Chemical profiling of DNA G-quadruplex-interacting proteins in live cells

  • Nat Chem. 2021 Jul;13(7):626-633. doi: 10.1038/s41557-021-00736-9.
Xiaoyun Zhang  #  1 ,  Jochen Spiegel  #  2 ,  Sergio Martínez Cuesta  1  2  3 ,  Santosh Adhikari  1 ,  Shankar Balasubramanian  4  5  6
Affiliations
  • 1. Department of Chemistry, University of Cambridge, Cambridge, UK.
  • 2. Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK.
  • 3. Data Sciences and Quantitative Biology, Discovery Sciences, AstraZeneca, Cambridge, UK.
  • 4. Department of Chemistry, University of Cambridge, Cambridge, UK. [email protected].
  • 5. Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK. [email protected].
  • 6. School of Clinical Medicine, University of Cambridge, Cambridge, UK. [email protected].
  • # Contributed equally.
PMID: 34183817 DOI: 10.1038/s41557-021-00736-9
Abstract

DNA-protein interactions regulate critical biological processes. Identifying proteins that bind to specific, functional genomic loci is essential to understand the underlying regulatory mechanisms on a molecular level. Here we describe a co-binding-mediated protein profiling (CMPP) strategy to investigate the interactome of DNA G-quadruplexes (G4s) in native chromatin. CMPP involves cell-permeable, functionalized G4-ligand probes that bind endogenous G4s and subsequently crosslink to co-binding G4-interacting proteins in situ. We first showed the robustness of CMPP by proximity labelling of a G4 binding protein in vitro. Employing this approach in live cells, we then identified hundreds of putative G4-interacting proteins from various functional classes. Next, we confirmed a high G4-binding affinity and selectivity for several newly discovered G4 interactors in vitro, and we validated direct G4 interactions for a functionally important candidate in cellular chromatin using an independent approach. Our studies provide a chemical strategy to map protein interactions of specific nucleic acid features in living cells.

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