Thermostable inorganic pyrophosphatase

All RNA samples were prepared by T7 RNA polymerase (RNAP)-based in vitro transcription following well-established protocols [65 (link)]. In brief, transcriptions were carried out in 40 mM Tris-HCl (pH 8 at 37 °C), 1 mM spermidine, 0.01% Triton-X100, 80 mg/mL polyethylene glycol, 0.3 μM DNA templates (Integrated DNA Technology), 1 mM DTT, 2 U/µL thermostable inorganic pyrophosphatase (New England Biolabs, Ipswich, MA, USA), 5–15 mM ribonucleotide 5′-triphosphates (rNTPs), 5–15 mM MgCl₂, and 0.1 mg/mL T7 RNAP. The reactions were first optimized for appropriate rNTP and MgCl₂ concentrations at the small (50 µL) and medium (500 µL) scales before carrying out large-scale (5 mL) reactions. All transcriptions proceeded for 3 h at 37 °C. After transcription, the samples were extracted with acid phenol:chloroform, ethanol precipitated, purified by preparative denaturing polyacrylamide gel electrophoresis, and electroeluted. The samples were then dialyzed five times against UltraPure water and folded by heating for 2 min at 95 °C, snap-cooling on ice, and slowly equilibrating to room temperature.

10 µL of detection mixture was prepared by mixing 3 µL Thermopol buffer (B9004, New England Biolabs), 0.4 µL BST 2.0 WarmStart (M0538, New England Biolabs), 1.2 µL dNTPs mix (10 mM stock, Promega), 1.13 µL Syto82 dye (S11363, Thermo Fisher Scientific), 0.08 µL Thermostable inorganic pyrophosphatase (M0296, New England Biolabs), 0.16 µL of primer mix (20 µM stock, 5′-T*C*GCAACATCCTATATCTGC-3′ and 5′-T*G*AGCTTTGACAATACTTGA-3′) and 4.03 µL nuclease free water. 1.25 µL PPL reaction sample was added to 10 µL of detection mix. Samples were incubated at 50 °C for 60 min in a CFX384 Touch Real-Time PCR Detection System (Bio-Rad). The fluorescence read-out was taken every minute in the Cal Orange 560 setting.

The sequence of all DNA molecules and expected RNA transcript sequences were chosen to minimize the occurrence of alternative secondary structures checked by the DNA and RNA folding program NUPACK (29 (link)). The DNA and RNA sequences used in this study are listed in Supplementary Section S1. (See Figure S1 for sequence domains and predicted secondary structures.) All DNA oligonucleotides and the short RNA signal iMG were purchased from Integrated DNA Technologies (USA). The T7 RNA polymerase (Cellscript, Madison, WI, USA; #C-AS2607), 10× transcription buffer and thermostable inorganic pyrophosphatase (New England Biolabs, Ipswich, MA, USA; #B9012S, #M0296S), NTP and RNase R (Epicentre, Madison, WI, USA; #RN02825, #RNR07250) were purchased. Malachite Green (MG) dye was purchased from Sigma (#M9015). Since pyrophosphatase is involved in regulating the byproduct inorganic pyrophosphate for our transcriptional circuits and is not directly involved in the dynamics, we neglect this enzyme in our models and do not call it an ‘essential enzyme’ for the circuit dynamics. The nominal concentrations of enzyme stocks quoted by the manufacturer were used: 10.5 μM for RNase R and 6 μM for T7 RNA polymerase (RNAP).

The immRNA 3p10LA9 (A9) was produced using T7 RNA polymerase, followed by purification with Hi-Trap Q HP column (Cytiva, MA, USA) (23 (link)). Briefly, the A9 DNA template (IDT, Singapore) was annealed 95°C for 5 min and slowly cooled to room temperature. Subsequently, the transcription was performed by adding 1 µM A9 template into reaction buffer (40 mM HEPES, pH7.5, 30 mM MgCl₂, 2 mM spermidine, 10 mM DTT, 5 mM GTP, 4 mM CTP/ATP/UTP, and 0.01% Triton-X100, pH 7.4) containing 400-600 nM T7 polymerase and 0.2 U/ml thermostable inorganic pyrophosphatase (New England BioLabs, MA, USA) for 24h at 37°C. RNA products were extracted using phenol/chloroform/isoamyl alcohol (v/v/v = 25/24/1, Sigma, MO, USA) and precipitated in 75% (v) ethanol and 0.1% (v/v) sodium acetate (0.3 M, pH 5.2). All precipitated RNAs were dissolved in HEPES buffer (10 mM, pH 7.4) and isolated by Hi-Trap Q-Hp column for purification. The obtained A9 RNA was precipitated, re-dissolved in RNase-free H₂O, and stored at -80°C for further use. All other RIG-I agonists (3p10L, 3p10LG9, SLR14, SLR14A9) were synthesized using the same method with different DNA templates. The sequence of all RIG-I agonists, along with their references are shown in Table 1 and the sequence of the DNA template used for their In Vitro Transcription (IVT) in Table 2.