5' Cap (Cap1)
- Translation initiation
- Self/non-self recognition
RNA and LNP Production
In vitro transcribed (IVT) RNA is rapidly being adopted as a powerful tool in both research and clinical settings. Incorporating a suite of proprietary technologies, VectorBuilder’s end-to-end platform delivers high-performance RNA and lipid nanoparticles (LNPs) with enhanced delivery and translation, supporting every stage of research, discovery, and development from IVT vector design to full validation.
Highlights
Innovative: Proprietary technologies covering IVT backbones, RNA synthesis, purification, and LNP formulation to deliver optimal performance.
Customizable: Wide range of RNA modification options, production scales, and QC assays for complete flexibility under expert guidance.
End-to-end: Seamless workflow covering every stage from design and production to full validation and GMP manufacturing for clinical applications.
Partner with VectorBuilder and take your RNA research further
with cutting-edge RNA and LNP solutions.
Workflow for IVT RNA and LNP Production
IVT vector
design & cloning
design & cloning
IVT RNA production
LNP encapsulation
Quality control (QC)
Functional validation
As fast as 4 weeks
- Talk to our experts for support in the design and optimization of your IVT RNA vectors
- Proprietary sequence optimization and UTRs for optimal gene expression
- Robust cloning, including transcription of 120 nt (and longer) polyA tail
- Good Vector Practice (GVP) applied at early design stages to ensure optimal quality and performance of your IVT RNAs
- Synthesis of up to 12,000 nt mRNA and saRNA, and 5,000 nt circRNA from ug to g scales; other small RNAs also available
- High capping efficiency (up to 99%) by co-transcriptional or enzymatic methods
- Various modified nucleotide options: m1Ψ, m5C, 5moU, m6A, etc.
- Proprietary purification process to efficiently remove impurities
- Cell-free and animal-free production available
- Wide variety of standard formulations (e.g. SM102, ALC-0315, MC3) available
- Encapsulation of various types of RNA/DNA molecules, including mRNA, saRNA, siRNA, Cas9 mRNA/sgRNA mix, circRNA, pDNA, etc.
- High encapsulation efficiency (up to 100%)
- Low (<0.1) polydispersity index (PDI)
- Antibody-conjugated LNPs and custom formulations available for therapeutic development
- A full spectrum of QC assays for both IVT RNA and LNP-encapsulated RNA and plasmids
- Optional QC assays fully customizable for individual projects
- Expert guidance to ensure QC assays are well-matched to your project
- Qualification of sequence optimization of various vector components (UTR, coding sequence, polyA, Kozak, etc.) in vitro and in vivo
- Assessment of drug efficacy and safety using animal models including rodents and non-human primates (NHPs)
- Development and validation of therapeutic IVT RNA for various applications, such as antigen presentation, antibody expression, CAR expression, and CRISPR
Explore our collection of high-quality, research-ready premade IVT RNA and LNP-RNA.
Technical Information
IVT RNA overview
IVT RNA has become a powerful platform in genetic medicine, offering safe, flexible, and efficient gene delivery without the risk of genomic integration. Each RNA modality offered at VectorBuilder brings unique advantages: IVT mRNA enables rapid and transient gene expression for personalized use across therapeutic applications including vaccines, protein replacement, CAR-T, and CRISPR; self-amplifying RNA (saRNA) extends expression duration and potency through built-in replication mechanisms, making it a promising platform for next-generation vaccines; and circular RNA (circRNA), with its covalently closed structure, avoids degradation and supports sustained expression for long-term therapeutic effects. Each modality is further detailed below.
IVT mRNA
The following diagram depicts the fundamental components of mRNA, with each component playing critical roles in regulating gene expression. There are a variety of methods for assembling these components in vitro and VectorBuilder can help assess the best method for achieving optimal expression, yields, and purity for your project. You can also read the Vector Academy article The Basics of mRNA Therapeutics for a more detailed overview of IVT mRNA.
Coding sequence
- Translation efficiency
Poly(A) tail
- Stability
- Translation efficiency
- mRNA export from nucleus
5'/3' Untranslated Region (UTR)
- mRNA localization, translation, and stability
Figure 1. The structure and function of mRNA components.
IVT saRNA
saRNA is able to replicate itself in the host cell, with this self-amplification capability reliant on the replicase enzyme encoded in its first open reading frame. As illustrated in Figure 2 below, the nonstructural protein (nsP) genes, with sequence derived from Alphavirus, are translated from the saRNA to form a polyprotein replication complex (replicase), which is responsible for RNA replication. Among them, nsP1 functions as the capping enzyme with methyltransferase and guanylyltransferase activities, while nsP2 possesses RNA helicase and triphosphatase activities. Finally, nsP3 mediates protein-protein interactions and nsP4 serves as an RNA-dependent RNA polymerase. The replicase complex synthesizes a full-length negative-sense RNA, acting as a template for downstream RNA replication. Additionally, conserved sequence elements (CSEs) and a subgenomic promoter (SGP) are utilized as 5′ and 3′ UTRs for the transgene, enabling replicase-mediated amplification and transcription of the transgene. Due to the added size of the replicase gene, saRNA is often more difficult to produce in vitro than traditional mRNA. Therefore, it is critical to establish a robust production pipeline to ensure production of consistent high-quality saRNA.
Figure 2. Mechanism of saRNA replication.
IVT circRNA
VectorBuilder has developed an optimized production platform for the generation of high-yield and high-purity circRNA based on the Group I permuted intron-exon (PIE) self-splicing technology (Figure 3). The generation of circRNA by this mechanism results in minimal impurities and scar sequence. Of note, the translation of circRNA is IRES-dependent. The IRES recruits the ribosome to initiate translation internally on a transcript independent of its 5' end.
Figure 3. Mechanism of circRNA self-splicing and generation in vitro.
IVT RNA purification
Through extensive R&D, VectorBuilder has developed proprietary purification technologies to ensure the market-leading purity of our IVT RNAs:
| Primary Pure | Fast HiPure | HiPure | GMP HiPure | |
|---|---|---|---|---|
| Schematic |  |
 |
 |
For clinical applications, please check our GMP manufacturing page. |
| Applicable for | mRNA | mRNA & saRNA | mRNA & saRNA | |
| Mechanism | Negatively charged RNA molecules (both polyadenylated and unpolyadenylated) are purified with positively-charged carboxyl magnetic beads. | RNA molecules are purified with spin columns containing oligo(dT)-coated magnetic beads that specifically bind polyadenylated RNA. | RNA molecules are purified with oligo(dT) affinity chromatography that specifically binds polyadenylated RNA. | |
| Recommended use | Early-stage research and development | Delicate in vitro and in vivo experiments | Preclinical studies and scale-up processes | |
| Scale | 0.1 - 3 mg | 0.1 - 10 mg | 1 mg - 1 g |
Quality control
VectorBuilder offers a full spectrum of QC assays for IVT RNA, as well as LNP-encapsulated RNA and plasmids. Default QC items (marked with √) are always performed while optional QC items are performed depending on individual project needs.
IVT RNA QC
| Attribute | QC Assay | Research-Grade | GMP-Like | GMP-Grade | |
|---|---|---|---|---|---|
| Identity | mRNA sequence | Sanger sequencing | √ | √ | Check our GMP manufacturing page. |
| mRNA length | Denaturing agarose gel electrophoresis | √ | √ | ||
| Capillary gel electrophoresis (CGE) | Optional | √ | |||
| General/physical property | mRNA concentration | UV spectrophotometry | √ | √ | |
| RiboGreen assay | Optional | √ | |||
| Appearance | Visual inspection | Optional | √ | ||
| Potency | Gene expression | In vitro translation followed by Western blot | Optional | Optional | |
| Cell transfection | Optional | Optional | |||
| Safety | Sterility | Bioburden test | Optional | √ | |
| Mycoplasma | Culture method | Optional | √ | ||
| qPCR | Optional | Optional | |||
| Endotoxin | Kinetic chromogenic assay (KCA) | Optional | √ | ||
| Purity | mRNA integrity | Denaturing agarose gel electrophoresis | √ | √ | |
| Capillary gel electrophoresis (CGE) | Optional | √ | |||
| A260/280 | UV spectrophotometry | √ | √ | ||
| Capping efficiency | LC-MS | Optional | √ | ||
| PolyA analysis | LC-MS | Optional | √ | ||
| Residual dsRNA | Dot blot assay | Optional | √ | ||
| Residual plasmid DNA | qPCR | Optional | √ | ||
| Residual protein | NanoOrange assay | Optional | √ | ||
| Residual solvents | Gas chromatography | Optional | Optional | ||
| Circularization efficiency (for circRNA) | Denaturing agarose gel electrophoresis | √ | Optional | ||
| Capillary gel electrophoresis (CGE) | Optional | √ | |||
LNP-encapsulated RNA and plasmids QC
| Attribute | QC Assay | Research-Grade | GMP-Like | GMP-grade |
|---|---|---|---|---|
| Appearance | Visual inspection | √ | √ | Check our GMP manufacturing page. |
| Concentration | RiboGreen assay | √ | √ | |
| Encapsulation efficiency | RiboGreen assay | √ | √ | |
| Particle size | Dynamic light scattering (Zetasizer) | √ | √ | |
| Polydispersity index (PDI) | Dynamic light scattering (Zetasizer) | √ | √ | |
| Surface charge (Zeta potential) | Dynamic light scattering (Zetasizer) | √ | √ | |
| Encapsulated RNA integrity | Capillary gel electrophoresis (CGE) | Optional | √ | |
| Endotoxin | Kinetic chromogenic assay (KCA) | Optional | √ | |
| pH | pH paper | Optional | √ | |
| Sterility | Bioburden test | Optional | √ |
Experimental Validation
Our proprietary technologies span IVT backbones, RNA synthesis, purification, and LNP formulation, delivering optimal performance across every stage of development.
Our RNA production platform technologies enhance RNA expression and functionality, improve end-product quality and purity, and enable next-generation RNA modalities (e.g. circRNA).
UTR optimization
Figure 4. Optimizing UTRs of IVT mRNAs enhances translation. HEK293T cells were seeded on 12-well plates and transfected with 1 ug of mRNA comprising various 5' and 3' UTR combinations regulating Gaussia luciferase expression. Gaussia luciferase activity was measured from cell culture medium at 6 h, 24 h, 48 h, 72 h, and 96 h post-transfection
UTR engineering
Figure 5. Incorporation of a miR-122 targeting site into the 3’ UTR reduces liver-specific gene expression. Mice were intravenously injected with 10 ug of LNP-mRNA coding for HiExpress™ Firefly Luciferase, with or without a miR-122 targeting site in the 3’ UTR. Mice were sacrificed 6 hours post-injection and luminescence in harvested tissue was measured, indicating that miR-122 is highly and specifically expressed in the liver and the inclusion of miR-122 targeting sites led to reduced luciferase expression in liver tissue.
Codon optimization
Figure 6. Coding sequence optimization in IVT mRNAs enhances gene expression in vitro and in vivo. (A) HEK293T cells were transfected with 0.5 ug of mRNA coding for optimized HiExpress™ Firefly Luciferase and other luciferase. Luciferase activity was measured at 6 h, 24 h, 48 h, and 72 h post-transfection. (B) Luciferase activity measured in C57BL/6 mice injected intramuscularly with 30 ug of LNP-mRNA at 6 h, 24 h, and 48 h post-injection. (FLuc WT: wild-type firefly luciferase, FLuc WT (co): codon-optimized wild-type firefly luciferase, FLuc2: Luc2 firefly luciferase)
Poly(A) integrity
Figure 7. Poly(A) tail size analysis. Poly(A) tails were cleaved from IVT mRNA using ribonuclease T1 and isolated by oligo dT affinity chromatography. Isolated poly(A) tails were analyzed by (A) Urea-PAGE gel electrophoresis and (B) LC-MS. Deconvoluted spectrum was generated from 120 pmol of poly(A) tails with an expected size of 120 nt. (C) Size distribution of poly(A) tails with an expected size of 120 nt. Error bars represent standard deviation from triplicates. Weighted average length is 123 nt.
Poly(A) tail optimization
Figure 8. The effect of poly(A) tail length and structure on translational efficiency of mRNA. HEK293T cells were transfected with IVT EGFP mRNA with the same Cap1 and UTRs but different poly(A) tail lengths as listed above.
Nucleotide modification
Figure 9. Modified nucleotides increase gene expression and reduce dsRNA impurities. (A) Expression of firefly luciferase in HEK293T cells transfected with mRNA with or without modified nucleotides, N1-Methylpseudouridine (m1Ψ) and 5-Methylcytosine (m5C). Luciferase activity was measured at 6 h, 24 h, and 48 h post-transfection. Error bars indicate standard deviations. (B) Equal amounts (750 ng per dot) of magnetic bead-purified EGFP IVT mRNA with or without nucleotide modification (m1Ψ) was assessed by dot blot assay for dsRNA impurities.
IVT mRNA capping strategies
Figure 10. IVT mRNA capping efficiency analyzed by LC-MS. Highly efficient capping can be achieved either using (A) co-transcriptional or (B) enzymatic approaches.
Customized dsRNA removal strategies
Figure 11. Double-stranded RNA (dsRNA) removal efficiency of different purification processes. dsRNA is a by-product of IVT and a major trigger of undesired immunogenicity. Equal amounts (1500 ng per dot) of hSpCas9 IVT mRNA purified by different processes was assessed by dot blot assay to estimate dsRNA impurities, demonstrating that extra purification steps (e.g. IP-PR) may be necessary to achieve an ultra-purification scale with very low levels of dsRNA. Abbreviations: HIC, Hydrophobic interaction chromatography; IP-RP, Ion-pair reversed-phase liquid chromatography.
High-quality production
Figure 12. Denaturing agarose gel result indicating production of high-integrity IVT RNA (>10,000 nt).
circRNA stability
Figure 13. Comparative analysis of precursor and circular RNA (circRNA). Precursor and circRNA coding EGFP were incubated with varying concentrations of RNase A then analyzed by gel electrophoresis, showing that RNase A will digest all linear but not circRNA.
Structural characterization of LNP-mRNA
Figure 14. High-resolution cryo-TEM micrographs of LNP-mRNA. Scale bar = 100 nm.
LNP homogeneity
Figure 15. Particle size and Zeta potential distribution analysis by DLS. (A) PDI and (B) Zeta potential were determined by DLS, which measures the intensity differences of fluctuated light due to motion of particles, indicating homogeneous LNP mixtures.
Long-term LNP-mRNA performance
Figure 16. Long-term cryopreservation of anti-CD31 antibody-conjugated LNP-mRNA. (A) Firefly luciferase (FLuc)-expressing LNP-encapsulated mRNA was preserved under two conditions: 4 °C with no additives, and -80 °C in 7.5% sucrose solution for 153 days. Particle size (pink bars) and PDI (teal dots) of both groups were compared to the original parameters. (B) Imaging of Fluc mRNA expression 6 hours post-injection. Female ICR mice were intravenously injected with PBS or tested LNP-mRNAs. (C) Encapsulation efficiency of LNP-mRNA post-cryopreservation. Freshly prepared FLuc LNP-mRNA was used as the control. (D) Comparison of RNA integrity post-cryopreservation.
LNP delivery
Figure 17. Comparison of in vitro mRNA delivery using LNP and transfection reagent. EGFP mRNA was delivered to cells either via LNP encapsulation or a commercial transfection reagent. (A) Flow cytometry analysis of EGFP expression in Jurkat and HEK293T cells and (B) fluorescence imaging of HEK293T cells at 24 hours post-transfection. MFI: median fluorescence intensity.
saRNA expression
Figure 18. Comparison of EGFP expression in HEK293T cells after transfection with saRNA or traditional mRNA.
circRNA expression
Figure 19. Comparison of mRNA- and circRNA-mediated expression. HEK293T cells transfected with circRNA-EGFP exhibited sustained expression compared to traditional mRNA and non-treated control (NC). Exposure: 100 ms. Magnification: 100x.
mRNA-LNP delivery
Figure 20. LNP-encapsulated mRNAs support enhanced in vivo expression. C57BL/6J mice (8 weeks) were injected intramuscularly with LNP-encapsulated mRNA encoding for luciferase. (A) Luciferase activity visualized by live imaging at 6 h, 24 h, and 48 h post-injection. (B) Serum quantification of pro-inflammatory cytokines, IL-6 and TNF-⍺, 48 h post-injection (SE). (C) IFN-γ ELISpot assay of splenocytes derived from Balb/C mice 14 days post i.m. injection of 30 ug LNP-encapsulated mRNA coding for viral antigen A, viral antigen B, or control PBS.
saRNA-LNP delivery
Figure 21. saRNA exhibits prolonged in vivo translation compared to standard mRNA. Expression of firefly luciferase (Fluc) from 30 µg of LNP-encapsulated mRNA was compared to 10 µg of its saRNA counterpart. While Fluc expression from the traditional mRNA was no longer detectable after 48 hours, saRNA expression persisted for up to 8 days following intramuscular injection.
Resources
FAQ
| mRNA | circRNA | saRNA | |
|---|---|---|---|
| Structure | Linear; usually contains 5’ cap, 5’ UTR, ORF for GOI, 3’ UTR, and poly(A) tail | Circular; usually contains IRES and ORF for GOI | Linear; usually contains 5’ UTR, ORFs for replicase genes and GOI, 3’ UTR, and poly(A) tail |
| Cap | Requires 5’ Cap for stability and ribosome recruitment | No Cap; relies on IRES for ribosome recruitment | Requires 5’ Cap for stability and ribosome recruitment |
| RNA length (nt) | 100 ~ 12,000 | 1000 ～ 5000 | 7000 ~ 12,000 |
| Stability | Low | High | Low |
| Can modified nucleotides be added in production? | Yes | No | Yes |
| Expression level | Low | Medium | High |
| Expression duration | Short | Medium | Long |
| Immunogenicity | Low | Medium | High |
Cap 0 refers to N7-methylguanosine (m7G) that is added to the 5’ end of eukaryotic mRNAs via a 5’ to 5’ triphosphate linkage. This modification is added via a series of enzymatic reactions that occur co-transcriptionally and functions to regulate nuclear export, transcript stability, and promotes translation of the mRNA through recognition by eukaryotic translation initiation factor (eIF4E). Cap 1 refers to the addition of a methyl group to the 2’O on the first nucleotide (m7GpppNm) of the transcribed mRNA sequence in addition to the m7G cap. In mammalian cells, cap 1 structure is an important marker for mRNA to be recognized as self and not targeted by innate immunity. Adding cap 1 structure to synthesized mRNA has been demonstrated to enhance mRNA expression in vivo and reduce its immunogenicity.
Capping for in vitro transcribed RNA can occur either co-transcriptionally with cap analogs or post-transcriptionally via enzymatic reactions. We offer both capping methods, and the efficiency of them has been well validated using LC-MS. Depending on the client-preferred capping method, we will choose a compatible backbone for cloning the IVT mRNA vector from the beginning.
Cells contain cytosolic and endosomal RNA receptors that activate the immune response upon recognition of foreign RNA. Modified nucleotides are commonly found in endogenous cellular RNA. Incorporating certain modified nucleotides in IVT mRNA reduces its immunogenicity, alters secondary structure, and increases translation efficiency and half-life in a sequence-dependent manner. We provide a wide range of modified nucleotides, including the commonly used N1-Methylpseudouridine (m1Ψ) and 5-Methylcytosine (m5C). N1-Methylpsuedouridine and 5-Methylcytosine are naturally occurring nucleotides that were first identified in tRNAs, however, their use in coding mRNAs has only recently been appreciated. These methylated derivatives of uridine and cytosine can replace their canonical nucleotides in mRNA IVT and translation without altering traditional Watson-Crick base pairing. A major advantage to their use in mRNA therapeutics is their ability to alter recognition by RNA immune receptors thus mitigating unwanted immune effects and enhancing transcript stability and translation.
Featured citations
Phase Separation Enhanced PROTAC for Highly Efficient Protein Degradation
Yu, et al.
Biomacromolecules, 2024, doi: 10.1021/acs.biomac.4c00424
Products used:
Abstract: Biomacromolecular condensates formed via phase separation establish compartments for the enrichment of specific compositions, which is also used as a biological tool to enhance molecule condensation, thereby increasing the efficiency of biological processes. Proteolysis-targeting chimeras (PROTACs) have been developed as powerful tools for targeted protein degradation in cells, offering a promising approach for therapies for different diseases. Herein, we introduce an intrinsically disordered region in the PROTAC (denoted PSETAC), which led to the formation of droplets of target proteins in the cells and increased degradation efficiency compared with PROTAC without phase separation. Further, using a nucleus targeting intrinsically disordered domain, the PSETAC was able to target and degrade nuclear-located proteins. Finally, we demonstrated intracellular delivery of PSETAC using lipid nanoparticle-encapsulated mRNA (mRNA-LNP) for the degradation of the endogenous target protein. This study established the PSETAC mRNA-LNP method as a potentially translatable, safe therapeutic strategy for the development of clinical applications based on PROTAC.
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