Skip to content

mRNA Detection using an RNA editing-based reporter assay

As gene therapy continues to advance toward clinical mainstream, so does the demand for precise, scalable, and reliable methods to assess the performance of therapeutic vectors. Traditional tools like RT-PCR or protein-based assays are often labor-intensive, inconsistent, or lack functional readouts. A recent study published in Molecules introduces a promising new approach: an RNA editing-based reporter assay that leverages endogenous ADAR enzymes to detect target mRNAs inside living cells.

This innovative platform promises to reshape how we monitor transcriptional activity across gene therapy vectors, including AAVs, lentivirus, plasmids, and circular mRNAs, and positions itself as a next-generation standard in potency testing.

 

What are ADARs?

Adenosine deaminases acting on RNA (ADARs) are enzymes found in human cells that catalyze the conversion of adenosine (A) to inosine (I) in double-stranded RNAs. Since inosine is read as guanosine (G), this A-to-G editing can alter RNA function and has been explored for therapeutic correction of genetic mutations.

In this assay, the research team ingeniously repurposed ADAR activity to switch an inactive reporter gene (blocked by a stop codon) into an active one, only when a target mRNA is present, essentially turning RNA editing into a readout tool.

 

ADAR-Based Reporter Assay for Detection of mRNA Expression

ADAR-mediated RNA editing to detect mRNA activity

Dual-reporter plasmids use ADAR-mediated RNA editing to detect mRNA activity. NanoLuc translation occurs only when editing converts a UAG stop codon to UGG.

 

In this study, researchers constructed dual-reporter plasmids to detect therapeutic mRNA activity via ADAR-mediated editing:

  1. Each plasmid encodes firefly luciferase (as a control) and NanoLuc luciferase downstream of a sensor RNA containing a UAG stop codon.

  2. In the presence of the target mRNA, the sensor hybridizes with the target mRNA, forming a double-stranded RNA structure.

  3. ADAR edits the UAG to UGG, allowing NanoLuc to be translated.

The ratio of NanoLuc to FireLuc activity becomes a quantitative readout of mRNA abundance.

Using HEK293T cells, the researchers showed that NanoLuc activation reliably reflects mRNA levels across plasmids, circular mRNA, lentivirus, and AAV. The response was sequence-specific, dose-dependent, and consistent across delivery formats. They designed sensors targeting the therapeutic gene (SMN), a reporter tag (GFP), and a 3′ UTR (WPRE).

 

Novelty behind this mRNA detection assay

  • Novel mechanism: Uses ADAR-mediated RNA editing as a built-in mRNA detection switch.

  • Highly specific: Sensors detect only the presence of the corresponding mRNA (e.g., SMN sensor only responds to SMN).

  • Quantitative and reproducible: The assay showed high accuracy (90–115% recovery), strong linearity (R² > 0.99), and low inter-assay variation (<15% RSD).

  • Universal platform: Applicable to plasmids, AAV, lentivirus, and circular mRNA vectors.

  • No need for overexpressed ADARs: Endogenous ADARs in 293T cells were sufficient.

  • Better than RT-PCR for function: Delivers a real-time, functional readout of gene expression rather than static transcript levels.

 

This RNA editing-based reporter assay opens doors for broader adoption in:

  1. Potency testing for gene therapy vectors

  2. Preclinical validation of new RNA therapeutics

  3. Screening assays for mRNA delivery efficiency

 

The simplicity of sensor design (just changing the target sequence) also makes it adaptable to any gene or vector platform.

 

Relevance of mRNA detection in Gene Therapy

The therapeutic efficacy of gene therapy products depends on the successful expression of the target gene. Yet, current quality control methods face challenges:

  • RT-PCR struggles with RNA degradation and requires complex preparation.

  • Protein-level assays can suffer from high background noise or lack suitable antibodies.

This new RNA editing-based assay offers a functional, scalable, and sequence-specific alternative.

 

CATUG's Role on RNA Quality Testing

CATUG Biotechnology contributed to this project by providing the circular GFP mRNA, a critical component in validating the platform's compatibility with non-viral delivery systems.

Whether you’re working on AAV-based therapeutics, mRNA vaccines, or novel gene editing platforms, CATUG can provide custom RNA constructs, potency assay consulting, and RNA-LNP formulation services tailored to your needs.

 

📩 Reach out to our team or explore our mRNA catalog

 

Reference

Yu L, Zhou Y, Wang G, et al. An In Vitro RNA Editing-Based Reporter Assay for Transcriptional Activity of Therapeutic Gene in Gene Therapy Products. Molecules. 2024;29(22):5312. Published 2024 Nov 11. doi:10.3390/molecules29225312