Philipp M. G. Löffler

A DNA-programmed liposome fusion cascade

Chemically engineered and functionalized nanoscale compartments are used in bottom-up synthetic biology to construct compartmentalized chemical processes. Progressively more complex designs demand spatial and temporal control over entrapped species. Here, we address this demand with a DNA-encoded design for the successive fusion of multiple liposome populations. Three individual stages of fusion are induced by orthogonally hybridizing sets of membrane-anchored oligonucleotides. Each fusion event leads to efficient content mixing and transfer of the recognition unit for the subsequent stage. In contrast to fusion-protein-dependent eukaryotic vesicle processing, this artificial fusion cascade exploits the versatile encoding potential of DNA hybridization and is generally applicable to small and giant unilamellar vesicles. This platform could thus enable numerous applications in artificial cellular systems and liposome-based synthetic pathways.

Sliding over the Blocks in Enzyme-Free RNA Copying – One-Pot Primer Extension in Ice

Template-directed polymerization of RNA in the absence of enzymes is the basis for an information transfer in the ‘RNA-world’ hypothesis and in novel nucleic acid based technology. Previous investigations established that only cytidine rich strands are efficient templates in bulk aqueous solutions while a few specific sequences completely block the extension of hybridized primers. We show that a eutectic water/ice system can support Pb2+/Mg2+-ion catalyzed extension of a primer across such sequences, i.e. AA, AU and AG, in a one-pot synthesis. Using mixtures of imidazole activated nucleotide 5′-monophosphates, the two first “blocking” residues could be passed during template-directed polymerization, i.e., formation of triply extended products containing a high fraction of faithful copies was demonstrated. Across the AG sequence, a mismatch sequence was formed in similar amounts to the correct product due to U·G wobble pairing. Thus, the template-directed extension occurs both across pyrimidine and purine rich sequences and insertions of pyrimidines did not inhibit the subsequent insertions. Products were mainly formed with 2′-5′-phosphodiester linkages, however, the abundance of 3′–5′-linkages was higher than previously reported for pyrimidine insertions. When enzyme-free, template-directed RNA polymerization is performed in a eutectic water ice environment, various intrinsic reaction limitations observed in bulk solution can then be overcome.

Bottom–Up Protocell Design: Gaining Insights in the Emergence of Complex Functions

All contemporary living cells are a collection of self-assembled molecular elements that by themselves are non-living but through the creation of a network exhibit the emergent properties of self-maintenance, self-reproduction, and evolution. Protocells are chemical systems that should mimic cell behavior and their emergent properties through the interactions of their components. For a functional protocell designed bottom-up, three fundamental elements are required: a compartment, a reaction network, and an information system. Even if the functions of protocell components are very simplified compared to those of modern cells, realizing a system with true inter-connection and inter-dependence of all the functions should lead to emergent properties. However, none of the currently studied systems have yet reached the threshold level necessary to be considered alive. This chapter will discuss the on-going research that aims at creating artificial cells assembled from a collection of smaller components, i.e., protocell systems from bottom-up designs.

Ethyl 2-(1,2,3,4-tetrahydro-spiro-[carba-zole-3,2'-[1,3]dioxolan]-9-yl)acetate.

In the title compound, C18H21NO4, the hydrogenated six-membered ring of the carbazole unit adopts a half-chair conformation. The dioxolane ring and ethylacetate substituent point to opposite sides of the carbazole plane. The ethylacetate substituent adopts an essentially fully extended conformation, and its mean plane forms a dihedral angle of 83.8 (1)° with respect to the carbazole mean plane. The molecules are arranged into stacks in which the carbazole planes form a dihedral angle of 4.4 (1)° and have an approximate interplanar separation of 3.6 Å.

Synthesis of Lipophilic Guanine N-9 Derivatives: Membrane Anchoring of Nucleobases Tailored to Fatty Acid Vesicles

Covalent or noncovalent surface functionalization of soft-matter structures is an important tool for tailoring their function and stability. Functionalized surfaces and nanoparticles have found numerous applications in drug delivery and diagnostics, and new functionalization chemistry is continuously being developed in the discipline of bottom-up systems chemistry. The association of polar functional molecules, e.g., molecular recognition agents, with soft-matter structures can be achieved by derivatization with alkyl chains, allowing noncovalent anchoring into amphiphilic membranes. We report the synthesis of five new guanine-N9 derivatives bearing alkyl chains with different attachment chemistries, exploiting a synthesis pathway that allows a flexible choice of hydrophobic anchor moiety. In this study, these guanine derivatives were functionalized with C10 chains for insertion into decanoic acid bilayer structures, in which both alkyl chain length and attachment chemistry determined their interaction with the membrane. Incubation of these guanine conjugates, as solids, with a decanoic acid vesicle suspension, showed that ether- and triazole-linked C10 anchors yielded an increased partitioning of the guanine derivative into the membranous phase compared to directly N-9-linked saturated alkyl anchors. Decanoic acid vesicle membranes could be loaded with up to 5.5 mol % guanine derivative, a 6-fold increase over previous limits. Thus, anchor chemistries exhibiting favorable interactions with a bilayers hydrophilic surface can significantly increase the degree of structure functionalization.