# Long-read single-molecule protein sequencing on an array of unfoldase-coupled nanopores

> **NIH NIH R01** · UNIVERSITY OF WASHINGTON · 2022 · $647,138

## Abstract

SUMMARY
We propose to develop the foundations of a platform for direct sequencing of native, full-length protein strands
using unfoldase-coupled nanopore array technology. In principle, this technology could be used to identify
protein primary sequence, in addition to certain post-translational modifications (PTMs) found in prokaryotic
and eukaryotic cells, with single-molecule resolution. It is a foundational advance over existing and other
next-gen proteomic technologies such as Edman degradation, mass spectrometry, fluorescent label
approaches, and immunoaffinity-based methods that suffer from limitations in read length, throughput,
sensitivity, labeling efficiency, and/or the availability of suitable affinity reagents. Nanopore sequencing of intact
protein strands overcomes these limitations because the ~1 nanometer-long sensor directly interacts with the
protein strand as it is linearly-driven through the pore by the unfoldase motor protein, manifesting
sequence-specific ionic current signals. Thus, complete sequence analysis of native protein molecules can be
achieved. This method is a natural technical extension of current nanopore sequencing platforms that use
molecular motors to control movement of nucleic acid strands through nanopores in DNA/RNA sequencing.
During the grant period, we will pursue three specific aims: 1) Establish baseline methods of controlled protein
translocation through nanopore sensor arrays using unfoldase motors; 2) Develop computational and
bioinformatic methods to translate raw nanopore signal data into protein sequence information (amino acid
calling and PTM detection); and 3) Establish techniques for analysis of native proteins and proteomic samples.
Our team of investigators is uniquely qualified to take on this project:
i) We pioneered the analysis of full-length protein strands using unfoldase-coupled nanopore sensors and
recently demonstrated that the Oxford Nanopore MinION nanopore array device can be used to directly detect
peptide strands and resolve single amino acid substitutions (Nivala).
ii) Co-investigators on this application have elucidated and exquisitely characterized the enzymatic
mechanisms of unfoldase motor activity through in vitro biochemical, single-molecule, and structural studies
(Martin), and have led the development of nanopore raw signal analyses for sequencing of nucleic acids,
including direct RNA sequencing, genome and transcriptome-wide detection of modified bases, and assembly
of a human genome using ultra-long DNA nanopore reads (Jain).
iii) Collaborators will provide access to enabling nanopore technology platforms and expertise, including
highly-parallel nanopore sensor arrays and customized nanopore proteins, and offer natural routes to
technology transfer (Oxford Nanopore), contribute to characterization and comparison of project results to
traditional analysis methods such as protein mass spectrometry (Guttman), and advise on compelling
technological applications that wi...

## Key facts

- **NIH application ID:** 10498020
- **Project number:** 1R01HG012545-01
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Jeffrey Matthew Nivala
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $647,138
- **Award type:** 1
- **Project period:** 2022-09-21 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10498020

## Citation

> US National Institutes of Health, RePORTER application 10498020, Long-read single-molecule protein sequencing on an array of unfoldase-coupled nanopores (1R01HG012545-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10498020. Licensed CC0.

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