한빛사 논문
Ji-wook Kim1-3,7, Daeha Seo4,5,7, Jung-uk Lee1-3,7, Kaden M Southard4,6, Yongjun Lim1-3, Daehyun Kim1-3, Zev J Gartner5,6, Young-wook Jun1,2,4,7,* & Jinwoo Cheon1-3,*
1Center for Nanomedicine, Institute for Basic Science (IBS), Seoul, Republic of Korea. 2Yonsei-IBS Institute, Yonsei University, Seoul, Republic of Korea. 3Department of Chemistry, Yonsei University, Seoul, Republic of Korea. 4Department of Otolaryngology, University of California, San Francisco, San Francisco, California, USA. 5Department of Emerging Materials Science, Daegu Gyeongbuk Institute of Science & Technology (DGIST), Daegu, Republic of Korea. 6Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA. 7These authors contributed equally to this work
*Correspondence to: Young-wook Jun or Jinwoo Cheon
Abstract
Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to answer a host of important biological questions. This protocol describes a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. The key components of this tool are a magnetoplasmonic nanoparticle (MPN) actuator that delivers defined spatial and mechanical cues to receptors through target-specific one-to-one engagement and a micromagnetic tweezers (μMT) that remotely controls the magnitude of force exerted on a single MPN. In our approach, a SNAP-tagged cell-surface receptor of interest is conjugated with a single-stranded DNA oligonucleotide, which hybridizes to its complementary oligonucleotide on the MPN. This protocol consists of four major stages: (i) chemical synthesis of MPNs, (ii) conjugation with DNA and purification of monovalent MPNs, (iii) modular targeting of MPNs to cell-surface receptors, and (iv) control of spatial and mechanical properties of targeted mechanosensitive receptors in live cells by adjusting the μMT-to-MPN distance. Using benzylguanine (BG)-functionalized MPNs and model cell lines expressing either SNAP-tagged Notch or vascular endothelial cadherin (VE-cadherin), we provide stepwise instructions for mechanogenetic control of receptor clustering and for mechanical receptor activation. The ability of this method to differentially control spatial and mechanical inputs to targeted receptors makes it particularly useful for interrogating the differential contributions of each individual cue to cell signaling. The entire procedure takes up to 1 week.
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