Single-particle combinatorial multiplexed liposome fusion mediated by DNA

Abstract

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 long running time and excessive materials cost. We herein present Single PARticle Combinatorial multiplexed Liposome fusion mediated by DNA (SPARCLD), for the parallelized, multi-step and nondeterministic fusion of individual zeptoliter 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 ssDNA (LiNA) and fluorescent barcoded by distinct ratio of chromophores. The stochastic fusion results in distinct permutation of fusion sequences for each autonomous nanocontainer. Real-time TIRF imaging allowed the direct observation of >16000 fusions and 566 distinct fusion sequences accurately classified using machine learning.The high-density arrays of surface tethered target nanocontainers ~42,000 containers per mm 2 offers entire combinatorial multiplex screens using only picograms of material.

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

“…1c, d ). We recorded time-dependent intensity fluctuations for each particle by integrating the intensity of each diffraction-limited spot 36 . The intensity changed in a stepwise manner (Fig.…”

Design, synthesis and membrane anchoring strength of lipidated polyaza crown ether DNA-conjugates (LiNAs) studied by DNA-controlled assembly of liposomes

“…The reported solution based assays have been successfully transferred to a polymer cartridge and been shown to work equally effective with all reagent freeze-dried on the polymer cartridge and a PCR detection limit of 1 nM of target. The presented study provides important data on the required anchoring strengths for lipidated oligonucleotides (LiNAs) with applications in liposome assembly, 24,28,29 liposome fusion 16,17,31,32 and formulation of therapeutic oligonucleotides in lipid nanoparticle formulations. 43,44…”

“…liposomes) with membrane-anchored lipidated oligonucleotides (LiNAs) with covalently incorporated lipophilic groups has been reported for the formation of liposomes assemblies, 7,8 anchoring of liposomes to supports 9–12 ( e.g. for the study of proteins and protein interactions), 9–11 liposome membrane fusion, 13–17 nanostructures, 18–20 detection of DNA hybridization 21–25 and light-harvesting systems. 26,27 LiNAs have been reported with different lipophilic building blocks, e.g.…”

Heterogeneous and Surface-Catalyzed Amyloid Aggregation Monitored by Spatially Resolved Fluorescence and Single Molecule Microscopy

“…For both samples, we identified groups of spherulites formed through either isotropic or anisotropic growth from the core of the spherulites (see videos in SI and Figure a,b for snapshots). Using homemade software based on Euclidian minimum spanning tree and machine learning clustering, − we calculated the growth rate of each spherulite morphology. We used a model-free procedure, with the aim of comparing the values between the different growth pathways and experimental conditions.…”