Assembly and fusion between different populations of lipid nanoparticles was mediated by membrane-anchored lipidated nucleic acid (LiNA) strands and observed using surface plasmon resonance (SPR) as a label-free real-time assay. Irreversible membrane fusion was distinguished from reversible assembly by enzymatical cleavage of dsDNA tethers in situ. The assay enables user-friendly monitoring and application of membrane fusion in the context of liposomal drug delivery or synthetic biology.

Lipid nanoreactors are biomimetic reaction vessels (nanoreactors) that can host aqueous or membrane-associated chemical and enzymatic reactions. Nanoreactors provide ultra-miniaturization from atto- to zeptoliter volumes per reaction vessel with the major challenge of encoding and spatio-temporal control over reactions at the individual nanoreactor or population level, thereby controlling volumes several orders of magnitude below advanced microfluidic devices. We present DNA-programmed lipid nanoreactors (PLNs) functionalized with lipidated oligonucleotides (LiNAs) that allow programming and encoding of nanoreactor interactions by controlled membrane fusion, exemplified for a set of carbohydrate mimetics with mono- to hexasaccharide azide building blocks connected by click-chemistry. Programmed reactions are initiated by fusion of distinct populations of nanoreactors with individually encapsulated building blocks. A focused library of triazole-linked carbohydrate-Cy5 conjugates formed by strain-promoted azide-alkyne cycloadditions demonstrated LiNA-programmed chemistry, including two-step reaction schemes. The PLN method is developed toward a robust platform for synthesis in confined space employing fully programmable nanoreactors, applicable to multistep synthesis for the generation of combinatorial libraries with subsequent analysis of the molecules formed, based on the addressability of the lipid nanoreactors.

Combinatorial high-throughput methodologies are central for both screening and discovery in synthetic biochemistry and biomedical sciences. They are, however, often reliant on large-scale analyses and thus limited by a long running time and excessive materials cost. We here present a single-particle combinatorial multiplexed liposome fusion mediated by DNA for parallelized multistep and non-deterministic fusion of individual subattolitre nanocontainers. We observed directly the efficient (>93%) and leakage free stochastic fusion sequences for arrays of surface-tethered target liposomes with six freely diffusing populations of cargo liposomes, each functionalized with individual lipidated single-stranded DNA and fluorescently barcoded by a distinct ratio of chromophores. The stochastic fusion resulted in a distinct permutation of fusion sequences for each autonomous nanocontainer. Real-time total internal reflection imaging allowed the direct observation of >16,000 fusions and 566 distinct fusion sequences accurately classified using machine learning. The high-density arrays of surface-tethered target nanocontainers (~42,000 containers per mm2) offers entire combinatorial multiplex screens using only picograms of material.

Nucleotide monomers that hold two nucleobases each, i.e. double-headed nucleotides, have been shown to form two sets of functional Watson-Crick base pairs when incorporated into dsDNA, and they hereby behave as dinucleotides. To form the basis for fully modified double-headed nucleic acids (DhNA), we have prepared three new DhNA monomers and can now demonstrate that the molecular information of 10 Watson-Crick base pairs can be condensed to highly stable 5-mer DhNA duplexes.

Double‐headed nucleotides are DNA building blocks that store, in principle, twice as much information as native nucleotides due to the incorporation of an additional functional nucleobase. Herein, we present the development of two new double‐headed nucleotides, UW and UZ, featuring a methylene‐linked 5‐aza‐7‐deazaxanthine (Z) and a propynyl‐linked pseudouracil (W), respectively, attached to the 2′‐position of arabinouridine. These analogs are evaluated in DNA duplexes for their ability to act as dinucleotides, and the base‐pairing specificities of the 2′‐nucleobases are compared to previous analogues. Although an improved discrimination was observed in the context of a cytosine mismatch using UZ, neither of the two new analogs gave rise to increased overall base‐pairing fidelity possibly due to the formation of stable wobble pairs. Furthermore, we present an improved synthetic strategy for the preparation of the corresponding diaminopurine analog (UD), in which the key fluoro‐to‐amino substitution is achieved post‐synthetically to avoid the use of doubly protected diaminopurines and complicated deprotections.