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Pseudouridine Modification Service

Introduction Nucleotides Modification Workflow What We Can Offer FAQ

Introduction

Pseudouridine (Ψ) is the most abundant RNA modification. Its synthetic variant N1-methyl-pseudouridine (m1Ψ) aids mRNA vaccines' clinical success by helping therapeutic mRNA evade innate immune PRRs, with immune silencing and enhanced translation benefits—this is our service basis.

For projects facing low mRNA stability, high immunogenicity, or insufficient protein yield, Creative Biolabs uses advanced in vitro transcription/purification to integrate m1Ψ. It replaces uridine to mimic natural RNA, overcoming immune responses, stabilizing mRNA, and delivering optimized mRNA for superior protein production and reduced toxicity.

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Pseudouridine Modification Service

Structure of uridine and Ψ, enzymes involved in catalysis, and methods of detection with specific antibodies and chemical modification with CMC. (OA Literature)Fig.1 The structure of uridine and Ψ and the chemical modification of CMC.1

The key to m1Ψ's efficacy lies in its structure. Pseudouridine (Ψ) is a C5-C1' linked isomer of uridine. Adding a methyl group at the N1 position to form m1Ψ provides further benefits. Mechanistically, this modification works by:

  1. Immune Evasion: The modified structure prevents the activation of key antiviral sensors, primarily TLR7 and TLR8, which would otherwise recognize the synthetic RNA as foreign and trigger the production of Type I Interferons (IFN-I). By suppressing this immune response, the cell is allowed to efficiently translate the therapeutic blueprint.
  2. Ribosome Fidelity and Speed: m1Ψ enhances the ribosome's ability to read the mRNA template. This modification is shown to increase the speed and accuracy of translation, resulting in significantly higher yields of the desired therapeutic protein compared to unmodified RNA.
  3. Physical Stability: The altered structure reinforces base stacking and overall RNA rigidity, enhancing resistance to nucleolytic degradation in vivo.

Therapeutic Advantages of m1Ψ-Modified mRNA

  • Zero Immunogenicity Risk: Enables higher and repeated dosing necessary for chronic conditions or multi-dose vaccine regimens.
  • Significantly Enhanced Translational Efficiency: Achieves therapeutic protein levels faster and sustains them longer, reducing the required dose of expensive mRNA.
  • Improved Safety Profile: By preventing the inflammatory response associated with unmodified RNA, the modified therapeutic provides a cleaner and safer clinical profile.

Key Applications of Pseudouridine Modification

  • Protein Replacement Therapy: Delivering genes for missing or dysfunctional proteins (e.g., in cystic fibrosis or hemophilia) by ensuring high, transient protein expression.
  • Cancer Immunotherapy and Vaccines: Generating potent, targeted T-cell and B-cell responses by efficiently delivering tumor-associated antigens without triggering a distracting inflammatory reaction.
  • Cell Reprogramming (e.g., iPSCs): Utilizing integration-free methods to introduce transcription factors for generating therapeutic cell lines, offering maximum safety over viral vector methods.

Workflow

Required Starting Materials

  1. DNA Template: Linearized DNA or plasmid with target sequence (e.g., therapeutic protein, antigen), flanked by T7 promoter and optimized 3' UTR/poly(A) sequence.
  2. Target mRNA Sequence: Specific mRNA sequence, including open reading frame and desired 5'/3' flanking regions.
  3. 5' Cap Preference: Specification of capping strategy (e.g., Cap 1 for maximal translation).
Consultation & Design
Sequence Optimization

Template Preparation and Linearization

Rigorously analyze/prepare starting DNA; linearize plasmid via enzymatic digestion for IVT.

m1Ψ In Vitro Transcription (IVT)

Run proprietary IVT with DNA template, RNA polymerase, and UTP-replaced m1Ψ triphosphate master mix.

Chemical Modifications
Synthesis & Purification

5' Capping and Poly(A) Tailing

Perform co-transcriptional (e.g., Cap 1) or post-transcriptional capping per client choice; analyze/trim/extend poly(A) tail.

Enzymatic Purification and Filtration

Clean crude mRNA enzymatically to remove impurities; conduct filtration/precipitation.

Quality Control & Validation
Delivery & Support

Quality Control and Final Analysis

Conduct extensive QC (m1Ψ fidelity, structural integrity, quantification).

Final Deliverables

  1. Certificate of Analysis (CoA): Detailed report including final concentration, integrity assessment, and purity (HPLC/Agarose Gel).
  2. Analytical Report: Data verifying the successful m1Ψ incorporation via techniques such as Mass Spectrometry or NMR spectroscopy.
  3. Endotoxin and Bioburden Data: Quantitative analysis of contaminants, ensuring the modified mRNA is suitable for sensitive in vivo or ex vivo applications.
Consultation & Design
Sequence Optimization

Estimated Timeframe

The typical timeframe for this service ranges from 4 to 8 weeks, depending on the scale of the synthesis (milligram to gram), the complexity of the template, and the level of required custom purification and QC.

What We Can Offer

Scalable, End-to-End Synthesis

A seamless, one-stop service covering research-grade microgram quantities through pilot-scale development to large-scale GMP-compliant production for clinical trials.

Customized Molecular Engineering

Full support for codon optimization, variable poly(A) tail length design, and selection of the optimal 5' capping method (e.g., Cap 1 analogs) to maximize the translational potential of your specific construct.

High-Yield Process Optimization

Efficient upstream (in vitro transcription) and downstream (purification) process development tailored for your sequence, guaranteeing maximum yield and over 95% purity of the modified mRNA.

GMP-Certified Quality Assurance

Implementation of rigorous, GMP-certified quality systems, including a well-established process flow that adheres to the Hazard Analysis Critical Control Point (HACCP) approach for therapeutic material safety.

Advanced Process Control

Integration of Quality-by-Design (QbD) principles and high-end Process Analytical Technology (PAT) to enable real-time monitoring and precise control over the m1Ψ incorporation efficiency and overall process stability.

Structural and Functional Verification

Guaranteed stability and fidelity of the starting template and the final mRNA product, verified using high-standard analytical tools, including Mass Spectrometry and functional in vitro translation assays, to confirm biological activity.

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Customer Reviews

  • [Exceptional Immune Evasion] Using Creative Biolabs' Pseudouridine Modification Service in our dendritic cell research significantly reduced TLR signaling and cytokine induction, enabling unprecedented cell transfection without viability issues. This immune silencing is essential for our in vivo vaccine models.

    Time: 3 Months Ago, Dr. Ronnie Geller

  • [Reliable Scale-Up] The consistent m1Ψ incorporation fidelity from Creative Biolabs greatly facilitated our transition from pilot studies to large-scale preclinical manufacturing. Their m1Ψ-modified mRNA is stable, pure, and consistent across batches—an issue we faced with in-house methods.

    Time: 7 Weeks Ago, Sara Hon

  • [Translational Boost] Comparing our standard uridine mRNA to Creative Biolabs' m1Ψ-modified version showed immediate, measurable increases in target protein half-life and total expression in non-dividing primary cells, confirming m1Ψ's role in enhancing gene replacement efficacy.

    Time: 5 Days Ago, Lena Voss

FAQs

Q: Is m1Ψ modification truly necessary if I use an advanced LNP formulation for delivery?

A: Yes, modification is critical. Landmark studies demonstrated that even with the same advanced LNP, unmodified mRNA had drastically lower efficacy. The LNP solves the delivery challenge, but m1Ψ solves the intrinsic immunogenicity problem within the cell, which is why we recommend combining both for maximal clinical viability.

Q: Can Creative Biolabs provide other modifications along with m1Ψ in a single construct?

A: Absolutely. Our platform is flexible. While m1Ψ provides the primary immune silencing benefit, we can incorporate other modifications (such as 5-Methylcytidine) and optimize the 5' and 3' ends to further fine-tune stability and translational yield.

Q: How do you verify the precise incorporation of m1Ψ in the final mRNA product?

A: We use high-resolution analytical techniques, including Mass Spectrometry and NMR spectroscopy, in addition to standard HPLC purity assessment, to verify the exact location and high-fidelity incorporation of m1Ψ. This rigorous QC ensures you receive a product that is confirmed to be structurally sound and non-immunogenic.

Q: Is m1Ψ-modified mRNA stable for long-term storage or shipment?

A: Yes, the m1Ψ modification structurally stabilizes the RNA against nuclease attack, contributing to overall improved stability. We deliver the product lyophilized or in optimized buffers, ensuring long-term integrity. Specific storage recommendations are provided in the Certificate of Analysis (CoA) to maintain quality throughout your project lifecycle.

Q: We are a small research lab. Does Creative Biolabs offer small-scale synthesis before we commit to a large clinical batch?

A: We fully support research and development at every scale. We offer customized synthesis batches ranging from microgram quantities for initial in vitro testing up to multi-gram quantities for large-scale preclinical and clinical trials. Please reach out to discuss your specific volume and purity requirements.

Creative Biolabs' Pseudouridine Modification Service provides the critical chemical engineering required to transform conventional IVT mRNA into a safe, highly stable, and translationally superior therapeutic agent. By leveraging the power of N1-methyl-pseudouridine (m1Ψ), we empower your drug discovery efforts across vaccines, gene therapy, and regenerative medicine.

Contact Our Team for More Information and to Discuss Your Project

Hot IVT Vectors

Cat. No Product Name Promoter
CAT#: GTVCR-WQ001MR IVTScrip™ pT7-mRNA-EGFP Vector T7
CAT#: GTVCR-WQ002MR IVTScrip™ pT7-VEE-mRNA-EGFP Vector T7
CAT#: GTVCR-WQ003MR IVTScrip™ pT7-VEE-mRNA-FLuc Vector T7
CAT#: GTVCR-WQ87MR IVTScrip™ pT7-VEE-mRNA-Anti-SELP, 42-89-glycoprotein Vector T7

Hot IVTScrip™ mRNA Transcript

Cat. No Product Name Type
CAT#: GTTS-WQ001MR) IVTScrip™ mRNA-EGFP (Cap 1, 30 nt-poly(A)) Reporter Gene
CAT#: GTTS-WK18036MR IVTScrip™ mRNA-Human AIMP2, (Cap 1, Pseudo-UTP, 120 nt-poly(A)) Enzyme mRNA
(CAT#: GTTS-WQ004MR) IVTScrip™ mRNA-Fluc (Cap 1, 30 nt-poly(A)) Reporter Gene
(CAT#: GTTS-WQ009MR) IVTScrip™ mRNA-β gal (Cap 1, 30 nt-poly(A)) Reporter Gene

Reference

  1. Herridge, Rowan P., et al. "Pseudouridine guides germline small RNA transport and epigenetic inheritance." Nature structural & molecular biology 32.2 (2025): 277-286. https://doi.org/10.1038/s41594-024-01392-6. Distributed under Open Access license CC BY 4.0, the figure was cropped.
All products and services are For Research Use Only and CANNOT be used in the treatment or diagnosis of disease.