Multiplexed direct detection of barcoded protein reporters on a nanopore array

Abstract

Genetically encoded reporter proteins are a cornerstone of molecular biology. 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. To address this, 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. By adapting a commercially available nanopore sensor array platform typically used for real-time DNA and RNA sequencing (Oxford Nanopore Technologies’ MinION), we show direct detection of NanoporeTER expression levels from unprocessed bacterial culture with no specialized sample preparation. These results lay the foundations for a new class of reporter proteins to enable multiplexed, real-time tracking of gene expression with nascent nanopore sensor technology.

Passively Sensing SARS-CoV-2 RNA in Public Transit Buses

“…Studies evaluating filter placement and size, as well as control experiments in simulated environments, could further validate the sensitivity of the method. City-wide deployments enabled by scalable detection methods, such as rapid diagnostic lateral flow detection for on-site detection enabled by miniaturized PCR devices 41 or sequencers, 49 could gather more data, enabling network analysis techniques to study probability of SARS-CoV-2 transmission on a neighborhood level. This method could be adapted and deployed to provide an early signal of community outbreak of SARS-CoV-2, or other viruses transmitted by respiratory droplets, when case counts are low in the population.…”

The emerging landscape of single-molecule protein sequencing technologies

“…Recently, Rodriguez-Larrea's group has discussed how protein refolding at the entry and exit compartments can oppose and promote protein translocation, respectively 72,73 , and the use of deep learning networks to analyze raw ionic current signals for accurate classification of single point mutations in a translocating protein 74 . In addition, Cardozo et al built a library of approximately 20 proteins that are orthogonally barcoded with an intrinsic peptide sequence and successfully read them with nanopore sensors 75 .…”

Herding cats: Label-based approaches in protein translocation through nanopore sensors for single-molecule protein sequence analysis

“…The “tug-of-war” state created by the terminal charged residues enabled slow translocation (>1 ms) of peptides through a FraC nanopore to read out PTMs and 6 distinct chemical tags on the peptides. Our group has recently shown that an Smt3 protein genetically tagged at its C-terminus with a polyGSD peptide can promote capture of the protein in a CsgG nanopore but does not readily drive unfolding and complete translocation ( Cardozo et al., 2021 ). This observation supports the conclusion that a more highly charged peptide or the addition of other mechanisms would be necessary to generate enough force to completely unfold and translocate stably folded proteins through a narrow nanopore.…”

“…This strategy has been adopted in efforts toward nanopore-based protein/peptide identification ( Restrepo-Pérez et al., 2019a ). Third, the emergence of commercial nanopore sequencers has enabled highly parallel nanopore analysis and high-throughput data collection on a nanopore array ( Cardozo et al., 2021 ; Zhang et al., 2021b ), which otherwise would not be realized by single-channel experiments. A large set of training data, coupled with state-of-the-art machine learning models, would boost accurate translation of complex raw signals into amino acid sequences and PTMs, analogous to the example of highly improved basecalling accuracy in nanopore DNA sequencing using deep learning ( Rang et al., 2018 ; Teng et al., 2018 ; Wick et al., 2019 ).…”

“…Single-molecule sensitivity, full-length readout, real-time measurement, and device portability is just as, if not more, crucial for proteomics than it is for genomics and transcriptomics ( Shi et al., 2017 ). Towards this end, nanopore sensors have been used for discrimination of peptides and proteins ( Asandei et al., 2017 ; Cardozo et al., 2021 ; Huang et al, 2017 , 2019 ; Nivala et al., 2014 ; Piguet et al., 2018 ; Robertson and Reiner, 2018 ), real-time measurement of protein–protein ( Thakur and Movileanu, 2019 ) and protein–ligand interactions ( Harrington et al., 2013 ; Movileanu et al., 2000 ), antigen−antibody binding assays ( Han et al., 2008 ; Madampage et al., 2010 ; Sexton et al., 2007 ), and aptamer-mediated protein detection ( Rotem et al., 2012 ; Soskine et al., 2012 ; Sze et al., 2017 ). Moreover, protein nanopores have shown promise in identifying amino acids and post-translational modifications (PTMs), taking a major step toward single-molecule protein sequencing.…”