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Nanopore Sensing

Nanopore sensors enable rapid, portable, and relatively inexpensive DNA sequencing. But they can do so much more! We are exploring new and non-traditional ways to use commercial nanopore sensor arrays for bespoke molecular sensing, synthetic biology and proteomics.

Nanopore Protein Sequencing

The ability to sequence single protein molecules in their native, full-length form would enable a more comprehensive understanding of proteomic diversity. Current technologies, however, are limited in achieving this goal. Here, we establish a method for the long-range, single-molecule reading of intact protein strands on a commercial nanopore sensor array. By using the ClpX unfoldase to ratchet proteins through a CsgG nanopore, we provide single-molecule evidence that ClpX translocates substrates in two-residue steps. This mechanism achieves sensitivity to single amino acids on synthetic protein strands hundreds of amino acids in length, enabling the sequencing of combinations of single-amino-acid substitutions and the mapping of post-translational modifications, such as phosphorylation. To enhance classification accuracy further, we demonstrate the ability to reread individual protein molecules multiple times, and we explore the potential for highly accurate protein barcode sequencing. Furthermore, we develop a biophysical model that can simulate raw nanopore signals a priori on the basis of residue volume and charge, enhancing the interpretation of raw signal data. Finally, we apply these methods to examine full-length, folded protein domains for complete end-to-end analysis. These results provide proof of concept for a platform that has the potential to identify and characterize full-length proteoforms at single-molecule resolution.

Read the full paper here

Molecular tagging with Porcupine

What are molecular tags? Molecular tags label physical objects using DNA or other molecules instead of conventional labels like RFID tags, QR codes, barcodes, and stickers. Molecular tags can be useful in cases where the tag needs to be small, lightweight, or invisible, or for tagging things that are too flexible or numerous.

Porcupine’s approach to molecular tagging is portable, quick to write and read new tags, and usable outside a lab setting, making it more accessible than existing molecular tagging methods. To write new tags, we combine tiny amounts of pre-prepared DNA solutions (molecular bits, or molbits), and to read tags, we use nanopore sequencing devices.

Porcupine starts with a digital tag, which is converted into a molecular tag using presence and absence (i.e., if a bit is set to 1, it is added to the molecular tag; if it is 0, it is left out). The tag is then prepared for nanopore sequencing and dehydrated in order to extend the tag’s shelf life, decrease readout time, and make tags robust to environmental contamination. When it’s ready to be read again, the tag is rehydrated and loaded onto a MinION flowcell. After sequencing for a brief period of time, we classify molbits directly from the raw nanopore signal, avoiding basecalling. We then identify the bits that are present and recover the digital tag.

More details on how we design the molecular tags, encode/decode data, and classify the molbits can be found in the publication.

Additional resources:

• Preprint
• Presentation at DEF CON Biohacking Village 2020 - YouTube
• GitHub
• Featured on Bioinformatics Chat podcast

Sensing protein barcodes (NanoporeTERs)

NanoporeTERs: Nanopore-addressable protein Tags Engineered as Reporters

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.

People

Jeff Nivala

Research Assistant Professor

Mattias Tolhurst

Daphne Kontogiorgos-Heintz

Kingsley Wong

Safwan Diwan

Oren Fox

Gwen Roote