Impact of cap structures on the performance of in vitro

Capping is a crucial modification of in vitro–transcribed mRNAs, enhancing their stability, facilitating translation initiation, and enabling self/non-self discrimination in therapeutic applications. At its most basic level, capping involves the addition of a 7-methylguanosine (^m7G) to the 5′ end of the mRNA generating a Cap-0 structure, which is often recognized as non-self in higher eukaryotes. The first transcribed base can also be methylated at the 2′ ribose position to generate the more common Cap-1 structure found on endogenous mRNAs in higher eukaryotes.

This tech note details a comprehensive evaluation of mRNA capping strategies, focusing on CleanCap^® technology and its impact on mRNA quality and protein expression both in vitro and in vivo.

We report the following in this tech note:

Evaluate mRNA quality using enzymatic and CleanCap capping methods

Present protein expression data for three CleanCap cap analogs

Compare in vivo protein expression of CleanCap M6 and enzymatic capping

Demonstrate the possibility of dose reduction with CleanCap M6 mRNA

TECHNICAL NOTE

**Impact of cap structures on the performance of in vitro–transcribed mRNAs**