Developing Multiplexed Molecular Assays for Synthetic Biology and DNA Data Storage with Nanopore Sensing Technology

Abstract

Multiplexed molecular tools are a powerful means of interrogating biomolecular systems. Multiplexed assays offer significant advantages over singleplex assays, including time, reagent costs, sample requirements, and the amount of data that can be generated. In molecular biology, genetically encoded reporter proteins have expanded the toolbox of researchers to track biological phenomena. While they are widely used to measure many biological activities, the current number of uniquely addressable reporters that can be used together for one-pot multiplexed tracking is small due to overlapping detection channels such as fluorescence. Similarly, in DNA data storage, the ability to selectively target data files (i.e “randomly access”) in a multiplexable manner would lessen decoding latency and cost and enable deployment of practical DNA data storage architectures. The primary focus of this dissertation is to develop new multiplexable molecular assays for synthetic biology circuits and DNA data storage random access readout using nanopore sensor arrays. To overcome genetically encoded reporter protein multiplexing limitations, we built an expanded library of orthogonally-barcoded Nanopore-addressable protein Tags Engineered as Reporters (NanoporeTERs), which can be read and demuxed by nanopore sensors at the single-molecule level. Subsequently, to improve upon previous random access architectures, we demonstrate a new random access approach in which files can be selected in multiplex using a CRISPR-Cas9 target address and then decoded using a nanopore sequencer. This work presents a new class of reporter proteins that permit multiplexed, real-time tracking of gene expression along with a new random access DNA data storage strategy that increases one-pot multiplexing and decreased time-to-decoding.