Department of Chemistry & Nanoscience Centre, University of Copenhagen, Copenhagen, Denmark.
Novo Nordisk Foundation Centre for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.
Nat Chem. 2022 May;14(5):558-565. doi: 10.1038/s41557-022-00912-5. Epub 2022 Apr 4.
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 mm) offers entire combinatorial multiplex screens using only picograms of material.
组合式高通量方法是合成生物化学和生物医学科学中筛选和发现的核心。然而，它们通常依赖于大规模分析，因此受到运行时间长和材料成本高的限制。在这里，我们提出了一种由 DNA 介导的单个粒子组合式多重脂质体融合方法，用于平行化单个亚毫微微升纳米容器的多步和非确定性融合。我们直接观察到了高效（>93%）且无泄漏的随机融合序列，用于表面连接的靶脂质体阵列与六个自由扩散的货物脂质体群体的融合，每个货物脂质体都用单独的脂质化单链 DNA 功能化，并通过独特的生色团比例进行荧光标记。随机融合导致每个自主纳米容器的融合序列发生明显的置换。实时全内反射成像允许直接观察超过 16000 次融合，并使用机器学习准确分类 566 个不同的融合序列。表面连接的靶纳米容器的高密度阵列（每个毫米约 42000 个容器）仅使用皮克数量的材料即可进行完整的组合式多重筛选。
