Circular RNA (circRNA) related Services
Circular RNAs (circRNAs) are linear RNAs in which the 3' and 5' ends have been covalently ligated to form a stable circle. This closed circular structure is highly resistant to nucleases, as they lack free ends. The half-lives of circRNAs are much longer than linear mRNAs, with many circRNAs having half-lives of >48–72 h in serum-containing media, in contrast to the minutes to hours that many capped/polyadenylated mRNAs have. As a result, circRNAs can be used for long-term protein expression and circumvent the need for 5' cap analogues or 3' poly(A) tails, leading to ease of manufacturing and decreased innate immune detection. From the perspective of drug development, circRNAs may have utility as a next-generation RNA drug that have a longer-term expression than mRNA therapeutics, while still having the potential to be non-permanent gene therapies. Synthetic circRNAs designed to express erythropoietin, factor IX, or monoclonal antibodies have shown levels of protein expression for at least one week after a single intramuscular or intravenous dose in preclinical mouse and non-human primate models. In the context of vaccines, circRNAs encoding viral antigens have been shown to induce robust germinal-center responses, resulting in high-affinity neutralizing antibodies with lower numbers of booster injections when compared to mRNA vaccines.
Fig.1 Schematic representation of known functions of circRNAs 1,2
We know that harnessing the therapeutic promise of circRNAs requires a partner with unparalleled expertise and comprehensive, end-to-end capabilities. Our circRNA Services platform positions us to be that single-source partner, enabling us to eliminate all barriers from idea to application. Guided by rational circRNA design and leveraging ultra-scalable in vitro production, ultra-high purity QC and in vivo functional validation, we help you realize your therapeutic ambitions by translating your concepts into high-quality, meticulously engineered circRNA therapeutics—ready to drive your streamlined journey from concept to pre-clinical success.
Our circRNA Service Workflow
Our GMP-compliant circRNA synthesis platform combines high-yield in vitro transcription (IVT) with post-transcriptional circularization to generate milligram-to-gram quantities of high-purity, translation-competent circRNA.
| Platform | Mechanism | Advantages | Typical Yield |
|---|---|---|---|
| Self-splicing ribozymes (Group I/II) | Autocatalytic cleavage-ligation | High purity, minimal by-products | 1–5 mg / 20 mL Rxn |
| Permuted intron-exon (PIE) system | Intramolecular ligation via spliceosome | Sequence flexibility, long ORF capacity | 2–10 mg / 20 mL Rxn |
Consultation & Strategy
We begin by exploring your project objectives in depth so we can suggest the best circRNA design and production approach.
CircRNA Design & Construction
we iteratively model thermodynamic stability, circularization efficiency, and translational output, supplying you with detailed design maps, cloning strategies, and risk-mitigation recommendations.
Pilot Production & QC
Whether you need a single high-expression circRNA or an entire combinatorial library, our experts ensure each construct is optimized for robust production, long half-life, and maximal biological activity.
Large-Scale Production & Purification
Rapid, scalable manufacturing takes place in our cutting-edge facility, powered by rigorously validated and highly reproducible protocols.
What We Do
CircRNA Design & Strategy
Your therapeutic vision sets the blueprint; we translate it into a circRNA that actually works where you need it. From day-one brainstorming to final release, every decision is made with your timeline, indication, and regulatory path in mind.
- IRES / Translation Control Selection: We help you choose the exact ribosome-entry strategy that matches your expression window. Need rapid-onset antibodies? We swap in the CrPV IGR IRES. Need weeks-long enzyme replacement? We tune the circRNA with a milder EMCV IRES or an optimized 5' UTR spacer. You receive annotated maps and in vitro translation curves within one week so you can green-light or pivot early.
- Transgene Optimization: Your Gene-of-Interest is re-coded in silico for human, mouse, or cynomolgus bias, while we silently remove cryptic splice sites, miRNA seeds, and rare codons that could blunt expression. The final sequence is delivered to you as a locked FASTA and GenBank file, ready for patent filing or regulatory submission without further editing.
- Regulatory Element Integration: We insert tissue-specific enhancers, WPRE-like stabilizing loops, or destabilizing AU-rich elements exactly where you need them. Want faster turnover for a cytokine circuit? We add an engineered destabilizing cassette. Need month-long persistence for a protein-replacement therapy? We embed stabilizing RNA triple-helix motifs. Each element is empirically tested in your cell line of choice, and the data set ships with your construct.
- Circularization Architecture: Whether you plan intrathecal injection, intramuscular LNP, or exosome-mediated delivery to tumors, we design the circRNA length, linker composition, and 3' tail to maximize encapsulation efficiency and reduce renal clearance. You receive a matrix of predicted half-lives and biodistribution scores so you can lock the final design before ordering tox material.
CircRNA Construction
We turn your sequence—or even your rough concept—into sequence-verified, translation-ready circRNA while you focus on the next milestone.
- Flexible Starting point: Send us a plasmid, a Word doc of bases, or even a rough ORF concept. We can start from your existing backbone, synthesize the entire cassette de novo, or simply circularize an mRNA you already have. You choose the route; we take care of the rest and return a ready-to-use construct with full IP traceability assigned to you.
- Seamless Circularization: Using your preferred system (Group I ribozyme, PIE, or T4 ligase-based splint ligation), we insert the exact 5'/3' linkers, IRES, and spacer sequences you need, then sub-clone into an antibiotic-free, antibiotic-marked, or minicircle backbone—whichever simplifies your downstream steps. You receive annotated GenBank and SnapGene files the same day the construct ships.
- Guaranteed Sequence Fidelity: Every intermediate and the final circular RNA are verified by 100 % Sanger sequencing across the entire expression cassette and junction sites. We also run RNase-R resistance and ddPCR circularization assays before anything leaves our lab. You open the tube knowing the sequence is identical to the one you approved and ready for animal dosing without re-screening.
CircRNA Production & Scale-Up
From your first in vitro screen to your IND-enabling tox study, we manufacture exactly the amount—and quality—of circRNA you need, precisely when you need it.
- Proven IVT & Circularization Platform: We run high-yield, T7-based in vitro transcription followed by your chosen circularization chemistry (Group I ribozyme, PIE, or splint ligation) in both 2 mL research tubes and 2 L bioreactors. Suspension HEK293 or CHO-derived lysates are optionally depleted of dsRNA and endotoxin in-line, giving you clinically relevant purity from day one. You receive a detailed batch record so you can trace every reagent lot and incubation time.
-
Scalable Solutions: We accommodate a wide range of production scales to match your needs:
- Research Grade: 50 µg–5 mg delivered in 7–10 days—perfect for quick in vitro proof-of-concept or small-animal pilot dosing.
- Pre-clinical Grade: 50 mg–2 g released under cGMP with full CMC documentation, sterility, and endotoxin data that slot directly into your IND sections 3.2.S.2 and 3.2.S.4. You simply tell us the dose and species, we back-calculate the gram quantity and ship it with stability-indicating data out to 12 months.
- Flexible Formats & Modification Options: We can incorporate any modified nucleotide at the exact ratio you specify and we formulate and lyophilize in single-use vials with residual moisture <1 %. Every option is quoted up front, so you control cost and risk, not us.
CircRNA Purification & Formulation
Purity is not a checkbox—it is the difference between a therapeutic that works in patients and one that stalls in tox. Every purification step we run is designed around one goal: giving you circRNA that is >95 % circular, dsRNA-free, and ready for the route you choose—on the date you stipulate.
- Gold-Standard Chromatography: Small discovery lots (≤5 mg) are polished by dual-mode ion-exchange (IEX) followed by size-exclusion to strip linear precursors and endotoxin to <0.1 EU/µg. Mid-scale pre-clinical lots (up to 2 g) move to a proprietary RP-HPLC step that resolves circular from linear species with single-nucleotide precision; you receive the chromatogram before we ship the tube. Our dsRNA-affinity column option guarantees residual immunostimulants below FDA draft guidance.
- Tailored Formulation: Default release is in sterile, endotoxin-tested PBS + MgCl2 + 0.001 % Pluronic F-68, but we'll dialyze into any buffer you specify: citrate for LNP encapsulation, HEPES for electroporation, or a lyophilization-ready trehalose matrix for global distribution. Each lot comes with pH, osmolality, and residual solvent certificates formatted for your IND CMC section.
- Risk-Free Packaging & Logistics: Vials are filled under nitrogen, bar-coded with your internal lot number, and shipped with temperature loggers so you can release the material the moment it arrives—no re-testing, no delays.
Toxicity & Safety Determination
Before your first animal receives a single microgram of RNA, we certify that every circRNA batch you hold in your hand is as clean as your protocol demands—and we hand you the documentation to prove it.
- Endotoxin Analysis: We run a quantitative chromogenic LAL assay on every lot and report the exact EU/µg, if your SOP caps endotoxin at 0.05 EU/µg, we dial our release spec to match. You receive the raw plate readout and a signed certificate, so no re-test is required on arrival.
- Sterility Testing: Using USP <71>-compliant 14-day incubation in both aerobic and anaerobic broth, we confirm absence of bacteria and fungi. We offer Bact/ALERT accelerated culture—results in 72 h without compromising your timeline.
- Mycoplasma Detection: Our qPCR panel detects down to 10 CFU/mL across eight common mycoplasma species. If your contract research site insists on an additional 28-day culture confirmation, we run that in parallel and forward the raw data packet—saving you an extra week of back-and-forth.
- Residual dsRNA & HCP Analysis: We quantify dsRNA (<0.1 %) and host-cell proteins (<10 ppm) by HPLC-fluorescence/ELISA and provide the calibration curves. In case your FDA pre-submission requires even more stringent limits, we re-optimize the purification train at no extra cost and deliver a batch that is 'future proof' for Phase I.
Circular RNA Analysis Services
- In Situ Hybridization Detection
When you need to visualize where your circRNA is working in the cell, our in situ hybridization (ISH) services give you precise localization data—so you can see exactly where your RNA is expressed. We design fluorescent or chromogenic probes tailored to your circRNA sequence, ensuring high specificity and sensitivity. You provide the sequence, and we handle the rest, from probe synthesis to validation in your target tissue or cell line. Whether you are working with neurons, cardiomyocytes, or tumor cells, our ISH protocol is optimized for your specific tissue. We use advanced fixation and permeabilization techniques to preserve cell structure while allowing probe access, so you get clear, interpretable results. Our ISH service includes quantitative image analysis, providing you with detailed reports on the intensity and distribution of circRNA expression. You receive high-resolution images and data tables that show expression levels in different cellular compartments, helping you understand the biology behind your circRNA.
- In Vitro Expression Validation
Before moving to in vivo studies, you need to know that your circRNA expresses the protein you designed it for. Our in vitro expression validation services give you that confidence. We offer expression validation in a wide range of cell lines, including HEK293, HeLa, primary human T cells, and more. You choose the cell line that best matches your therapeutic target, and we ensure optimal transfection conditions for your circRNA. Using flow cytometry, Western blot, or ELISA, we quantify the protein expression driven by your circRNA. We provide detailed reports on expression levels over time, so you can see how your circRNA performs under different conditions. Beyond simple expression, we can run functional assays to test the activity of the protein produced by your circRNA. Whether it's a luciferase reporter assay or a cytokine release test, we design and execute the experiments to give you comprehensive data on your circRNA's performance.
LNP Formulation Service
When you need to deliver your circRNA with precision and efficiency, our LNP formulation service ensures that your RNA reaches its target cells safely and effectively—every time.
- Customizable LNP Formulations
We understand that each circRNA project is unique, which is why we offer fully customizable LNP formulations tailored to your specific needs. Whether you're targeting liver cells, neurons, or immune cells, our team works with you to select the optimal lipid composition and surface modifications to maximize uptake and minimize off-target effects.
- Targeted Delivery: Our LNPs can be functionalized with tissue-specific ligands, such as N-acetylgalactosamine (GalNAc) for hepatocytes or RVG peptides for neurons. You provide the target tissue, and we design the LNP to deliver your circRNA precisely where it's needed.
- Scalable Production: From small-scale in vitro studies to large-scale preclinical and clinical batches, our LNP formulation service scales seamlessly with your project. We ensure consistent quality and performance across all production scales, so you can move confidently from bench to bedside.
-
Comprehensive Quality Control
We take the guesswork out of LNP formulation by providing rigorous quality control at every step. You receive detailed reports on particle size, polydispersity index (PDI), zeta potential, and encapsulation efficiency, ensuring that your LNP-circRNA complex meets the highest standards. Using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA), we measure the size and distribution of your LNP-circRNA complexes. You get precise data on particle diameter and PDI, so you know your formulation is uniform and stable. We quantify the amount of circRNA encapsulated within the LNP using a combination of UV-Vis spectroscopy and qPCR. You receive accurate measurements of encapsulation efficiency, ensuring that your LNP delivers the intended dose of circRNA. -
In Vitro and In Vivo Validation
We validate the performance of your LNP-circRNA complexes in both in vitro and in vivo settings, giving you comprehensive data to support your research and regulatory submissions. Our team tests the transfection efficiency of your LNP-circRNA complexes in a variety of cell lines, including HEK293, HeLa, and primary human cells. Using fluorescence microscopy and flow cytometry, we provide detailed reports on cellular uptake and circRNA expression levels. For preclinical studies, we offer in vivo validation in rodent models, and measure circRNA biodistribution, protein expression, and pharmacokinetics, providing you with critical data on the efficacy and safety of your LNP formulation.
Why Choose Us?
- Scientific Expertise: Our PhD-led circRNA scientists become an extension of your own R&D team. We turn your therapy idea into an evidence-based experimental design, identify potential roadblocks upfront, and deliver weekly status reports from your first discovery call so that you can make go / no-go decisions with certainty—without adding additional headcount or consuming internal hours.
- End-to-End Solutions: Whether you arrive with only a gene-of-interest FASTA file or a locked IND filing date, we slot seamlessly into whichever stage you occupy. We ship capped, circularized, and endotoxin-cleared material in 96 h. One project manager consolidates synthesis, LNP formulation, and GLP studies into a single Gantt chart that syncs with your regulatory calendar so your internal teams stay focused on downstream milestones.
- Uncompromising Quality: Every circRNA is manufactured with strict quality controls to ensure purity is to the highest standard and our lot-to-lot reproducibility is consistent. We aggregate the raw QC data into your desired deliverable- ready to file CoA, editable IND section 3.2.S.4, board deck- so your regulatory writers can copy/paste (not reformat).
- Customization & Flexibility: We know every project is different, so we don't start with off-the-shelf. Rather we tailor-make each construct for your specific circRNA of interest, tissue of interest, and experimental goals based on a vast panel of serotypes to find the best match for your project.
- Rapid Turnaround: Precision-driven workflows and proactive project management deliver your products exactly when promised, keeping every phase of your research running smoothly and on schedule.
What Our Clients Say
"We were staring at a six-month GLP tox deadline and only had a rough circRNA sequence. Creative Biolabs's team jumped on a call the same day, redesigned our 5' UTR for better IRES efficiency, and delivered 2 g of GMP-grade, endotoxin-cleared circRNA-LNP two weeks ahead of schedule—letting us book the animal facility early and shave a full month off our IND timeline. They didn't just meet our milestone; they protected our runway."
— Kevin Liu, PhD, Senior Scientist
"We're a seed-stage company with one shot at proving our circRNA cancer vaccine platform. Creative Biolabs didn't just quote a price; they sat in on our investor calls, redesigned the 5' UTR overnight to boost antigen expression 3-fold, and shipped a lyophilized master mix that survived a cross-country lab move. When our first GLP tox batch came back under-spec, they re-synthesized and re-tested at no extra charge—keeping our Series A timeline intact. It feels like having an in-house RNA team without the payroll."
— Dr. Anita Desai, Co-founder & CSO
"The in vivo data we needed for our cardiomyopathy program required week-long protein expression in large animals. Creative Biolabs's circRNA delivered measurable human Factor IX for 21 days after a single LNP injection—double the durability we'd seen with capped mRNA. More importantly, their scientists joined our steering-committee calls, suggested a muscle-specific IRES, and walked our regulatory team through the dsRNA removal data. The purity and reproducibility across three lots gave our board the confidence to green-light Phase I."
— Dr. Samuel Park, Principal Investigator
FAQ
Q: Why circRNAs are attractive for therapeutic or diagnostic projects
A: Circular RNAs are single-stranded RNAs in which the 3' and 5' ends are covalently ligated to form a closed loop. This structure confers exceptional nuclease resistance, giving circRNAs an in vivo half-life of 18–24 h versus 4–7 h for linear mRNA. CircRNAs can act as miRNA sponges, protein scaffolds, or—when an IRES is included—templates for translation, making them powerful tools for gene therapy, vaccine design, and biomarker discovery.
Q: How do you ensure high circularization efficiency and purity of circRNAs?
A: Each precursor RNA is synthesized by high-yield IVT and purified by RNase-R digestion followed by dual HPLC (anion-exchange + size-exclusion). Circularization efficiency is quantified by denaturing PAGE and confirmed by Sanger sequencing across the junction. Typical yields ≥95 % circularized product with ≤1 % precursor or concatemer. Endotoxin levels are <0.1 EU/µg for research grade and <0.05 EU/µg for GMP grade.
Q: How do you mitigate immunogenicity when using in vitro-transcribed circRNA?
A: Our approach begins at the nucleotide level by substituting canonical bases with modified nucleosides such as N1-methyl-pseudouridine, 5-methyl-cytidine and, when required, 2'-O-methyl-adenosine and ‑guanosine. These changes reduce recognition by RIG-I, PKR and TLR pathways without compromising translation efficiency. We further purify the IVT template with restriction enzymes and exonucleases to remove dsRNA contaminants that are the primary drivers of interferon responses. After circularization, the product is treated with a proprietary dsRNA-specific antibody matrix that precipitates any remaining double-stranded species, followed by diafiltration to eliminate small immunostimulatory RNA fragments. To document the absence of immune activation, every batch is subjected to an in vitro reporter assay in which human THP-1 monocytes are co-cultured with the circRNA at 1 µg/mL. Secreted IFN-β and IL-6 are quantified by ELISA, and we guarantee levels within twofold of untreated controls. Additionally, we perform an orthogonal cell-based assay using cells that emit secreted embryonic alkaline phosphatase upon IRF3 activation; luminescence values must remain below 150 relative light units above background. Finally, we can provide optional in vivo data in C57BL/6 mice after intramuscular injection of 10 µg circRNA-LNP complexes, demonstrating serum IFN-α levels below the assay detection limit of 6 pg/mL at 6 h post-dose.
Q: What happens if my circRNA fails to express the expected protein in my cellular model, and do you offer troubleshooting services?
A: Although our constructs are validated by in-house expression assays, biological variability in different cell lines can occasionally lead to lower-than-expected yields. When this occurs, we initiate a structured troubleshooting protocol at no additional cost within 30 days of delivery. First, we request raw data including transfection efficiency, cell viability, and any luciferase or fluorescent reporter readings you have collected. We then re-synthesize the identical sequence and repeat our QC under identical conditions to rule out manufacturing anomalies. If expression remains suboptimal, we perform a deep-sequencing scan of the back-splice junction to confirm the circular form is intact and not undergoing aberrant splicing or exonuclease attack. In parallel, we perform an in-silico re-analysis of the 5' and 3' UTRs for cryptic splice sites or miRNA target sequences that could be cell-type-specific. When necessary, we offer a complimentary redesign that replaces the UTRs with validated alternatives derived from highly expressed circRNAs in your cell lineage; turnaround for this redesign is 5 business days. Finally, if the issue persists, we can provide a small-scale LNP re-encapsulation with alternative ionizable lipids to address endosomal escape inefficiencies, again at no charge for the first iteration.
Q: Can circRNAs be stored at room temperature for short periods?
A: Lyophilized circRNA powders exhibit remarkable thermostability; independent accelerated-aging studies show that loss of circular integrity is <2 % after 4 weeks at 25 °C when protected from moisture and light. Nevertheless, we mitigate risk by employing a trehalose-based stabilizer matrix and argon back-filling within amber borosilicate vials sealed with butyl rubber stoppers. Each shipment includes a desiccant sachet and an irreversible time-temperature indicator that changes color if the internal temperature exceeds 8 °C for more than 30 cumulative hours. For LNP formulations, we ship on dry ice in validated insulated shippers that maintain –70 °C for at least 96 h, the package contains a USB temperature logger that records data every 15 min, and the file is automatically uploaded to our cloud portal so you can review the cold-chain profile upon receipt. If any excursion occurs, we replace the batch free of charge. In addition, we conduct real-time stability studies at –20 °C, 4 °C, and 25 °C for every product line, enabling us to grant shelf-life extensions based on empirical data rather than conservative estimates.
References
- Das, Arundhati, et al. "Emerging role of circular RNA–protein interactions." Non-coding RNA 7.3 (2021): 48. https://doi.org/10.3390/ncrna7030048.
- Distributed under Open Access license CC BY 4.0, without modification.
