What base modifications can be incorporated into my custom mRNA or long RNA?

TriLink offers a variety of modified rNTPs suitable for in vitro transcription. We have assessed transcription efficiency based on final product formation of a 1.9 kb transcript with the following triphosphates at 100% substitution using T7 polymerase.

NTP Transcription Efficiency
5-Aminoallyl-CTP (N-1065) +++
2-Amino-ATP (N-1001) +++
5-Br-UTP (N-1054) +
5-Carboxy-CTP (N-1084) ++
5-Carboxy-UTP (N-1091) n/a
5-Carboxymethylester-UTP (N-1096) ++
7-Deaza-ATP (N-1061) ++
5-Formyl-CTP (N-1085) +++
5-Formyl-UTP (N-1090) +
5-Hydroxy-CTP (N-1089) ++
5-Hydroxy-UTP (N-1092) ++
5-Hydroxymethyl-CTP (N-1087) ++
5-Hydroxymethyl-UTP (N-1086) +++
5-Iodo-UTP (N-1012) +
5-Methoxy-CTP (N-1094) ++
5-Methoxy-UTP (N-1093) ++
N6-Methyl-Amino-ATP (N-1083) ++
N6-Methyl-ATP (N-1013) +
5-Methyl-CTP (N-1014) +++
Pseudo-UTP (N-1019) +++
Thieno-CTP (N-1097) n/a
Thieno-GTP (N-1088) ++
1-Thio-ATP (N-8005) +
2-Thio-UTP (N-1032) +++

+++ = Greater than 75% efficiency compared to unmodified NTP
++ = Between 25-75% efficiency compared to unmodified NTP
+ = Less than 25% efficiency compared to unmodified NTP
n/a = No significant product formed with 100% substitution

In all cases, transcription was carried out at 37°C.

8 thoughts on “What base modifications can be incorporated into my custom mRNA or long RNA?

    • In our experience, N1-Methylpseudouridine-5′-Triphosphate has similar incorporation efficiency to Pseudouridine-5′-Triphosphate.

      +++ = Greater than 75% efficiency compared to unmodified NTP

    • Dear Pavan,
      TriLink has not done any experimentation to determine if E coli RNA polymerase would be able to incorporate these modified bases. T7 RNA polymerase is a phage polymerase and is thus likely to be more flexible in incorporating modified NTPs than E coli RNA polymerase.

      Best Regards,

  1. Hi,
    I’m interested in the 2’OMe modifeid nucleotides (N-1015-18) and also in 2’F and 1’Thio (N-1007 and N-8005).
    Have you tested if these modifications can be incorporated by transcription in vitro using T7 RNAP? if yes, in what efficiency?

    • Dear Moran,

      Thank you for your question. We see a modest amount of incorporation of 1-Thio-ATP (N-8005) using T7 polymerase (≤25%). However, preliminary studies have shown greater incorporation the other 1-Thio analogs (≥50%). For 2´F incorporation (N-1007), we recommend using T7 R&DNA™. Though we have not tested 2´ OMe analogs with wt T7 polymerase, this paper suggests that T7 polymerase with a Y639F/H784A double mutation is optimal for incorporation.

      Please let me know if you have further questions.


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