1 Single particle combinatorial multiplexed liposome fusion mediated by DNA 1

17 Combinatorial high throughput methodologies are central for both screening and discovery in synthetic 18 biochemistry and biomedical sciences. They are, however, often reliant on large scale analyses and thus 19 limited by long running time and excessive materials cost. We herein present Single PARticle Combinatorial 20 multiplexed Liposome fusion mediated by DNA (SPARCLD), for the parallelized, multi-step and non21 deterministic fusion of individual zeptoliter nanocontainers. We observed directly the efficient (>93%), and 22 leakage free stochastic fusion sequences for arrays of surface tethered target liposomes with six freely 23 diffusing populations of cargo liposomes, each functionalized with individual lipidated ssDNA (LiNA) and 24 fluorescent barcoded by distinct ratio of chromophores. The stochastic fusion results in distinct permutation 25 of fusion sequences for each autonomous nanocontainer. Real-time TIRF imaging allowed the direct 26 observation of >16000 fusions and 566 distinct fusion sequences accurately classified using machine learning. 27 The high-density arrays of surface tethered target nanocontainers ~42,000 containers per mm2 offers entire 28 combinatorial multiplex screens using only picograms of material. 29 30