Double-stranded RNA (dsRNA) is a major impurity of therapeutic mRNA produced by in vitro transcription. Impurity levels of this transcriptional by-product need to be tightly monitored in mRNA production lots to ensure efficient mRNA expression and to reduce the risk of undesired high immunogenicity[1-2].
Dot blot assays using SCICONS’s Anti-dsRNA monoclonal antibody J2 are a fast and easy method for the detection of dsRNA in both crude in vitro transcription mixes as well as purified mRNA[1-6]. Estimation of the amount of dsRNA impurity is feasible with a dsRNA control run in parallel (Fig. 1).
Figure 1: dsRNA is efficiently detected with a dot blot assay using SCICONS’s Anti-dsRNA monoclonal antibody J2 (according to Moradian et al. (2022)).
A) Equal amounts (1 µg) of Poly (A) ssRNA (negative control) and dsRNA 142 bp (positive control) along with a 4-fold dilution series of dsRNA 142 bp bp were blotted and subsequently be detected with SCICONS’s Anti-dsRNA monoclonal antibody J2.
B) dsRNA content of purified mRNA (1 µg per dot) with different Cap and nucleotide modifications was determined with a dot blot assay using SCICONS’s Anti-dsRNA monoclonal antibody J2. mRNA integrity was confirmed by denatured agarose gel electrophoresis.
ARCA: Anti reverse cap analog, MT: Methyltransferase, AnP: Antarctic phosphatase, ᴪ: Pseudouridine, me1ᴪ: N1-Methylpseudouridine, 5moU: 5-Methoxyuridine, 5meC: 5-Methylcytidine.
[1] Kariko et al. (2005) Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity 23 (2):165.
[2] Kariko et al. (2008) Incorporation of Pseudouridine yields superior nonimmunogenic vector with increased translational capacity and biological stability. Molecular Therapy 16 (11):1833.
[3] Baiersdörfer et al. (2019) A Facile Method for the Removal of dsRNA Contaminant form In Vitro-Transcribed mRNA. Mol Ther Nucleic Acids 15:26.
[4] Moradian et al. (2022) Chemical modification of uridine modulatesmRNA-mediated profinflammatory and antiviral response in primary human macrophages. Mol Ther Nucleic Acids 27:854.
[5] Piao et al. (2022) Double-stranded RNA reduction by chaotropic agents during in vitro transcription of messenger RNA. Mol Ther Nucleic Acids 26:618.
[6] Schönborn et al. (1991) Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res. 19:2993.
