Single-cell mechanogenetics using monovalent magnetoplasmonic nanoparticles

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.

Figure 1. Schematic illustration of magnetoplasmonic nanoparticles (MPNs)

A MPN is comprised of a magnetic-core (perturbation module), a plasmonic-shell (imaging and conjugation module), and a functionalized oligonucleotide (chemical targeting module).

Figure 2. Schematic workflow of the protocol (left panel), key design principles (right panel)

Synthesis of small and monodisperse MPNs is achieved by sequential nanoparticle growth of a magnetic core, dielectric inner shell, and plasmonic outer shell. Dense loading of Au_2nm seeds and sufficiently long incubation of the shell growth are essential for the uniform shell formation (Stage 1, Steps 1–39). A targeting functionality is introduced by conjugation of MPNs with thiolated DNA via Au-thiol chemistry. Monovalently conjugated MPNs are selectively isolated via charge-based valency discrimination with anion exchange HPLC (Stage 2, Steps 40–47). The MPNs can target cell surface receptors with a diverse set of receptors via modular targeting and Watson-Crick hybridization (Stage 3, Steps 48–56). Coupled with an external micro magnetic tweezers (µMT), the targeted MPNs can provide two different modes of perturbation to the receptors as a function of distance between the MPN and the µMT: The SFM and WFM modes can be used for spatial and mechanical control, respectively (Stage 4, Steps 57–64).

Figure 3. Growth kinetics of MPNs

(a) Photographs and (b) absorption spectra of reaction solution as a function of time. (c) Changes in plasmon resonance and measurements of full width at half maximum (FWHM) scattering as a function of time.

Figure 4. Controlled synthesis of MPNs

(a) TEM images of 13 nm Zn-doped iron oxide nanoparticles (M), silica-coated Zn-doped iron oxide magnetic nanoparticles (M-SiO₂), Au_2nm seed bound M-SiO₂, and gold coated M-SiO₂. Scale bars, 50 nm. (b) Structural parameters of MPNs shown in panel c–h. (c–e) Control of gold shell thickness; TEM images of MPNs (left) with 10 nm (c), 12.5 nm (d), and 15 nm (e) shell thicknesses and their size distribution (right). The 96, 64, and 24 pmol of Au_2nm seed bound 20 nm M-SiO₂ are used in Step 34, respectively. Scale bars, 200 nm. (f–h) M-SiO₂ size dependent plasmonic absorption shift of 50 nm MPNs. TEM images (top) of 50 nm MPNs with 20 nm (f), 25 nm (g), and 30 nm (h) M-SiO₂ core and their corresponding scattering images (bottom). Scale bars, 50 nm. (i) Absorption spectra of 50 nm MPNs with 20 nm (black), 25 nm (green), and 30 nm (orange) M-SiO₂ core.

Figure 5. Monovalent DNA conjugation of MPNs

(a) Schematic illustration of oligonucleotide conjugation and purification of monovalent MPNs. Monovalent MPNs can be isolated via charge-based valency discrimination in an anion exchange HPLC column. To enhance colloidal stability and increase separation yield, thiolated PEG is used as an additional coating. (b) Oligo-length dependent HPLC elution profiles of MPNs. 30, 60, 80 and 100 bases DNA oligos are conjugated to 40 nm MPNs.

Figure 6. Dark-field microscope and micro-magnetic tweezer (µMT) setup

(a) A schematic illustration of µMT and dark-field microscopy setup. Steel needle (tip radius: 5 µm) - NdFeB magnet (diameter 1 cm) assembly is mounted to the xyz translation stage. A 4 mm ellipsoidal dot mirror is installed at a filter cube set for a dark-field illumination. (b) A photograph of experimental setup. Scale bar, 20 µm.

Figure 7. Distance dependent force generation of MPNs

Force acting on particles was plotted (gray dots) and was fitted with a power law (black solid line). TGT DNA force sensor and Stokes’ drag force are used to measure the applied force at SFM and WFM, respectively Each data point represents the mean value of multiple repetitions (n= 10 for SFM, n=30 for WFM). Error bars represent ±s.d.

Figure 8. Experimental setup for colloidal synthesis of magnetic nanoparticles

Figure 9. Mechanogenetic control

(a) Spatial control of actin polymerization using WFM mode of MPNs. (left) Schematic illustration of MPN induced spatial segregation of VE-cadherin and actin polymerization. (right) Spatial segregation of VE-cadherin receptors (green) and resulting actin polymerization (red) are observed by time-lapse fluorescence imaging of SNAP-VE-cadherin-mEmerald and Lifeact7-mCherry signals, respectively. Scale bars, 4 µm. (b) Mechanical control of single cell gene transcription using SFM mode of MPNs (9 pN). (left) Schematic illustration of UAS-Gal4 fluorescence reporter system and transcriptional activation by MPNs. (right) A representative optical microscope image of single cell mCherry expression. Scale bars, 4 µm.