During the preparation of mRNA via in vitro transcription (IVT) for mRNA vaccines and therapeutics, double-stranded RNA (dsRNA) is generated as a byproduct. This dsRNA can be recognized by intracellular nucleic acid receptors such as TLR3, MDA5, or RIG-I, which in turn trigger innate immune inflammatory responses.
Hzymes has newly launched the RIG-I dsRNA Detection Kit, designed to rapidly evaluate the immunogenicity of dsRNA in samples.

How Does RIG-I Precisely Recognize and Transduce Signals from dsRNA?
1. Structure of RIG-I-like Receptors
In higher organisms, pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns (PAMPs). RIG-I-like receptors (RLRs), located in the cytoplasm, are responsible for detecting RNA viruses and inducing immune responses. They play a critical role in both innate and adaptive antiviral immunity. The RLR family includes three members:
• Retinoic acid-inducible gene I (RIG-I)
• Melanoma differentiation-associated gene 5 (MDA5)
• Laboratory of Genetics and Physiology 2 (LGP2)
Their protein structures are similar to those of the Dicer family of endoribonucleases found in mammals, containing a DExDH-box type RNA helicase domain with ATPase activity. This domain, along with the adjacent C-terminal domain (CTD), is essential for RNA binding【1】.

Additionally, the CTDs of RIG-I and LGP2 function as repressor domains to keep the receptors in an inactive conformation when not bound to RNA. Upstream of the helicase domain, RIG-I and MDA5 possess two N-terminal caspase activation and recruitment domains (CARDs), which mediate signal transduction through interaction with the CARD domain of the mitochondrial antiviral signaling protein (MAVS). LGP2 lacks the CARD domain and thus cannot interact with MAVS; instead, it may function as a regulatory molecule in the signaling pathways mediated by RIG-I and MDA5【2】.

2. Mechanism of Specific Recognition
Structural Features Recognized in dsRNA
1.dsRNA adopts an A-form helix, which is distinct from the typical B-form helix of dsDNA. The major groove of dsRNA is narrower and deeper, while the minor groove is wider and shallower. The unique configuration of the phosphate backbone and the exposed 2’-hydroxyl group in the minor groove can be specifically recognized by conserved protein motifs. Notably, the narrow major groove contains sequence-specific information—commonly used by proteins to interact with dsDNA—but it prevents similar interactions with dsRNA. Therefore, protein-dsRNA interactions are mainly mediated by the minor groove and phosphate backbone, and typically do not depend on the RNA sequence【3】.
2.RIG-I primarily recognizes short double-stranded RNA with 5′-triphosphate or 5′-diphosphate modifications, which are characteristic PAMP features of viral RNA【4】. Due to the presence of various RNA editing enzymes, mRNAs in humans and other higher eukaryotes have a 7-methylguanosine (m7G) cap (Cap0) at the 5’ end and may also contain Cap1 (N1-2'-O-M) or Cap2 (N1-2-2'-O-M) modifications. These 2’-O-methyl modifications help prevent self-RNA from being mistakenly recognized by RIG-I.

Signal Transduction
In vivo: When host cells are in a resting state, the CARD domains of RIG-I and MDA5 are bound to their own repressor domains, rendering them inactive. Upon viral infection, the CTDs of RIG-I and MDA5 recognize viral dsRNA, hydrolyze ATP to release energy, and undergo conformational changes. This activates their CARD domains, which then activate the CARD-containing adaptor protein MAVS, promoting the release of interferons.

Dynamic Mechanism of Recognition【5】

Figure 2. RIG-I Activation Pathway

1.Initial Binding:
The CTD binds the 5’-triphosphate ends of RNA, while the helicase domain binds the double-stranded region to form the initial complex.
2.ATP-Dependent Conformational Change:
Upon RNA binding, the helicase domain hydrolyzes ATP, inducing a conformational change that shifts RIG-I from a closed (inactive) to an open (active) state.
3.Exposure of CARD Domains:
The conformational change exposes the CARD domains, allowing them to bind to MAVS on the mitochondria. This forms a multimeric complex that activates downstream signaling pathways (such as NF-κB and IRF3), ultimately inducing type I interferons and inflammatory cytokines.

Based on the recognition function of the receptor protein, an in vitro detection strategy was designed. In the absence of a ligand, RIG-I’s ATPase activity is autoinhibited. Binding to dsRNA activates its ATP hydrolysis activity, which correlates with dsRNA content. This biochemical activity forms the basis for dsRNA detection.

New Product Launch
Hzymes – RIG-I dsRNA Detection Kit

1. Core Principle
When RIG-I binds to dsRNA, its ATPase activity is triggered, hydrolyzing ATP into ADP and Pi. This product evaluates the immunogenicity of dsRNA in samples by measuring the amount of ADP generated during the RIG-I/sample reaction.
2. Product Advantages
• Direct Immunogenicity Assessment via Receptor Activation In Vitro
A breakthrough from traditional detection methods, directly evaluating sample immunogenicity based on receptor activation levels.
• High Sensitivity for Short-chain RNA
Highly sensitive detection of short dsRNA sequences, superior performance for short fragments.
• Complementary Evaluation with ELISA
Works in conjunction with ELISA, offering a comprehensive assessment of sample immunogenicity.

3. Data Presentation
Specific Recognition

Exclusively recognizes dsRNA, with negligible recognition of ssRNA, ssDNA, or dsDNA.

Recognition of Short dsRNA
RIG-I 

ELISA antibody

RIG-I shows superior recognition of short dsRNA compared to antigen-antibody recognition in ELISA.

Comparison of Immunogenicity Detection Results Using Different Methods

Comparison of Immunogenicity Assessment Results by Different Methods		ELISA	IL-6：pg/ml	IFN-a：pg/ml	RIG-I
mEPO	WT-37℃	0.075%	211.82	85495.37	0.281%
	20-30-37℃	0.027%	24.36	1155.89	0.085%
CLDN6	WT-37℃	0.027%	150.82	23587.80	0.312%
	20-30-50℃	0.003%	21.42	33.33	0.059%
Random sequence1000nt	WT-37℃	0.047%	638.73	42480.43	0.110%
	20-30-37℃	0.006%	115.66	Below the limit of detection	0.077%

References
[1] Yoneyama M, Kikuchi M, Natsukawa T, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol, 2004, 5(7): 730 -737.
[2] 王长万, 侯法建, RIG-Ⅰ样受体介导的RNA识别及其功能机制, 生命的化学 , 2024, 44(9): 1682-1692.
[3] Peisley A, Hur S. Multi-level regulation of cellular recognition of viral dsRNA. Cell Mol Life Sci. 2013 Jun;70(11):1949-63.
[4] Paola M. Barral, Devanand Sarkar, Zao-zhong Su, et al. Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: Key regulators of innate immunity. Pharmacology & Therapeutics, 2009, 124(2): 219-234.
[5] Kowalinski E, Lunardi T, McCarthy AA, et al. Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA. Cell. 2011 Oct 14;147(2):423-35.