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Given mRNA's potential in treating various ailments like cancer, genetic disorders, and infectious diseases, it's garnered widespread interest for therapeutic use. However, despite its promise, there are several challenges in practically developing and applying mRNA synthesized through in vitro transcription (IVT). One significant hurdle is the generation of unwanted byproducts, such as double-stranded RNA (dsRNA), during the IVT process. These dsRNA impurities, if present, could trigger unnecessary immune responses when administered in vivo. Hence, it's crucial to detect and remove them to ensure the safety and effectiveness of mRNA therapies. At BOC Sciences, our focus is on providing professional RNA transcription services globally. We also offer dsRNA detection services to maintain the highest standards of quality and safety in mRNA therapy.
What is dsRNA?
Double-stranded RNA (dsRNA) consists of two RNA strands that pair up with each other in an antiparallel way. This is different from single-stranded RNA (ssRNA). dsRNA has its own distinct structure and functions, which can activate innate immune responses strongly. When it comes to mRNA IVT (in vitro transcription), unwanted dsRNA impurities might appear as byproducts of the transcription process, causing issues for further applications.
dsRNA Virus
dsRNA viruses have genomes composed of double-stranded ribonucleic acid (RNA), serving as templates for the viral RNA-dependent RNA polymerase (RdRp). This enzyme transcribes a positive-strand RNA, acting as messenger RNA (mRNA), which is then translated into viral proteins by the host cell's ribosomes. Additionally, the positive-strand RNA can be replicated by RdRp to generate a new double-stranded viral genome. These viruses are classified into two phyla, Duplornaviricota and Pisuviricota (specifically Duplopiviricetes), belonging to the Orthornavirae kingdom and Riboviria realm. It is noteworthy that dsRNA viruses do not share a common ancestor but evolved independently from different positive-sense, single-stranded RNA viruses.
dsRNA Formation Mechanisms in In Vitro Transcription
The generation of dsRNA byproducts during RNA transcription can occur through two mechanisms: runoff transcripts serving as templates for RNA-dependent RNA polymerase activity, leading to extension at the 3' end, and RNAP switching to the non-template strand, producing antisense RNA molecules. These byproducts, formed by complementary sequences within runoff transcripts or promoter-independent synthesis, can contribute to dsRNA formation. The identification of dsRNA products depends on whether extension occurs in a sense or antisense direction, and analysis may require natural conditions due to the similarity in size between antisense molecules and runoff products.
BOC Sciences' dsRNA Detection Service
At BOC Sciences, we understand the importance of detecting and minimizing dsRNA contaminants in mRNA IVT reactions. Our specialized dsRNA Detection Service offers comprehensive solutions for assessing product quality and safety. Leveraging state-of-the-art techniques and expertise in nucleic acid analysis, we provide accurate and reliable dsRNA detection services tailored to meet the specific needs of our clients. Whether you require qualitative screening or quantitative analysis of dsRNA contaminants, our experienced team is committed to delivering timely and actionable results.
dsRNA Assay Methods We Offering
| dsRNA Assay Methods | Description |
| Enzyme-Linked Immunosorbent Assay (ELISA) | This is a commonly used method for dsRNA detection, which can use nylon membranes or commercial ELISA kits. When using nylon membranes, the sample volume needs to be optimized to ensure consistent diffusion among samples and reduce the impact on result accuracy. Based on sandwich ELISA, capture antibodies are immobilized on microplate wells to form a solid-phase antibody. dsRNA calibration standards and samples are added to the wells, followed by detection antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies. After washing, color development is initiated, and absorbance values are measured using a plate reader. The absorbance values correlate with the amount of dsRNA in the samples. |
| Immunoblotting Method | Immunoblotting employs dsRNA-specific antibodies to detect the dsRNA content in IVT reactions. The J2 antibody is an anti-dsRNA antibody that specifically recognizes and binds to dsRNA. It identifies the presence of dsRNA in samples based on specific signals (qualitative detection). Suitable standard controls are simultaneously prepared to quantitatively analyze the residual dsRNA in the samples, thereby enhancing detection sensitivity. This method relies on the antibody's specific recognition of dsRNA, but mRNA secondary structures or modified nucleotides may alter the antibody's recognition of dsRNA structure, requiring specialized research. * Note: The sensitivity of antibody detection depends on the length of the dsRNA region, and sensitivity adjustments are needed to ensure dsRNA detection. |
| Gel Electrophoresis Method | dsRNA byproducts formed in sense or antisense to runoff transcripts, which are shorter than the runoff transcripts, can be distinguished from the target transcripts under denaturing gel conditions. Runoff transcripts or antisense strands synthesized using DNA templates or non-template chains serve as templates. The length of the resulting dsRNA, which forms from their pairing, matches that of the runoff transcripts and cannot be effectively separated by denaturing gel electrophoresis. However, this separation can be achieved using active gels. Both gel electrophoresis methods are suitable for qualitative detection of different byproducts, but they have relatively low resolution and cannot precisely quantify residual dsRNA. |
| MDA5-Specific Binding Method | Cytoplasmic immune receptor MDA5 is a RIG-I-like receptor (RLR) that specifically recognizes dsRNA, thereby activating the body's antiviral immune response. Thus, in vitro detection strategies can be designed based on the binding between MDA5 and dsRNA. Cryo-electron microscopy (cryo-EM) can observe MDA5-dsRNA complexes containing short filaments. Analysis of filament structures shows that each MDA5 molecule spans 14-15 RNA base pairs. However, this analysis method is limited by the requirements of low-temperature EM analysis equipment. Furthermore, MDA5 is an ATP-dependent helicase, and its binding to dsRNA activates MDA5's ATP hydrolysis activity. This activity can be detected by standard biochemical assays. Therefore, in vitro assays for ATP hydrolysis in the presence of MDA5 may be a good alternative method for assessing the extent of immune response elicited by target mRNA. |
| Sequencing or Mass Spectrometry Analysis | For in-depth study of the nucleotide sequences of dsRNA, RNA-seq or full molecular mass spectrometry analysis can be performed. Both approaches have limitations: RNA-seq analysis may be affected by biased RNA structure ligation, and mass spectrometry analysis provides heterogeneity distribution of selected RNAs but cannot provide sequence information for RNA 3' ends. Therefore, both analysis methods are not universally applicable and cannot be used in a high-throughput manner for screening multiple sequences. |
Methods for Miniming dsRNA in Our In Vitro Transcription Services
In mRNA IVT reactions, BOC Sciences is well aware of the critical importance of minimizing the formation of dsRNA byproducts to generate high-quality therapeutic mRNA. Therefore, BOC Sciences takes the following measures during mRNA IVT steps to reduce dsRNA contamination.
- Optimization of Reaction Conditions: Adjusting parameters such as magnesium concentration, temperature, and reaction time can help minimize dsRNA formation while maintaining RNA yield.
- Use of Modified RNA Polymerases: Employing heat-stable RNA polymerases or modified enzyme formulations can reduce the generation of dsRNA contaminants during IVT reactions.
- Sequence Optimization: Designing mRNA templates with minimal potential for dsRNA formation can help mitigate contamination issues. Strategic modifications to the template sequence can prevent the annealing of complementary regions and reduce dsRNA byproduct formation.
The detection and minimization of dsRNA contaminants in mRNA IVT reactions are critical steps in the development of safe and effective mRNA therapeutics. BOC Sciences' dsRNA Detection Service offers a comprehensive solution for assessing product quality and ensuring regulatory compliance, supported by our expertise, cutting-edge technology, and commitment to excellence in nucleic acid analysis.
Case Study
Case study 1 Exploring the Mechanical Properties of dsRNAs through Molecular Dynamics Simulations.
Schematic diagram of mechanical characterization of double-stranded RNA (dsRNA). (Gonzalez, A.M.; et al, 2019)
This case uses state-of-the-art computational techniques to perform atomic molecular dynamics simulations to explore the mechanical behavior of various RNA double-stranded bodies under external forces. Their results show that, similar to dsDNA, the mechanical properties of dsRNA are highly dependent on the nucleotide sequence. Notably, the researchers observed significant differences in the stretching and twisting responses of RNA and DNA double-stranded bodies under force, which is attributed to the unique sequence-dependent characteristics of dsRNA. They found that the elastic response of dsRNAs is primarily influenced by the high localized flexibility of the pyrimidine-purine step, a feature that is independent of sequence context. Overall, this study emphasizes the importance of understanding the sequence-dependent mechanical properties of dsRNAs, provides valuable insights for future nanotechnology applications, and enhances our understanding of RNA biology.
FAQ
1. What is the function of the dsRNA?
dsRNA functions in RNA interference (RNAi), antiviral defense, gene regulation, experimental gene manipulation, and as intermediates in viral replication.
2. What is an example for dsRNA?
An example of dsRNA (double-stranded RNA) is the RNA molecule produced by some viruses during their replication cycle, such as the RNA viruses in the Reoviridae family.
3. Where is dsRNA found?
Double-stranded RNA is found in many parts of the body. They can be found in some viruses and used as genetic material. In addition, double-stranded RNA also performs regulatory functions within cells, such as during RNA interference. This RNA can trigger gene silencing or inhibit the expression of specific genes. At the same time, double-stranded RNA can also appear as an intermediate product in some biological cells, such as during transcription or RNA modification.
4. How is dsRNA formed in IVT?
When transcribed in vitro, the process of forming double-stranded RNA (dsRNA) typically involves the synthesis of complementary sequences of two RNA strands using RNA polymerase. When these complementary RNA strands are synthesized, they can form double-stranded structures under certain conditions. This approach is commonly used in laboratory techniques, such as using DNA templates and RNA polymerases to generate RNA, or synthesizing two complementary RNA fragments and combining them to form double-stranded RNA.
5. Why Detect dsRNA in In Vitro Transcription?
Detecting dsRNA contaminants in mRNA IVT reactions is crucial for ensuring the safety, efficacy, and regulatory compliance of mRNA therapeutics. Contaminants can trigger innate immune responses, potentially causing adverse effects in vivo, while also interfering with therapeutic function or inducing off-target effects. By identifying and minimizing dsRNA contaminants, the safety and efficacy of mRNA-based therapies can be improved, meeting regulatory standards and streamlining the approval process.
Reference
- Gonzalez, A.M.; et al. Sequence-dependent mechanical properties of double-stranded RNA. Nanoscale. 2019,11: 21471-21478.
* Only for research. Not suitable for any diagnostic or therapeutic use.
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