Quantification of RNA for cellular fingerprinting with solid-state nanopores

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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.