Editing Capped mRNA (section)

= mRNA Synthesis with Modified Nucleotides (E2060) =

Modified mRNAs containing 5mCTP and Pseudo-UTP have been shown to suppress RNA-mediated innate immune activation in vivo. The HiScribe T7 ARCA mRNA Kit (with tailing) is capable of incorporation of 5mCTP and Pseudo-UTP, into mRNA. Up to 2.5 mM total modified nucleotides can be added into the transcription reaction without impacting the mRNA yield significantly. Other modified UTP or CTP may also be used but RNA yield will vary depending on the properties of the nucleotides. Modified GTP and ATP should not be used because they will interfere with capping and tailing efficiency. Please note modified nucleosides are not supplied with the kit.<br/><br/>The protocol below uses 1.25 mM 5mCTP and 1.25 mM Pseudo-UTP, generating mRNA containing 50% 5mCTP and 50% Pseudo-UTP.



#Thaw the necessary kit components, mix and pulse-spin in microfuge to collect solutions to the bottoms of tubes. Assemble the reaction in the following order:

'''Composition of NTP/cap analog mixture ('''



{|
|-
| colspan="1" rowspan="1" | '''Component'''
| colspan="1" rowspan="1" | '''Stock concentration (mM)'''
| colspan="1" rowspan="1" | '''Final concentration (mM)'''
| colspan="1" rowspan="1" | '''Volume (µl)'''
|-
| colspan="1" rowspan="1" | ATP (from MEGAscript T7 Kit)
| colspan="1" rowspan="1" | 75
| colspan="1" rowspan="1" | 7.5
| colspan="1" rowspan="1" | 4
|-
| colspan="1" rowspan="1" | GTP (from MEGAscript T7 Kit)
| colspan="1" rowspan="1" | 75
| colspan="1" rowspan="1" | 1.875
| colspan="1" rowspan="1" | 1
|-
| colspan="1" rowspan="1" | Me-CTP (from Trilink)
| colspan="1" rowspan="1" | 100
| colspan="1" rowspan="1" | 7.5
| colspan="1" rowspan="1" | 3
|-
| colspan="1" rowspan="1" | Pseudo-UTP (from Trilink)
| colspan="1" rowspan="1" | 100
| colspan="1" rowspan="1" | 7.5
| colspan="1" rowspan="1" | 3
|-
| colspan="1" rowspan="1" | 3´-O-Me-m<sup>7</sup>G(5´)ppp(5´)G RNA cap structure analog
| colspan="1" rowspan="1" | 10
| colspan="1" rowspan="1" | 2.5
| colspan="1" rowspan="1" | 10
|-
| colspan="1" rowspan="1" | Total volume
| colspan="1" rowspan="1" | 23
|}

'''IVT Reaction Mix'''

{|
|-
| colspan="1" rowspan="1" | '''Component'''
| colspan="1" rowspan="1" | '''Final concentration'''
| colspan="1" rowspan="1" | '''Amount (µl)'''
|-
| colspan="1" rowspan="1" | Nuclease-free water
| colspan="1" rowspan="1" | Variable
|-
| colspan="1" rowspan="1" | <s>RNase Inhibitor</s>
| colspan="1" rowspan="1" | <s>40 U</s>
| colspan="1" rowspan="1" | <s>1</s>
|-
| colspan="1" rowspan="1" | NTP/cap analog mixture (above)
| colspan="1" rowspan="1" | 23
|-
| colspan="1" rowspan="1" | PCR product
| colspan="1" rowspan="1" | 1 µg
| colspan="1" rowspan="1" | Variable
|-
| colspan="1" rowspan="1" | 10x reaction buffer
| colspan="1" rowspan="1" | 1x
| colspan="1" rowspan="1" | 2
|-
| colspan="1" rowspan="1" | 10x T7 RNA polymerase enzyme mix
| colspan="1" rowspan="1" | 1x
| colspan="1" rowspan="1" | 2.5-4
|-
| colspan="1" rowspan="1" | Total volume
| colspan="1" rowspan="1" | 40
|}





{|
|-
| width="33%" | Nuclease-free water
| width="33%" | to 20 µl
| width="33%" | 
|-
| 2X ARCA/NTP Mix
| 10 µl
| 1 mM GTP, 4 mM ARCA,<br/>
1.25 mM CTP, 1.25 mM<br/>UTP, >1.25 mM ATP final

|-
| 5mCTP, 10 mM
| 2.5 µl
| 1.25 mM 5mCTP final
|-
| Pseudo-UTP, 10 mM
| 2.5 µl
| 1.25 mM Pseudo-UTP final
|-
| Template DNA
| X µl
| 1 μg
|-
| T7 RNA Polymerase Mix
| 2 µl
| 
|-
| Total reaction volume
| 20 µl
| 
|}

#Follow standard mRNA synthesis protocol.