Method for preparing correctly oriented cap-structured mRNA using ARCA[1]
This protocol is for reference only. Please adjust according to actual needs.
(1) Reagent preparation: dissolve ARCA in water or appropriate buffer to prepare a 0.5mM stock solution. Prepare an NTP mixture containing 0.5mM ATP, 0.5mM CTP, 0.5mM UTP and 0.1mM GTP (ARCA to GTP molar ratio 5:1). Prepare SP6 or T7 RNA polymerase, DNA template, RNase inhibitor and transcription buffer.
(2) In vitro transcription reaction system construction: add 600ng DNA template, 1U/μL RNase inhibitor, 0.5mM ATP/CTP/UTP, 0.1mM GTP and 0.5mM ARCA to the transcription buffer. Add SP6 or T7 RNA polymerase.
(3) mRNA synthesis: incubate at 37°C for 1 hour. Add 2μL DNase and incubate at 37°C for 20 minutes to remove template DNA. Purify the RNA product to obtain ARCA cap-structured mRNA with correct orientation.
(4) Translation verification of ARCA-capped mRNA: transfect ARCA-capped mRNA into HEK293 cells (DMEM+10%FBS; 37°C; 5%CO2), or add rabbit reticulocyte lysate and incubate at 30°C for 60 minutes. Evaluate translation efficiency by luciferase luminescence values. Detect remaining mRNA proportion in HEK293 cells by RT-qPCR to evaluate stability.
Precautions:
(1) To prevent RNA degradation, use RNase-free consumables throughout the experiment and perform operations on ice.
(2) For your safety and health, wear laboratory coats and disposable gloves during operation.
References:
[1] Kocmik I, Piecyk K, Rudzinska M, et al. Modified ARCA analogs providing enhanced translational properties of capped mRNAs. Cell Cycle. 2018;17(13):1624-1636.
