APPRAISE is a computational method for predicting the receptor binding propensity of engineered proteins based on structural modeling. This tool, leveraging the most recent breakthroughs in computational structure prediction (e.g., AlphaFold2), may help protein engineers to evaluate protein/peptide-based therapeutics for targeted drug delivery. You can try APPRAISE in your browser using Google Colab. (bioRxiv Manuscript, GitHub repository)

Related publication:

• Ding, X.*, Chen, X., Sullivan, E. E., Shay, T. F., & Gradinaru, V.* (2024). APPRAISE: Fast, accurate ranking of engineered proteins by receptor binding propensity using structural modeling. Molecular Therapy. * Corresponding authors. Link PDF

The brain is protected by a tight barrier called the blood-brain barrier (BBB) that prevents most macromolecules from accessing the brain. However, bioengineers have identified a panel of brain-transducing gene delivery vectors based on Adeno-associated virus (AAV), a non-pathogenetic virus that symbioses with primates, using a protein engineering technique called directed evolution. I developed and applied computational pipelines based on AlphaFold to to help understand why and how these viral vectors can utilize protein receptors on the BBB as gateways toward the brain. These studies may help biomedical scientists to make therapeutics that can target the brain efficiently and specifically.

Related publications:

• Shay, T. F.†*, Sullivan, E. E.†, Ding, X.† (co-first), Chen, X., Kumar, S. R., Goertsen, D., ... & Gradinaru, V.* (2023). Primate-conserved Carbonic Anhydrase IV and murine-restricted Ly6c1 are new targets for crossing the blood-brain barrier. Science Advances. † Equal contribution. Link  News
• Jang, S., Shen, H. K., Ding, X., Miles, T. F., & Gradinaru, V.* (2022). Structural basis of receptor usage by the engineered capsid AAV-PHP. eB. Molecular Therapy-Methods & Clinical Development. Link

Expand the size of AAV capsids. We aimed to increase the cloning capacity of AAV capsids by expanding the sizes of single particles (3-minute video by Caltech Neuroscience).

Other collaborative projects on AAV capsid engineering. I was involved in multiple capsid engineering projects and contributed with my expertise in structural biology and biophysics.

Related publications:

• Chen, X., Wolfe, D. A., Bindu, D. S., Zhang, M., Taskin, N., Goertsen, D., ... & Gradinaru, V.* (2023). Functional gene delivery to and across brain vasculature of systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates. bioRxiv. Link
• Seo, J. W., Ingham, E. S., Mahakian, L., Tumbale, S., Wu, B., Aghevlian, S.,Shams S., Baikoghli M., Jain P., Ding X., Goeden N. ... & Ferrara, K. W.* (2020). Positron emission tomography imaging of novel AAV capsids maps rapid brain accumulation. Nature communications. Link
• Ravindra Kumar, S., Miles, T. F., Chen, X., Brown, D., Dobreva, T., Huang, Q., Ding X., Luo Y. ... & Gradinaru, V.* (2020). Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nature methods. Link

Structure-guided directed evolution of channelrhodopsins

2016 Spring

Advisor: Dr. Frances Arnold (Arnold lab, Nobel Prize 2018)

Co-mentor: Dr. Claire Bedbrook

We engineered a genetically encoded tool for optical control of neuronal activity by structure-guided recombination.

Related publication:

• Bedbrook, C.N., Rice, A.J., Yang, K.K., Ding, X., Chen, S., LeProust, E.M., Gradinaru, V. and Arnold, F.H., (2017). Structure-guided SCHEMA recombination generates diverse chimeric channelrhodopsins. Proceedings of the National Academy of Sciences. Link

Technology development for sensitive detection and analysis of neoantigen-specific T cells

2015 Winter

Advisor: Dr. James Heath (Heath lab, now Heath lab)

Co-mentor: Dr. Songming Peng

I contributed to the development of a technology for extraction and analysis of neoantigen-specific T cells from cancer patients.

Related publication:

• Peng, S., Zaretsky, J.M., Ng, A.H., Chour, W., Bethune, M.T., Choi, J., Hsu, A., Holman, E., Ding, X., Guo, K. and Kim, J.,..., Baltimore, D., Ribas, A., Heath, J.R. (2019). Sensitive detection and analysis of Neoantigen-specific T cell populations from tumors and blood. Cell reports. Link

Protein engineering for non-invasive neural modulation

2015 Fall

Advisor: Dr. Mikhail Shapiro (Shapiro lab)

Co-mentor: Dr. Jérôme Lacroix & Di Wu

We explored a few designs of genetically encoded tools for acoustic modulation of neuronal activity.

Related publication:

• Acknowledged for contributing to initial experiments in Wu, D., Baresch, D., Cook, C., Ma, Z., Duan, M., Malounda, D., ... & Shapiro, M. G. (2023). Biomolecular actuators for genetically selective acoustic manipulation of cells. Science Advances. Link

Advisors: Dr. Lei Liu, Dr. Yiming Li

Collaborator: Dr. Zhipeng Wang

School of Life Sciences, Tsinghua University

Teaching is the best way of learning. Together with Zhipeng Wang, a chemistry PhD student, I wrote parts of two introductory minireviews on chemical biology topics after serious literature research. These topics are NOT our specialties, but we got a better understanding of the fields through reading and writing the reviews.

Related publications:

• Wang, Z., Ding, X., Li, S., Shi, J., & Li, Y. (2014). Engineered fluorescence tags for in vivo protein labelling. RSC Advances. Link
• Wang, Z. P., Ding, X. Z., Wang, J., & Li, Y. M. (2015). Double-edged sword in cells: chemical biology studies of the vital role of cytochrome c in the intrinsic pre-apoptotic mitochondria leakage pathway. RSC Advances. Link

Advisor: Dr. Michael Lin (Lin lab)

Co-mentor: Dr. François St-Pierre (now St-Pierre lab)

Departments of Bioengineering and Pediatrics, Stanford University

Brain processes information by electrical signals. It would be very helpful for us to understand how this happens if we can precisely record electrical activity in individual neurons or even whole neuronal circuits. Genetically encoded voltage indicators are tools that enable optical detection of the membrane potential of genetically defined neuronal circuits through fluorescence microscopy. At Stanford, I worked on a genetically encoded voltage indicator named Accelerated Sensor of Action Potentials (ASAP). ASAP is a fusion protein made of two modular components: 1) a membrane-localized voltage-sensitive domain and 2) an engineered GFP. The unique architecture of ASAP made its fluorescent intensity responsive to membrane voltage. Original ASAP sensors produced fast, millisecond kinetics, but was with limited fluorescence response to action potentials. In this project, I developed and validated a screening method to be used to search for improved sensors. I constructed 87 variants of the original sensor and screened them with this assay. Through the screening, I found a few promising variants with 50%-100% larger fluorescence responses.

The visit was sponsored by Stanford Undergraduate Visiting Researcher (UGVR) program.

Related publication:

• Yang, H. H.†, St-Pierre, F.†, Sun, X., Ding, X., Lin, M. Z., & Clandinin, T. R. (2016). Subcellular Imaging of Voltage and Calcium Signals Reveals Neural Processing In Vivo. Cell. †:Equal contribution. Link

Advisor: Dr. Haitao Li (Dr. Haitao Li)

Co-mentor: Dr. Xiaonan Su

School of Medicine, Tsinghua University

Histone post-translational modifications, or histone PTMs, play important roles in epigenetic regulation, and they are proposed to constitute a ‘histone code’. The role of a histone effector is to recognize the specific histone PTMs (or read the histone code) and recruit downstream proteins to take action. Previous reports showed that Spindlin-1 reads the trimethyl-lysine4 on histone H3 (H3K4me3). The structure of Spindlin-1 had already been solved before my project. However, the structure of free protein could not tell the molecular mechanism that Spindlin-1 recognizes the modification. The goal of this project was to solve the complex structure of Spindlin-1 bound to H3 peptide with trimethyl-lysine4 modification. We started from molecular cloning and expressing the protein in E.Coli, and established protocols for purifying the protein, and then screened for the crystallization conditions. After getting the co-crystal of the complex and solved the structure, I continued on biophysical characterization of the interaction with Isothermal Titration Calorimetry(ITC) and some mutant studies. Later structural studies showed a concurrent recognition of H3K4me3 and H3R8me2a by Spindlin-1.

This was my first formal scientific research project. The training was sponsored by National Training Programs of Innovation and Entrepreneurship for Undergraduate (China), and Tsinghua Talented Program ("Xuetangban" Program).

Related publication:

• Su, X.†, Zhu, G.†, Ding, X., Lee, S. Y., Dou, Y., Zhu, B., ... & Li, H. (2014). Molecular basis underlying histone H3 lysine–arginine methylation pattern readout by Spin/Ssty repeats of Spindlin1. Genes & development. †:Equal contribution. Link  News