The synthesis of CAPs was described in the Supporting Information. The mRNA and replicon were synthesized following the previously reported method.[8a ] The LNP formulations were prepared based on the previous reports.[13 ] Briefly, the ethanol phase was composed of CAP lipids MC3, or ALC‐0315 with other helper lipids dissolved in ethanol at a certain molar ratio; Aqueous phased was prepared by diluting firefly luciferase (FLuc) mRNA, Dmc1 mRNA, or saRNA in a citrate buffer (pH 3). The LNPs were prepared by a rapid mixing method via a pipetting technique or a microfluidic device and were then purified by dialysis. Particle size and zeta potential were quantified by NanoZS Zetasizer (Malvern). The entrapment efficiency was measured by the RiboGreen assay. The morphology of LNPs was characterized by a Cryo‐EM (Thermo Scientific Glacios) device as we described previously.[13 ]
Cryo em
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
Cryo-EM is a powerful imaging technique used to visualize the structure of biological molecules, such as proteins, at the atomic level. It involves rapidly freezing samples to cryogenic temperatures, which preserves their native structure, and then using an electron microscope to capture high-resolution images. This method allows for the analysis of complex biomolecular complexes and structures that were previously challenging to study using other techniques.
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Lab products found in correlation
4 protocols using cryo em
1
Synthesis and Characterization of Lipid Nanoparticles
2
Cryo-EM Analysis of Apolipoprotein Vesicles
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The morphology of apoVs was observed by Cryo-EM (Thermo Fisher, USA) as we previously described [16 (link), 68 (link)]. In brief, after sample preparation, images of apoVs were collected at a dose rate of 40 e/pixel/s, exposed for 1 s. The pixel size at the object scale was 1.584 Å (nominal magnification 92 K) and 2.557 Å, with the defocus set at about -3 μm.
3
CryoEM Visualization of Micelle Structures
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CryoEM (FEI Tecnai
12) was used to confirm the size of the micelles as well as observe
their shape. Samples were prepared at room temperature at 1 mg/mL
DOX-bound unimers in 0.01 M HEPES. The samples were then manually
added to holey-carbon-coated copper grids. A FEI Vitrobot then blotted
and plunged the samples into liquid ethane. The samples were transferred
to liquid nitrogen until imaged.
12) was used to confirm the size of the micelles as well as observe
their shape. Samples were prepared at room temperature at 1 mg/mL
DOX-bound unimers in 0.01 M HEPES. The samples were then manually
added to holey-carbon-coated copper grids. A FEI Vitrobot then blotted
and plunged the samples into liquid ethane. The samples were transferred
to liquid nitrogen until imaged.
4
Cryo-TEM and Cryo-ET of Protein Complexes
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The study was carried out with Titan Krios 60–300 TEM/STEM (FEI) CryoEM, equipped with TEM direct electron detector Falcon II (FEI), Cs image corrector (CEOS, Heidelberg, Germany) and VPP [38 (link)], at accelerating voltage of 300 kV. The accumulated total dose for VPP Cryo-TEM was ~30e/Å2, the defocus value was ~0.5 µm, and the VPP phase shift was ~π/2. For CET study, 15 datasets of the sample were collected automatically with FEI Tomography software in low-dose mode, with 18000x magnification, and the defocus value was in the range between –3 and –5 μm, using bidirectional tilt scheme (0°, –2°, …, -58°, –60°, 2°, 4°, …, 58°, 60°). The accumulated total dose was of 61e/Å2.
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