Cleancap

mRNAs were transcribed from linearized plasmids encoding firefly Luc, codon-optimized mEPO, EGFP, or hiNOS using T7 RNA polymerase (MEGAscript T7 Transcription Kit, Thermo Fisher Scientific, Darmstadt, Germany). The mRNAs were transcribed to contain the 5′ UTR derived from the tobacco etch virus 5′ leader RNA (TEV).³⁴ (link) Further, a 100-nt-long poly(A) tail interrupted by a short linker (A30LA70) was transcribed from the corresponding DNA templates. For the generation of nucleoside-modified mRNAs, uridine 5’-triphosphate (UTP) was replaced with the triphosphate derivative of m1Ψ (m1ΨTP) in the transcription reaction. Capping of the IVT mRNAs was performed co-transcriptionally using the trinucleotide cap1 analog CleanCap (TriLink, San Diego, CA, USA). After DNase digestion, the synthesized mRNA was isolated from the reaction mix by precipitation with half volume of 8 M LiCl solution (Sigma-Aldrich, Hamburg, Germany), and finally the pellet was dissolved in nuclease-free water. The RNA concentration was determined using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific). Aliquots of denatured IVT mRNAs were analyzed by electrophoresis in non-denaturing 1.4% agarose gels containing 0.005% (v/v) GelRed nucleic acid gel stain.³⁵ (link) RiboRuler High Range RNA Ladder (Thermo Fisher Scientific) was loaded as a molecular weight marker.

mRNAs were transcribed from linearized plasmids encoding firefly luciferase, or full-length spike, or RBD of SARS-CoV-2 spike by the MEGAscript® T7 Transcription Kit (Cat# AMB1334-5, Life Technologies) through a modified protocol. Briefly, in order to reduce the mRNA immunity and increase its stability in vivo as COVID-19 mRNA vaccine did^{1 (link),2 (link)}, the uridine-5′-triphosphate (UTP) from the T7 Transcription Kit was replaced by m1Ψ- 5′-triphosphate (Cat# N-1081, TriLink). Meanwhile, mRNA was also capped co-transcriptionally in vitro with the trinucleotide cap1 analog CleanCap (Cat# N-7413, TriLink). After 6 h of reaction, mRNAs were precipitated by lithium chloride (LiCl) and washed twice with 75% ethanol in nuclease and endotoxin free water (Cat# W3440, Teknova). Then, the mRNA pellet was dissolved in nuclease and endotoxin free water and further subjected to cellulose purification in the presence of 16% ethanol to remove dsRNA^{74 (link)}. The concentration and purity of mRNAs was measured by nanodrop and by agarose gel electrophoresis, respectively. mRNAs were aliquoted and stored at −20 °C.

Triphosphate-derivatives of N-methylpseudouridine (Hongene) were used to generate modified nucleoside containing RNA. All RNAs were capped using Cleancap™ (Trilink Biotechnologies). All IVT reagents except enzymes listed in (Table 1) were preheated to 37°C, mixed in a 1.5 mL plastic tube in Thermomixer^TMC (Eppendorf) and incubated at 37°C with shaking at 300 rpm. The reaction was quenched after 180 min by addition of 5 mM EDTA, pH 8.0 for its subsequent purification by the different methods.

Monocytes were plated in RPMI, supplemented with 10% heat-inactivated FBS, and 1% P/S at 1 × 10⁶ cells/mL in an ultra-low attachment plate for 3 h prior to transfection. Monocytes were collected and cell pellets were washed 1× PBS followed by resuspension in T buffer (Fisher Scientific, Waltham, MA, USA) at a concentration of 5 × 10⁷ cells/mL. Cells were then loaded into a 10 uL Neon pipette tip and electroporated using a Neon Transfection System (Fisher Scientific, Waltham, MA, USA) with E5 protocol (1700 volts, 20 millisecond bandwidths, 2 pulses) unless indicated otherwise. Electroporated monocytes were then immediately transferred to 24-well plates containing 1 mL of complete monocyte medium at 5 × 10⁵ cells/ml and placed in the tissue culture incubator.
A similar procedure was followed for experiments using chemically modified Cas9 (Cas9) mRNA instead of Cas9 RNPs. In these experiments 1 μg of sgRNA was combined with either 1.5 μg SpCas9 mRNA (CleanCap^®, Trilink Biotechnologies, San Diego, CA, USA) or 1.5 μg coBE4 mRNA (Trilink Biotechnologies, San Diego, CA, USA) prior to addition to cells.