Modified mRNA encoding an enhanced monomeric red fluorescent protein (Em: 610 nm) with dual detection capabilities:

• Delivery tracking: Cy3 fluorophore (Ex/Em 550/570 nm) enables real-time visualization of mRNA cellular uptake
• Expression monitoring: RFP fluorescence (Ex/Em 584/610 nm) confirms successful protein translation

Incorporates N¹-methyl-pseudouridine (m¹Ψ) for enhanced translational efficiency and reduced immunogenicity.

• 5' Capping:
  • ARCA (m⁷G(5')ppp(5')G, Cap 0) or
  • CleanCap AG (m⁷G(5')ppp(5')(2'-OMeA)pG, Cap 1)
• Nucleotide Modification: 1:3 ratio of Cy3-UTP to m¹Ψ-5'-triphosphate
• 3' Tail: 180 nt poly(A) sequence with stabilizing elements
• Sequence Length: ~1,100 nt (including optimized UTRs)

• Quantification of mRNA delivery efficiency
• Optimization of transfection protocols
• Time-course studies of protein expression
• Co-transfection controls with other mRNA constructs
• Visualization of intracellular mRNA trafficking

• Concentration: 1.0 ± 0.2 µg/µL (A₂₆₀ quantification)
• Purity: >90% full-length by denaturing agarose gel electrophoresis
• Endotoxin: <0.1 EU/µg
• Fluorescence Intensity: Standardized against NIST-traceable fluorophore standards

For HEK293 cells (24-well format):

1. Dilute 0.5-1 µg mRNA in 50 µL Opti-MEM®
2. Mix with 1.5-2 µL MessengerMAX™ reagent
3. Incubate 15-20 min at room temperature
4. Add complex to cells in complete medium
5. Monitor Cy3 signal (4-24 hr) and RFP expression (24-72 hr)

Full protocol: MessengerMAX™ Transfection Protocol

• Cap integrity verified by RNase H cleavage assay
• Poly(A) length confirmed by Northern blot analysis
• Fluorescence intensity standardized using control samples
• Sterility tested by bacterial culture

• Short-term: -20°C (up to 1 month)
• Long-term: -80°C (stable for 24 months)
• Shipping: Dry ice (stable for 72 hours)
• Note: Aliquot to avoid freeze-thaw cycles