Which dyes are available and are there specific dyes that are most compatible with active translation? Is there one specific nucleotide that can accommodate the dye better than others with respect to translation?

Bulky dyes may block the ribosome during translation and we have seen lower translational efficiencies with more substitutions. We recommend not to fully substitute, but only substitute 10-50%. For our catalog products, we use a 25% substitution of the dye-labeled nucleotide. Please contact us for any additional information.

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.