Noti hf

Plasmids for IVT were designed using the full-length nucleotide sequences of RSV F and RSV G from RSV A2 (GenBank M74568.1). The coding region was followed by a 3′ untranslated region derived from the mouse alpha globin sequence. Sequences were codon optimized and inserted in a pMA-7 vector (Thermo Fisher Scientific, GeneArt) to be used as a template for mRNA synthesis. Plasmids were linearized with Not-I HF (New England Biolabs) overnight prior to IVT using a T7 mScript kit (Cellscript) following the manufacturer’s instructions. ATP, GTP, and CTP were used alongside m1Y-5′-triphosphate (TriLink). RNAs were capped using 2′-O-Methytransferase followed by enzymatic addition of a poly-A tail, both according to the mScript kit instructions. The capped and tailed mRNAs were then purified using an RNeasy kit (Qiagen), treated with Antarctic Phosphatase for 2 h (New England Biolaboratories), and purified again. Vero cells were transfected using a Neon electroporation system (Invitrogen) into a 24-well plate were transfected with 1 µg of synthetic mRNA encoding RSV F or RSV G, respectively, according to the manufacturer’s protocol. 20 h post-transfection, cells were stained with SBA-488 at 4 °C immediately before fixation and immunostaining without permeabilization.

cDNAs of ASCC1, ASCC2, ASCC3, and cGAS were bought from Horizon, were amplified with primers adding attB1 and attB2 sequences (Table S3) and were cloned into the pDONR223 vector using the gateway BP recombinase system (Thermo Fisher Scientific, 11789020). All cGAS mutants were generated using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, E0554S) with specific primers (Table S3) and verified by sequencing. pDONR223 constructs were recombined into the pFRT/ TO/FLAG/HA-DEST or pFRT/TO/GFP-DEST destination vector using Gateway LR Clonase II Enzyme mix according to the manufacturer’s protocol (Thermo Fisher Scientific, 11791020). pGEX6p-1 cGAS WT was generated using In-Fusion® HD Cloning Kit (Takara Bio USA, 102518). pGEX6p-1 cGAS 8his (c-terminus) was generated using the Q5® Site-Directed Mutagenesis Kit (New England Biolabs, E0554S). pGEX6p-1 hPrimpol1 was generated using BamHI-HF (New England Biolabs, R3136S) and NotI-HF (New England Biolabs, R3189S) restriction enzymes

The type A outer surface protein C (OspC) gene of Borrelia burgdorferi B31 (GenBank accession #AAC66329.1) was designed to include the human tyrosinase signal peptide (MLLAVLYCLLWSFQTSAGHFPRA; GenBank accession #AH003020) at the N-terminus (30 (link), 31 (link)). The coding region was optimized for expression in mice and commercially synthesized in a pUC57 plasmid vector by Bio Basic Inc. (Markham, ON). The OspC gene containing the signal sequence was sub-cloned into a pVAX1 plasmid vector (ThermoFisher, Ottawa, ON), using NotI-HF and EcoRI-HF restriction enzymes (New England Biolabs, Whitby, ON). Large-scale amplifications of the pVAX1-OspC plasmid were generated using the QIAGEN Plasmid Giga Kit (Montreal, QC) according to the manufacturer’s instructions. The genetic sequences were validated by Sanger Sequencing prior to nanoparticle encapsulation.

To cut out the transgene from the plasmid backbone, a restriction digest reaction was set up as a 50 μL reaction in a PCR tube as follows:

Reagent	Volume/concentration
Restriction enzyme (HindIII-HF, NotI-HF, New England Biolabs)	1 μL of each
10X CutSmart® buffer (New England Biolabs)	5 μL
Plasmid DNA	1 μg
dH2O	Remaining volume (up to 50 μL)

The reaction was run for four hours at 37 °C on a thermocycler