A: Double-stranded RNA (dsRNA), generated as a byproduct during in vitro transcription (IVT), is an unwanted contaminant in an mRNA prep. Here are a few key reasons why dsRNA should be removed:
- Immunogenicity: dsRNA is highly immunogenic when introduced into cells. It can trigger a strong innate immune response in cells resulting in diminished or lack of mRNA expression. Intracellular pattern recognition receptors recognize dsRNA, a hallmark viral infection, as non-self RNA, leading to inflammation and other immune reactions.
- Purity of mRNA: For applications including mRNA vaccines, gene product replacement therapy or other therapeutics, it is crucial to have high-purity mRNA. dsRNA contaminants can co-elute with mRNA, complicating the purification process and reducing the efficacy of the final product.
- Side Effects: The presence of dsRNA in therapeutic mRNA formulations can cause adverse side effects in patients due to the immune system’s response to what it perceives as a viral threat.
- Regulatory Compliance: Regulatory bodies require stringent purity standards for biopharmaceutical products. Removing dsRNA helps meet these standards and ensures the safety and efficacy of the product.
A: The simplest method is to use the Min-Immune™ Gold dsRNA Removal Kit provided by CELLSCRIPT™. It is a novel enzymatic solution for the removal of dsRNA contamination present in RNA samples. Various other methods, such as reverse-phase HPLC (RP-HPLC), hydroxyapatite chromatography and cellulose chromatography, are used to remove dsRNA from mRNA preparations. However, these methods are labor intensive, expensive, require specialized equipment and often significantly reduce the final product (single-stranded RNA) yield.
A: No, by leveraging common laboratory equipment and techniques, the treatment provides a simple and cost-effective solution for researchers.
A: No, the method is applicable to most IVT RNA and mRNA samples including capped/uncapped, poly(A)-tailed/untailed, canonical NTP content/modified NTP-analog content and RNA lengths of any size.
A: RNAs that contain inherent dsRNA regions that are required for RNA functionality and are also of sufficient length to be recognized by the Min-Immune™ Gold RNase III enzyme should not be treated with this kit.
A: A standard 1X kit reaction treats 60 µg of RNA, so one 25-reaction size kit can treat 1.5 mg of RNA. The reaction can be scaled up or down to treat as much or as little RNA as desired.
A: Final treated RNA reaction products are commonly analyzed by denaturing agarose gel electrophoresis to assay the integrity of the RNA and by immunoblotting the RNA using a dsRNA-specific antibody to assay for residual dsRNA content. In both assays, one must compare to the untreated control RNA sample.
A: Residual dsRNA content, as indicated by the immunoblot assay, can be derived from two sources, 1) remaining dsRNA byproduct from the transcription reaction, meaning the Min-Immune™ Gold treatment step was insufficient or partially inhibited, or 2) the RNA molecule itself contains some inherent regions of dsRNA structure that are not recognized by the Min-Immune™ Gold RNase III enzyme but are recognized by the dsRNA-specific antibody. In case #1, the RNA sample can be purified and retreated. Subsequent treatments of the same RNA sequence could be modified by increasing the incubation time or increasing the Min-Immune™ Gold RNase III enzyme content. In case #2, the RNA may be suitable for downstream applications regardless of the result since the contaminating dsRNA byproduct of transcription has been removed, or the dsRNA region could be engineered out of the RNA sequence by selective point mutations.
