RESUMO
Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.
Assuntos
DNA , Sequenciamento de Nucleotídeos em Larga Escala , Microscopia de Fluorescência , Imagem Individual de Molécula , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imagem Individual de Molécula/métodos , DNA/química , DNA/genética , Microscopia de Fluorescência/métodos , Proteína 9 Associada à CRISPR , Análise de Sequência de DNA/métodos , Biblioteca Gênica , Sistemas CRISPR-CasRESUMO
Assemblies of 2-styrylbenzothiazole containing an 18-crown-6 ether fragment with perchlorates of amino acids ClO4-NH3+(CH2)nCOOH (n = 2, 10) were studied by UV, NMR spectroscopy, time-resolved fluorescence spectroscopy and quantum-chemical calculations. The obtained data showed that complex formation of the crown-containing 2-styrylbenzothiazole with amino acids occurs through mono- or ditopic coordination. The formation of a ditopic complex influences the E-Z photoisomerization reaction of 2-styrylbenzothiazole.