Lacey carbon film

To assess CpG-A incorporation into SNAs, agarose gel electrophoresis was performed using 1% agarose (Sigma) with SYBR Safe DNA gel stain in 0.5× tris/borate/EDTA (TBE) (ThermoFisher Scientific) under 120 V for 1 h. To assess dual-CpG loading, fluorophore-labeled oligonucleotides (fluorescein-CpG-A and Cy5-CpG-B) were used, and agarose gel electrophoresis was performed using 0.5% agarose in 1× TBE without further staining. Dynamic light scattering (DLS) and zeta potential measurements were performed using a Malvern Zetasizer Nano with ~10 nM samples by particle. CryoTEM was performed with a Hitachi HT7700 TEM with a Gatan cryo-transfer holder, and imaging was performed under a 120 kV accelerating voltage. CryoTEM samples were prepared by loading 4 μL of sample (100 μM CpG SNA stock solution or 1.33 μM liposome solution) onto 300-mesh copper TEM grids with lacey carbon films (Electron Microscopy Sciences) using a FEI Vitrobot Mark IV with its chamber equilibrated at 4 °C and 100% humidity. The samples were blotted for 5 s and plunged into liquid ethane before they were transferred and stored in liquid nitrogen. FRET measurements were performed using a Synergy plate reader (Biotek) with 10 nM samples by oligonucleotides. FRET efficiency (E) was calculated as
$$E=1-\frac{F_{\text{DA}}}{F_{\text{D}}}$$
where F_DA and F_D are the donor fluorescence intensities with and without an acceptor, respectively.

Cryo-TEM samples were prepared by depositing 3 μL sample on a 200 mesh Cu grids with lacey carbon films (Electron Microscopy Sciences). All TEM grids were surface plasma treated for 20 s using a Gatan Solarus Advanced Plasma Cleaning System 950 prior to use. An automated vitrification robot (Leica EM GP) was used for plunge vitrification in liquid propane. Cryo-TEM studies were performed on the JEOL JEM 2100F operated at 200 kV, 2k × 2k Gatan CCD camera. Gatan Digital Micrograph. ImageJ was used for TEM image analysis.