CleanCap® Reagent M6

You may need to extend total IVT reaction time past 4 hours to achieve maximum RNA yields.

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.

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).

CleanCap analog is a co-transcriptional small molecule, serving as a trinucleotide primer. It is extended by the RNA polymerase during IVT, enabling high capping efficiency of >95%.

Following the provided the standard protocol, we typically obtain crude RNA yields of 4 to 5 mg/mL from co-transcriptional capping with CleanCap technology.

Yes, please inquire about them from the SKU table provided above or email sales@trilinkbiotech.com.

We have a simple licensing structure for innovators looking to use CleanCap technology in their mRNA drug development. Please find details on the licensing overview page.

Co-transcriptional mRNA capping is a process where mRNA is capped with a cap analog during in vitro transcription as the mRNA is being synthesized. Compared to traditional enzymatic capping methods which occurs post transcriptionally, this process often provides higher efficiency, with fewer steps and less hands-on time.

The CleanCap co-transcriptional capping method caps mRNA with a CleanCap analog during synthesis in a single reaction ("one-pot" process). CleanCap analogs have a naturally occurring cap-1 structure, which is found in higher eukaryotes, reducing immunogenic responses and improving both mRNA stability and functionality.

Please refer to our CleanCap technology's patent fact sheet.

A limited use label license is covered under the terms of sale for research use of CleanCap products and technology.

Please visit our licensing webpage, reach out to your TriLink business development manager, or email licensing@trilinkbiotech.com.

TriLink's commercial licensing framework does not apply running royalties on a per mRNA product sales basis.

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.

They both utilize co-transcriptional capping but differ in the following ways.

Cap structure: ARCA forms a cap-0 structure, which is not recognized as "self" in higher eukaryotes and can trigger unwanted immune responses. CleanCap analogs, on the other hand, form a Cap-1 structure, which is naturally found in higher eukaryotes and thus better controls reactogenicity and improves protein translation.

Transcriptional start site: ARCA's dinucleotide structure (^m7GpppG) means that transcription inherently starts with guanosine (G). CleanCap analogs are trinucleotides (^m7GpppA_mG for mRNA and ^m7GpppA_mU for saRNA) and bind to the +1 and +2 nucleotides downstream of the T7 promoter, incorporating an AG start site in mRNA and an AU start site in saRNA.

GTP competition: ARCA, as a guanosine dimer, competes with free GTP during the in vitro transcription reaction, which can lower both efficiency and yield of capped RNAs. CleanCap analogs, with their AG or AU start site, avoid competition with GTP, eliminating the need for NTP starvation and improving both capping efficiency and RNA yield.