The expression and spatial profile of multiple RNA species at high precision in single cells is key information for understanding cellular behaviors and functions. Fluorescence microscopy is a powerful tool for imaging RNAs, but its multiplexing ability is limited by the number of spectrally distinct fluorophores. Here, we present a DNA-sequence-encoded fluorescence barcoding method, termed sequence-encoded amplicon (SeqEA), enabling highly multiplexed imaging of RNAs in single cells with single-molecule and single-nucleotide resolution. SeqEA exploits a thermodynamically tuning DNA hybridization approach for precisely encoding rolling circle amplicons with discrete fluorescence intensities as fluorescence barcodes, allowing for multiplexed tagging single-molecule RNAs. As a proof of principle, we have demonstrated its ability for simultaneously visualizing nine mRNA species and evaluated the coordination and spatial pattern of breast-cancer-associated oncogene expression. SeqEA provides a single-cell analysis platform for investigating the gene expression network and molecular mechanism of disease development.

在单个细胞中高精度地表达多个RNA种类和空间分布是了解细胞行为和功能的关键信息。荧光显微镜是用于RNA成像的强大工具，但是其多重能力受到光谱上不同的荧光团数量的限制。在这里，我们介绍了一种称为序列编码扩增子（SeqEA）的DNA序列编码荧光条形码方法，可实现单分子和单核苷酸分辨率的单细胞中RNA的高度多重成像。SeqEA利用一种热力学调谐的DNA杂交方法来精确编码具有离散荧光强度的滚动环扩增子作为荧光条形码，从而可以对单分子RNA进行多重标记。作为原理上的证明，我们已经展示了其同时可视化9种mRNA并评估与乳腺癌相关的癌基因表达的协调性和空间模式的能力。SeqEA提供了一个单细胞分析平台，用于研究基因表达网络和疾病发展的分子机制。