In vitro synthesis of RNA is catalyzed by bacteriophage (SP6, T7 or T3) RNA polymerases using linear DNA as a template. Ribonucleotide triphosphates (NTPs) are the building blocks of these in vitro transcription reactions.
Some naturally occuring RNAs such as messenger RNAs (mRNAs) or long non-coding RNAs (lncRNAs) contain a variety of modifications e.g. 5‘-end guanosine methylation (5‘-Cap), 3‘-end adenosine (poly(A)) tail or several internal epigenetic modifications which markedly influence their biological function (e.g. translation efficiency, stability or immunogenicity).
These structural features can be introduced during in vitro transcription via correspondingly modified NTPs and cap analoga to obtain biologically functional synthetic RNAs.
|Modified NTPs||Unmodified NTPs||Cap Analoga|
|5-Methyl-CTP (100 mM)||NTP bundle |
(4x 100 mM ATP, CTP, GTP, UTP)
|m7GP3G (100 mM)|
|N4-Acetyl-CTP (100 mM)|
|Pseudo-UTP (100 mM)||ATP (100 mM)||m7,3‘-OGP3G (ARCA) (100 mM)|
|N1-Methylpseudo-UTP (100 mM)||CTP (100 mM)|
|5-Methoxy-UTP (100 mM)||GTP (100 mM)|
|2-Thio-UTP (100 mM)||UTP (100 mM)|
|N6-Methyl-ATP (100 mM)|