brief communications
Multiplexed direct detection of barcoded protein reporters on a nanopore array
Nicolas Cardozo1,2 na1,
Karen Zhang ORCID: orcid.org/0000-0003-1177-635X1 na1,
Kathryn Doroschak ORCID: orcid.org/0000-0003-4806-51641 na1,
Aerilynn Nguyen1,
Zoheb Siddiqui1,
Nicholas Bogard ORCID: orcid.org/0000-0001-9231-93823,
Karin Strauss ORCID: orcid.org/0000-0002-8327-54771,4,
Luis Ceze ORCID: orcid.org/0000-0002-1377-62171 &
Jeff Nivala ORCID: orcid.org/0000-0002-8210-54171
Nature Biotechnology volume 40, pages 42–46 (2022)Cite this article
Assay systems
Detection of specific proteins using nanopores is currently challenging. To address this challenge, we developed a collection of over twenty nanopore-addressable protein tags engineered as reporters (NanoporeTERs, or NTERs). NTERs are constructed with a secretion tag, folded domain and a nanopore-targeting C-terminal tail in which arbitrary peptide barcodes can be encoded. We demonstrate simultaneous detection of up to nine NTERs expressed in bacterial or human cells using MinION nanopore sensor arrays.
A nanopore interface for higher bandwidth DNA computing
Karen Zhang
, Yuan-Jyue Chen
… Jeff Nivala
Fig. 1: NTERs.
Fig. 2: Classification and multiplexed detection of NTER expression levels with a MinION.
Data are available upon request and on can be found on github.com/uwmisl/NanoporeTERs.
Codes are available upon request and can be found on github.com/uwmisl/NanoporeTERs. Custom MinION MinKNOW runscripts can also be obtained from Oxford Nanopore Technologies upon request.
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We thank additional members of the Molecular Information Systems Lab for helpful discussion and feedback on this work. The OsmY expression plasmid was generously provided by C. Bryan and L. Carter (Institute for Protein Design, University of Washington). We also thank A. Heron and R. Gutierrez (Oxford Nanopore Technologies) for providing the configurable MinION run script and discussions on its use, and M. Jain (UCSC) for a custom Matlab script that facilitated visualization of the raw MinION data. This work was supported in part by NSF EAGER Award no. 1841188 and NSF CCF Award no. 2006864 to L.C. and J.N., an NIH/NCI Cancer Center Support Grant (no. P30 CA015704) Pilot Award and NSF Award 2021552 to J.N. and a sponsored research agreement from Oxford Nanopore Technologies.
These authors contributed equally: Nicolas Cardozo, Karen Zhang, Kathryn Doroschak.
Paul G. Allen School of Computer Science and Engineering, University of Washington, Seattle, WA, USA
Nicolas Cardozo, Karen Zhang, Kathryn Doroschak, Aerilynn Nguyen, Zoheb Siddiqui, Karin Strauss, Luis Ceze & Jeff Nivala
Molecular Engineering PhD Program, University of Washington, Seattle, WA, USA
Nicolas Cardozo
Department of Electrical and Computer Engineering, University of Washington, Seattle, WA, USA
Nicholas Bogard
Microsoft Research, Redmond, WA, USA
Karin Strauss
Kathryn Doroschak
Aerilynn Nguyen
Zoheb Siddiqui
Luis Ceze
Jeff Nivala
N.C., K.Z., A.N. and N.B. performed wet laboratory experiments. K.Z. and K.D. developed the data analysis pipeline and performed computational analyses. Z.S. implemented the machine learning approach. N.B., K.S., L.C. and J.N. supervised the project. J.N. conceived and directed the project. All authors contributed to writing and editing of the manuscript.
Correspondence to Jeff Nivala.
A provisional patent has been filed by the University of Washington covering aspects of this work (Patent Application no. 17/283,007). K.S. is an employee of Microsoft. J.N. is a consultant to Oxford Nanopore Technologies. The remaining authors declare no competing interests.
Peer review information Nature Biotechnology thanks Yi-Tao Long, Meni Wanunu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–13, Notes and References.
Cardozo, N., Zhang, K., Doroschak, K. et al. Multiplexed direct detection of barcoded protein reporters on a nanopore array. Nat Biotechnol 40, 42–46 (2022). https://doi.org/10.1038/s41587-021-01002-6
Accepted: 28 June 2021
Issue Date: January 2022
Yuan-Jyue Chen
Nanopore-detectable reporter proteins
Arunima Singh
Nature Methods (2021)