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RNA Chemistry

RNA-Specific Terminal Caps

Reference guide for mRNA capping (Cap 0/1/2, ARCA, CleanCap), terminal phosphate states (ppp/pp/p), NAD caps, and key 3′ RNA caps. Designed for therapeutic mRNA, vaccines, CRISPR guides, and innate-immunity studies.

Overview

5′ caps protect RNA from exonucleases and tune translation and innate sensing. Cap 1 is standard for therapeutic mRNA, while 5′-triphosphate RNA (pppRNA) is intentionally used to activate RIG-I in immunology. Non-canonical caps such as 5′-NAD appear in bacteria and can be leveraged in synthetic biology. At the 3′ end, cyclic- and mono-phosphate caps modulate ligation and stability workflows.

Stability

m⁷G caps (Cap 0/1/2) increase 5′ stability and translation efficiency.

Immunogenicity

ppp/pp RNA triggers RIG-I; Cap 1 reduces innate sensing for in vivo use.

Manufacturing

Co-transcriptional reagents (CleanCap™) and ARCA simplify GMP workflows.

mRNA Caps (5′)

Canonical eukaryotic-style caps supporting translation and stability. Choose Cap 1 for most therapeutic programs; Cap 2 can further reduce innate sensing in some systems.

Product/Modification Function Applications Notes / Code
Cap 0 (m⁷GpppN) 5′–5′ m⁷G triphosphate cap; translation initiation; base protection. In vitro translation, basic mRNA research. Translation ↑ [Cap0]
Cap 1 (m⁷GpppNm) Cap 0 + 2′-O-Me at first nucleotide; reduces innate sensing. Therapeutic mRNA, vaccines, LNP programs. Immune ↓ [Cap1]
Cap 2 (m⁷GpppNmN) Cap 1 + 2′-O-Me at the second nucleotide; may further reduce innate sensing. Advanced therapeutics; immune-silent designs. Immune ↓↓ [Cap2]
ARCA (Anti-Reverse Cap Analog) Cap analog that prevents reverse incorporation; boosts translation. Co-transcriptional capping for IVT mRNA. Orientation-safe [ARCA]
CleanCap™ (series) Co-transcriptional capping reagents; high efficiency Cap 1/2. GMP mRNA manufacturing, high yield capping. Co-Tx [CleanCap]
Hide Technical Notes
  • Recommendation: Default to Cap 1 for in vivo delivery; evaluate Cap 2 in sensitive models.
  • Poly(A) length, UTRs, and base chemistry (Ψ/5-MeC/2′-OMe) co-determine translation and immunogenicity.
  • QC: Verify capping efficiency (LC-MS/enzymatic assays); confirm dsRNA content < threshold.

Terminal Phosphate States (5′)

Uncapped or processed 5′ ends used for innate-immunity probes or enzymology workflows.

Product/Modification Function Applications Notes / Code
5′-Triphosphate (pppN) Strong RIG-I agonist; modest protection vs. exonucleases. Immune stimulation, vaccine adjuvant studies. RIG-I ↑ [5′-ppp]
5′-Diphosphate (ppN) Decapping intermediate; immune-stimulatory. Innate sensing research. Innate ↑ [5′-pp]
5′-Monophosphate (pN) Processed RNA; ligation-ready for many enzymes. Small RNA cloning, turnover studies. Ligation [5′-p]
Show Technical Notes
  • Safety: Avoid 5′-ppp in therapeutic products unless immunostimulation is intended; enzymatically cap or phosphatase-treat.
  • Enzymes: RppH/PNK workflows convert ends between ppp/pp/p for cloning and library prep.

Non-Canonical 5′ Caps

Alternative metabolites used as 5′ caps in bacteria/archaea or synthetic biology contexts.

Product/Modification Function Applications Notes / Code
5′-NAD Cap Nicotinamide adenine dinucleotide covalently linked at 5′. Prokaryotic RNA regulation; synthetic circuits; innate sensing. Non-canonical [NAD-Cap]
Show Technical Notes

NAD capping can alter degradation pathways; evaluate compatibility with ligases and cap-binding proteins prior to workflow design.

3′ RNA Caps

Common 3′ terminal states used for stability or library construction.

Product/Modification Function Applications Notes / Code
2′,3′-Cyclic Phosphate (>&!) Blocks extension; forms during RNase-mediated cleavage. RNA ligation workflows (RtcB), degradation mapping. Block [2′3′-cPO₄]
3′-Phosphate Prevents polymerase extension; convertible to 3′-OH. Blocking, controlled ligation, cloning prep. Block [3′-PO₄]
3′-Amino Provides reactive handle; also blocks natural extension. Conjugation, surface coupling, probe construction. Conjugation [3′-NH₂]
3′-Biotin Affinity capture and detection; terminal block. Pull-downs, ELISA/ISH, library selections. Affinity [Bio-3′]
Show Technical Notes
  • Ligation tips: 2′,3′-cPO₄ is compatible with RtcB; for standard ligases convert to 3′-OH.
  • Blocking: 3′-PO₄ / 3′-NH₂ stop extension and 3′ exonucleases in many systems.

Design & Strategy

Therapeutic mRNA Defaults

  • Use Cap 1, optimized UTRs, poly(A) 80–120 nt, and modified bases (Ψ/5-MeC) as required.
  • Minimize dsRNA impurities; apply HPLC or cellulose purification as needed.
  • Confirm capping efficiency >95% before LNP formulation.

Immunology & Tools

  • Choose 5′-ppp/pp RNA to probe RIG-I pathways; include sequence-matched Cap 1 controls.
  • For ligations, normalize 5′/3′ end states with PNK/RppH/RTCB workflows.

Chemistry & QC

Chemistry

  • Co-transcriptional capping (CleanCap™ series) and enzymatic capping services.
  • Terminal conversions between ppp/pp/p; 3′ end conversions (cPO₄/PO₄/OH).
  • Conjugations: 3′-biotin, 3′-amine; 5′-handles on guides as requested.

QC & Documentation

  • Capping efficiency assays; HPLC/LC-MS identity & purity; dsRNA quantitation.
  • Endotoxin, microbial limits, moisture/salt, and residual DNA as scoped.
  • COA with OD/µmol, method parameters, impurity profile.

FAQ

Cap 1 vs Cap 2—when to choose?

Cap 1 is the default for human therapeutics. Test Cap 2 if innate sensing remains elevated or in specific cell types where additional 2′-O-methylation helps.

Can I intentionally keep 5′-ppp?

Yes, for RIG-I activation studies. For any therapeutic product, remove ppp (or re-cap) to reduce immunogenicity and increase stability

Do 3′ caps affect ligation?

Yes. 2′,3′-cPO₄ requires RTCB-type ligases; convert to 3′-OH for standard T4 ligase reactions.

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