西亚试剂：Multiplex single-molecule interaction profiling of DNA-barc

发布时间：2025-10-18

In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2, 3, 4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods5 is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, wher barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables ‘library versus library’ screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

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