dsRNA:Key quality control items for mRNA vaccines and drugs

Genetic Engineering & Biotechnology News. 2019 Dec 19; 39(12):56-58.

Sci Adv. 2020 Jun 24;6(26): eaaz6893.

During IVT (In Vitro Transcription), T7 RNA polymerase may use RNA or DNA (template or non-template strands) as templates, generating various dsRNA byproducts.

dsRNA can be recognized by multiple receptor proteins, triggering human immune responses.

RIG-I：one of the primary immune effector proteins

The immune response comprises innate immunity (non-specific) and adaptive immunity (specific), which work synergistically to defend against pathogens. In innate immunity, host cell-specific receptors recognize pathogen-associated molecular patterns (PAMPs), inducing the production of type I interferons (IFNs) and inflammatory cytokines. These receptors are called pattern recognition receptors (PRRs), including:

toll-like receptors，TLRs

RIG-I like receptors，RLRs

NOD-like receptors，NLRs

C-type lectin receptors，CLRs）

cytosolic DNA receptors，CDRs）

HEK-Lucia™ RIG-I cells stably express high levels

of human RIG-I and respond strongly to cytosolic RIG-I ligands such

as 3p-hpRNA and 5’ppp-dsRNA

A549-Dual™ cells express numerous pattern recognition receptors (PRRs), including the RIG-I-like receptor (RLR) RIG-I [1, 2], and the Toll-like receptors (TLRs) TLR2 [3], TLR3 [4, 5] and TLR5 [6] but not TLR4 [3].

RLRs are critical cytoplasmic PRRs that recognize dsRNA viruses and are essential for antiviral innate and adaptive immunity. The RLR family includes three members:

RIG-I (Retinoic Acid-Inducible Gene I)

MDA5 (Melanoma Differentiation-Associated Gene 5)

LGP2 (Laboratory of Genetics and Physiology 2)

J Virol. 2014 Aug;88(15):8194-200.

1：Structure of the RIG-I like receptors (RLRs). RIG-I and MDA5 contain two N-terminal CARD domains (caspase activation and recruitment domains), a central DExH helicase domain, and a C-terminal repressor domain (RD) with a CTD (C-terminal domain).

RIG-I dsRNA Quantification Kit

Hzymes’ RIG-I dsRNA Quantification Kit is a cutting-edge tool designed for the sensitive, specific, and direct quantification of double-stranded RNA (dsRNA)—a critical impurity in mRNA vaccines and RNA-based therapeutics. It provides a unique way to evaluate immunogenic dsRNA by mimicking how the human immune system naturally responds to dsRNA contamination.

Why Is dsRNA Detection Important in mRNA Drug Development?

During in vitro transcription (IVT), enzymes like T7 RNA polymerase can unintentionally generate dsRNA byproducts. These contaminants can activate innate immune receptors such as RIG-I, triggering an inflammatory response in humans.

For manufacturers of mRNA drugs and vaccines, removing and accurately measuring dsRNA is essential to ensure product safety, efficacy, and regulatory compliance.

What Makes RIG-I Special for dsRNA Detection?

RIG-I (Retinoic Acid-Inducible Gene I) is a cytoplasmic pattern recognition receptor (PRR) that naturally detects short viral dsRNA (30–300 base pairs). When RIG-I binds to dsRNA, it activates ATPase activity, initiating a signaling cascade that results in type I interferon release.

Hzymes harnesses this natural immune mechanism to build a powerful, biology-driven detection system that is more representative of real immune activation compared to traditional methods like ELISA.

How the RIG-I dsRNA Quantification Kit Works

Key Features and Workflow

Binding & Activation: Mix your sample with RIG-I enzyme and ATP to trigger the ATPase reaction.
ATP Removal: Add Reagent 1 to eliminate leftover ATP.
ADP Detection: Add Reagent 2 to convert ADP to ATP.
Luminescence Reading: Use a luciferase/luciferin reaction to measure the newly formed ATP with a microplate reader.
Quantification: Determine dsRNA levels from a standard curve.
The entire reaction can be performed in microplate format with as little as 5 µL per well, making it ideal for high-throughput workflows.

Performance You Can Trust

Ultra-Sensitive: Detects dsRNA at concentrations as low as 1 pg/μL
Highly Specific: RIG-I distinguishes dsRNA from other nucleic acids with minimal cross-reactivity
Strong Correlation: Demonstrated excellent agreement with ELISA and immune response levels (r² = 0.98729)
Short dsRNA Detection: Outperforms ELISA in detecting short dsRNA fragments

Why Choose Hzymes’ RIG-I Kit Over Traditional ELISA?

Applications in Biopharmaceutical Quality Control

mRNA vaccine development
Gene therapy raw material QC
Immunogenicity testing
dsRNA contamination profiling
Cell-based assay support

Get Started with Hzymes’ RIG-I dsRNA Quantification Kit

If you’re working in biotech, pharma, or RNA-based drug development, Hzymes’ RIG-I kit is the ideal solution for your dsRNA testing needs. It complements ELISA, provides superior sensitivity for short dsRNA, and offers deeper insight into the immune activation potential of your samples.