High-throughput single-cell co-sequencing of small and large RNAs to identify molecular circuitry in cancer

项目摘要 小RNA（smRNAs）是重要的非编码调节因子，广泛参与人类癌症的发生和发展。 进展smRNAs的功能和作用机制在肿瘤研究中得到了广泛的研究。他们是 也被作为新的治疗靶点进行研究，但迄今为止的结果并不令人满意，部分原因是 miRNA-mRNA调控网络的复杂性与人类癌症中的细胞异质性。因此，在本发明中， 一种新的工具可以在大量的单细胞中共同分析smRNAs和大RNA（lgRNAs）， 解决这一长期存在的挑战，为smRNA研究增添新的维度， smRNAs参与癌症生物学的新机制，并使其在癌症中的潜在应用成为可能 诊断和治疗。PIs Fan和Lu之间的密切合作首次证明了 时间，来自相同单细胞的smRNA和lgRNA的共测序（Wang等人，自然通讯， 2019年）。它进一步表明，在单细胞水平上具有配对的smRNA和lgRNA谱可以揭示 miRNA介导的基因调控和控制miRNA表达和细胞间 异质性然而，这种单细胞smRNA-lgRNA共测序技术是手动过程， 低通量和高成本，由于统计能力不足， 询问高度异质性肿瘤细胞。在这个应用中，我们提出了这种先进的发展， 本发明涉及一种用于提供高通量单细胞smRNA/lgRNA共测序（scSLRco-seq）技术的方法。它 采用了一种新的滑移转移微器件与新的分子条形码方案相结合， 用于同时分析1000个单细胞中的smRNA和lgRNA的下游生物化学。 具体而言，我们将（目标1）开发一种微流体交叉流图案化方法，以创建2，500个DNA条形码 阵列的小和大RNA的空间耦合捕获和条形码，（Aim 2）开发了一种新的滑动- 转移芯片，以整合用于高通量scSLRCo-seq的多步骤生物化学工作流程，以及（Aim 3） 使用人类和小鼠骨髓性白血病模型验证该技术。这项新技术解决了 缺乏高通量单细胞smRNA/lgRNA共分析的能力，填补了单细胞组学的空白 领域，使研究新的问题以前不可能回答。它代表了一个重大的飞跃， 并将在癌症研究和应用中广泛使用。

PROJECT SUMMARY Small RNAs (smRNAs) are important non-coding regulators broadly implicated in human cancer initiation and progression. The functions and mechanism of smRNAs have been widely studied in cancer research. They are also being investigated as new therapeutic targets but the results so far are unsatisfactory, due in part to the complexity of miRNA-mRNA regulatory network coupled with cellular heterogeneity in human cancers. Thus, new tools that can co-profile smRNAs and large RNAs (lgRNAs) in large numbers of single cells can help address this long-standing challenge, add a new dimension to smRNA research, empower the discovery of new mechanisms of smRNAs to participate in cancer biology, and enable potential applications to cancer diagnostics and therapeutics. A close collaboration between PIs Fan and Lu has demonstrated, for the first time, co-sequencing of both smRNAs and lgRNAs from the same single cells (Wang et al., Nature Comm., 2019). It further showed that having paired smRNA and lgRNA profiles on the single-cell level can reveal miRNA-mediated gene regulation and new mechanisms for controlling miRNA expression and intercellular heterogeneity. However, this single-cell smRNA-lgRNA co-sequencing technology is a manual process with low throughput and high cost, limiting its potential for cancer research due to insufficient statistic power to interrogate highly heterogeneous tumor cells. In this application, we propose the advanced development of this technology to deliver a high-throughput single-cell smRNA/lgRNA co-sequencing (scSLRco-seq) technology. It employs a novel slip-transfer microdevice in combination with novel molecular barcoding scheme and downstream biochemistry for simultaneous analysis of smRNAs and lgRNAs in 1000’s of single cells. Specifically, we will (Aim 1) develop a microfluidic cross-flow patterning method to create 2,500 DNA barcode arrays for spatially-coupled capture and barcoding of small and large RNAs, (Aim2) develop a novel slip- transfer chip to integrate multi-step biochemistry workflow for high throughput scSLRCo-seq, and (Aim3) validate this technology using human and mouse myeloid leukemia models. This novel technology addresses the lack of capability for high-throughput single-cell smRNA/lgRNA co-profiling, filling a gap in single-cell omics field and enabling the study of new questions previously impossible to answer. It represents a major leap in the field and will find wide-spread use in cancer research and applications.