Hi
It is possible to use dUTP instead of UTP in a classic T7 RNA Pol in vitro transcription?
I want to make some more (mRNA) ISH probes, but the dye i want to use (for tau-STED) is not available for UTP, but dUTP (and they are cheap, which is sadly quite important). There are some older Paper about the topic, i did not find any "new" ones and nobody was using fluophore linked dNTPs.
thx in advance
https://academic.oup.com/nar/article/23/11/1845/2400478
dNTPs are not suitable substrates for T7 RNA polymerase. At their core nucleic acid polymerases use the same enzymatic structures for catalyzing the polymerization, but steric gating provides a strong selectivity for dNTPs or NTPs. Using the non-preferred substrate will result in a several thousand fold decrease in activity/catalytic efficiency (see Article The control of the discrimination between dNTP and rNTP in D...
).
I second that - modifications at the sugar moiety are generally not as well accepted as base-modifications. Fluorophore-linked NTPs usually don't really pose a problem for commercially available polymerases, dNTPs, however, are a different story.
However, if you're willing to put in some more work, check out this publication (it goes a bit further than the one you've cited): Article A Y639F/H784A T7 RNA polymerase double mutant displays super...
Certain mutations allow for incorporation of sugar-modified nucleotides. Hope this helps!
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