The Chen Group
Nanopore Technologies at UMass Amherst
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We are a nanopore development group at the University of Massachusetts Amherst.
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Research in the Chen group focuses on membrane proteins, specifically pore-forming proteins (PFP). We aim to understand the folding, assembly, dynamics and functional mechanism of PFPs. In parallel, we use what we learn about PFPs to develop biotechnological applications for detecting biomarkers and screening drugs to treat infectious diseases and cancer. To achieve our goal, the research in Chen’s lab uses a combination of approaches in molecular biology, biochemistry and biophysics.
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Our Research
New nanopore tweezers tools to monitor protein structural dynamics at single molecule level.
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In this project, we are developing a novel nanopore-based approach to observe the subtle changes in the structures of enzymes in real-time. Our method, dubbed “single molecule nanopore tweezer” is a label-free, single-molecule approach that can continuously monitor enzymes' dynamic structural changes over long period of time (hrs) while still maintain high temporal resolution (100 us).
Flavivirus are major mosquito-borne pathogens infecting millions of people worldwide each year. Currently there is no antiviral therapy available for treating West Nile, Dengue and Zika viral infections. The flavivirus two-component NS2B/NS3 protease is a highly conserved serine protease that is essential for viral replication, making it an attractive drug target. We will build a nanopore tweezers tool set that is readily tunable for trapping, tracking, and analyzing various flaviviral proteases to determine the structural dynamics, and binding thermodynamics and kinetics profiles of NS2B/NS3 interacting with various inhibitors. This work will provide unprecedented kinetic information on the function-structural dynamics relationship of NS2B/NS3 complex and mechanisms of substrate binding and inhibition, as well as establish a new paradigm for high-throughput drug screening that is independent of enzymatic activity.
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In a separate research area, protein kinases are attractive drug targets because of their pivotal roles in regulating the majority of cellular pathways. Kinases have dynamic structures that change significantly as they perform their catalytic function. Insight into their conformational and energetic landscape of the catalytic function regulation will facilitate the drug design. Traditional ensemble structural techniques, such as X-ray cryptography, cryo-EM or NMR, provide atomic view of kinases’ conformational states. We will apply nanopore tweezers to deliver detailed kinetics of conformational states of kinases for constructing a comprehensive biophysical model for the ligand binding, γ-phosphate transfer, regulation and inhibition.
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Related publications:
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Li F, Fahie MA, Gilliam K, Pham R# and Chen M*: Mapping the energy landscape of Abl kinase by using ClyA nanopore tweezers. Nat Commun, 2022, DOI:10.21203/rs.3.rs-1153833/v1.
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Shorkey S, Du J, Pham R#, Strieter ER and Chen M*: Real-time and label-free measurement of deubiquitinase activity with a MspA nanopore. ChemBioChem, 2021, 22(7), 2688-2692.
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Li X, Lee K, Chen J and Chen M*: Different anomeric sugar bound states of MBP resolved by a ClyA nanopore tweezer. ACS Nano 2020, 14(2): 1727–1737.
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Engineering OmpG nanopores for protein sensing.
For early disease diagnostics, highly sensitive detection of protein-related biomarkers is pivotal to enable timely therapeutic outcomes, while for point-of-care testing and personalized health care/management, rapid, accurate and simple detection methods are desirable. In a scenario such as an epidemic outbreak, it is imperative to quickly create a sensor to help monitor and contain highly infectious agents. We have created a serial of OmpG nanopore-based protein sensors. We are to extend our OmpG platform for sensing a wide variety of disease-related proteins and micro-organisms.
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Related publications:
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Fahie MAV, Li F, Palmer C#, Yoon C# and Chen M*: Modifying the pH sensitivity of OmpG nanopore for improved detection at acidic pH, Biophysical J, 2022, 121(5): 731-741.
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Perez-Rathke A§, Fahie MA§, Chisholm C, Liang J* and Chen M*: Mechanism of OmpG pH-dependent gating from loop ensemble and single channel studies. J Am Chem Soc 2018, 140 (3): 1105–1115.
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Fahie M, Chisholm C and Chen M*: Resolved single-molecule detection of individual species within a mixture of anti-biotin antibodies using an engineered monomeric nanopore, ACS Nano 2015, 9(2):1089-98.
Nanopore DNA/RNA Sequencing.
Nanopore DNA/RNA sequencing is based on measuring changes in the ionic current as a single DNA/RNA molecule translocate through a nanopore. The commercialized nanopore sequencer, MinION holds many advantageous features, including long read length, real-time analysis, high portability, and moderate costs. Despite recent advances, the technology still exhibits a relatively high error rate with raw sequences compared to standard Sanger sequencing methods. In this project, we aim to understand the factors determining current signals. The newly acquired knowledge will also guide us to engineer biological nanopores for enhanced DNA/RNA sequencing technologies with high precision.
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Related publications:
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Tabatabaei KS§, Pham B§, Pan C§, Liu J, Chandak S, Shorkey S, Hernandez AG, Aksimentiev A*, Chen M*, Schroeder CM*, Milenkovic O*: Expanding the Molecular Alphabet of DNA-Based Data Storage Systems with Nanopore Readouts. Nano letters, 2022, doi/10.1021/acs.nanolett.1c04203.
Peptide and protein sequencing.
We aim to develop a nanopore protein sequencing technology that will directly read the sequence of the amino acids of proteins as an intact polypeptide chain. We are particularly interested in using the method to study post-translational modification.
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Related publications:
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Yu L, Kang X, Li F, Mehrafrooz B, Makhamreh A, Fallahi A, Aksimentiev A, Chen M, Wanunu M: Unidirectional Single-File Transport of Full-Length Proteins Through a Nanopore, 2022, submitted, bioRxiv, DOI: 10.1101/2021.09.28.462155.