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.
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.
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.