CleanCap® Reagent M6 - (N-7453)
CleanCap® Reagent M6 [CleanCap m6AG (3’ OMe)], is designed for the co-transcriptional capping of mRNA to produce an mRNA with base modified Cap 1. Cap 1 mRNAs have superior in vivo activity compared to Cap 0 mRNAs produced by legacy capping methods. The addition of a methyl group on position 6 of the first adenosine (m6A) may further increase protein expression relative to CleanCap AG or CleanCap AG (3’OMe). It has been hypothesized that the m6A modification adjacent to the 7-methylguanosine cap can positively influence mRNA stability by preventing enzyme-mediated decapping (Mauer et al. 2017)
CleanCap M6 uses the same 5’ AG initiation sequence as CleanCap AG and CleanCap AG (3’OMe).
CleanCap has been shown to provide > 95% capping efficiency and typical crude yields of 4 mg to 5 mg per mL of transcription.
CleanCap M6 can be used in conjunction with wildtype bases or TriLink’s catalog of modified NTPs, such as N1-Methylpseudouridine-5’-Triphosphate (N-1081), Pseudouridine-5’-Triphosphate (N-1019), and 5-Methoxyuridine-5’-Triphosphate (N-1093)
|Purity||≥95% by AX-HPLC|
|Extinction Coefficient||34,381 Lmol-1cm-1 at 257 nm ± 3 nm in 100mM Sodium Phosphate Buffer pH 7.0|
|Molecular Formula||C34H48N15O24P4 (free acid)|
|Molecular Weight||1173.73 g/mole (free acid)|
|Recommended Storage||-20°C or below|
|Other Name(s)||CleanCap ® Reagent m6AG (3’OMe) for co-transcriptional capping of mRNA, m7(3’OMeG)(5’)ppp(‘5)m6(2’OMeA)pG|
|Application||CRISPR, In vitro Transcription, Recombinases, TALENS, Transposases, Vaccine Development, Zinc Finger Nucleases|
|Base Analog(s)||Adenosine, Guanosine|
|Nucleotide Category||Cap analogs|
|Cap Analogs||CleanCap Cap Analogs|
Yes, CleanCap M6 initiates on "AG" templates at the +1 and +2 position downstream of the TATA box.
Yes, the lower pH is critical for maintaining capping efficiency and reaction kinetics in IVT. Please follow appropriate safety measures while preparing and handling the 10x reaction buffer. Once mixed, the 10x solution can be stored for up to 1 month at at –20 C. For best results avoid excessive freeze thaws.
Yes, both the pH and high concentration of CleanCap M6 are necessary to achieve high capping efficiency with the m6A modification. Using lower concentrations of CleanCap M6 will result in lower cap initiation and therefore more uncapped products from IVT.
Less than 8 mM cap analog is not advised.
You may need to extend total IVT reaction time past 4 hours to achieve maximum RNA yields.
On our 5'UTR we have tested pulse-feed additions at 1 hour and 1.5 hours into initial reaction and observed ~2% decrease in capping efficiency. Early spike in capping efficiency still passed our 95% or higher specification.
Starting the reaction with 9 mM each NTP with 1x buffer and 10 mM cap results in little to no RNA formation. The reaction will not be balanced with enough magnesium to begin polymerization.
Starting the reaction with 9 mM each NTP and 2x reaction buffer to increase magnesium will also add extra components from the 10x reaction buffer that inhibit the reaction from reaching the desired yield. You may observe 3 mg/mL RNA from this reaction set up.
For best results we recommend storing the individual NTPs and 10x reaction buffer at 4C during the initial reaction incubation to avoid freeze thaw and prepare the spike-in mix ~5 minutes before use (addition to reaction tube).
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