LZCap^® DNA Template Design

LZCap^®AU(3'Acm) is suitable for AG-initiated sequences. As shown in the figure below, the T7 promoter (underlined) followed by the AG sequence can effectively initiate transcription.

Protocol

1． Thaw components required for the experiment on ice.

2． Refer to the following reaction system to configure the transcription system at room temperature.

Modified N1-Me-pUTP can be used in place of wild-type UTP. The modified N1-Me-pUTP reduces the immunogenicity of mRNA.  Henovcom can also provide modified nucleotide N1-Me-pUTP (Cat. No.: HN1002 ).

Notes:

1)     LZCap^®AU(3'Acm) is suitable for T7 promoter transcription vector with 5 ‘AU 3’initiated sequences, which needs to be considered when constructing the vector.

2)     The reagents, consumables and containers used in the experiment are free of RNase contamination.

3)     It is recommended to use a linearized DNA template for transcription.

4)     When modified nucleotides were used in place of wild-type nucleotides, the final concentration of the reaction was unchanged.

5)     If the PCR product is used as the transcription initiation DNA template, the amount of DNA template can be reduced by half.

3． Mix the prepared reaction solution, centrifuge briefly, and incubate at 37°C for 2-3 hours. If the transcript length is less than 100nt, increase the reaction time to 4-8 h.

General Safety Profile

Shipping and Storage

This product could be stored at -25~-15 ℃ for two years.

Precautions

1. For your safety and health, please wear personal protective equipment (PPE), such as laboratory coats and disposable gloves, when operating with this product.
2. For research use only.

F&Q

1. How do you design the LZCap?

Enzymes have a relatively "specific" recognition of substrates. Therefore, when designing a new cap structure, on one hand, we need novel structural modifications for patent purposes, but on the other hand, we strive to maintain similarity with natural/known structures as much as possible. The natural structure has a ribose 3' OH, which can be modified (e.g., methylation). Based on this consideration, we chose to add a carbon at the 3' position for patent novelty, followed by an NH to mimic the hydrogen bonding of OH, and then an acetyl group to reduce the basicity of NH and enhance its hydrogen bonding capability. The activity of LzCap is better than methylated natural cap, possibly due to increased hydrogen bonding. Compared to the methyl and methoxy groups, the acetyl amino group may also increase van der Waals interactions between the substrate (cap) and the initiating factor (enzyme).

2. Is the acetyl amino group stable?

The acetyl amino group is already sufficiently stable. It is much more stable than the 7-methylated position and the phosphodiester bond, which are the least stable parts of the cap.

Please contact us for more details about the LZCap licensing.