In vitro synthesis of RNA is catalyzed by bacteriophage (SP6, T7 or T3) RNA polymerases using linear DNA as a template. Ribonucleoside 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 guanosinemethylation (5‘-Cap), 3‘-end adenosine (poly(A)) tail or several internal base 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.
Kits for enzymatic RNA Synthesis and poly(A) tailing are available as well.
| Modified NTPs | Unmodified NTPs | Cap Analoga | Proteins |
|---|---|---|---|
| 5-Methyl-CTP (100 mM) | NTP bundle (4x 100 mM ATP, CTP, GTP, UTP) |
m7GP3G (100 mM) | T7 RNA Polymerase (HC) |
| N4-Acetyl-CTP (100 mM) | m7,3‘-OGP3G (ARCA) (100 mM) | T7 P&L RNA Polymerase (HC) | |
| Pseudo-UTP (100 mM) | ATP (100 mM) | m7,3‘-OGP3(2'OMe)ApG (100 mM) | T7 3M RNA Polymerase (HC) |
| N1-Methylpseudo-UTP (100 mM) | CTP (100 mM) | RNAse Inhibitor - recombinant | |
| 5-Methoxy-UTP (100 mM) | GTP (100 mM) | ||
| 2-Thio-UTP (100 mM) | UTP (100 mM) | ||
| N6-Methyl-ATP (100 mM) |
