Citationsformater