Quantification of RNA for cellular fingerprinting with solid-state nanopores

利用固态纳米孔对 RNA 进行细胞指纹定量

• 批准号: 2626154
• 金额: --
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2626154
• 关键词: Quantification; RNA; cellular; fingerprinting; solid

Nanopores have become a versatile sensing technique for the single molecule analysis of DNA, RNA and proteins. Single molecules are detected by measuring changes in ionic current flow as they pass through a nanometre scale pore separating two chambers containing salt solution. In this project we will develop a new technique for the identification and counting of native RNA molecules for diagnostics and fundamental transcriptomics research. The core of the project will build on the techniques on design and assembly of DNA-RNA hybrid structures learned during the Midi project. Initially, we will use RNA samples to develop larger libraries of DNA staple that allow to distinguish 100 different RNA target molecules. It will be necessary to understand the physics of the translocation process and the underlying folding of DNA-RNA hybrid structures. The solid-state nanopore sensing will allow for rapid screening of the stability of the RNA-DNA hybrids as well as RNA structural elements. The latter are under intense investigation with other, lower throughput single-molecule techniques like optical tweezers. We will also investigate the kinetics of structure formation in liquid droplets in microfluidic environments. The proper formation of the RNA-DNA hybrid structures in pico-femtoliter droplets is unexplored and will enable the reduction of sample volumes to allow for single-cell measurements. Single-cell transcriptomics is a fast-growing area where the need to use reverse transcription and amplification hinders the direct analysis of native RNA structures. With the integration of of solid-state nanopore sensing into microfluidic systems, the project in the third year will detect RNA targets in cellular extracts with single cell resolution.

纳米孔已成为DNA、RNA和蛋白质单分子分析的通用传感技术。单个分子是通过测量离子电流的变化来检测的，因为它们通过一个纳米级的孔，分隔两个含有盐溶液的房间。在这个项目中，我们将开发一种新的技术来鉴定和计数天然RNA分子，用于诊断和基础转录组学研究。该项目的核心将建立在在Midi项目中学到的DNA-RNA混合结构的设计和组装技术的基础上。首先，我们将使用RNA样本来开发更大的DNA订书库，以便区分100个不同的RNA目标分子。有必要了解DNA-RNA杂交结构的易位过程和潜在折叠的物理学。固态纳米孔传感将允许快速筛选RNA-DNA杂交物以及RNA结构元件的稳定性。后者正在与其他产量较低的单分子技术(如光镊子)进行紧张的研究。我们还将研究微流体环境中液滴中结构形成的动力学。目前还没有探索在微飞升液滴中正确形成RNA-DNA杂化结构的方法，这将使样品体积减少，以便进行单细胞测量。单细胞转录学是一个快速发展的领域，需要使用逆转录和扩增来阻碍对天然RNA结构的直接分析。随着将固态纳米孔传感技术集成到微流控系统中，该项目将在第三年以单细胞分辨率检测细胞提取物中的RNA靶标。