Dear all,

thanks for all your comments and Khalid for your article! Finally, I found some time to take a closer look at the topic and this is what I can tell you so far:

Most monomeric RNAPs (like T7 RNAP) as well as DNAPs are members of a structural superfamily resembling an "half open right hand" sharing strong similarities in active site architecture and reaction mechanism. Substrate (i.e. r/dNTP) discrimination for DNAPs is accomplished by a "steric gate" (i.e. a bulky amino acid residue that would clash with the 2'OH-group of incoming rNTPs; e.g. E615 in Taq-pol., E480 in T7DNAP or E710 in Klenow Fragment) while RNAPs positively select for the 2'OH via hydrogen bonding (e.g. with Y639 in T7RNAP).

Accordingly, T7RNAP Y639F readily incorporates dNTPs into RNA transcripts but shows significantly decreased activity in vitro when only the 4 dNTPs are present (Sousa et al 1995). Likewise in 1995, Kostyuk et al reported that S641 would act as a "substrate specificity switch" and exchanging it with Ala would turn the T7 RNAP into an efficient DNAP. However, this was not reproduced by Huang and coworkers as stated in their 1997 paper and also the Moscow group around Kochetkov did not mention these earlier results about the S641A single mutant in their later works. So, I guess, unfortunately this would have been too convenient for us to be true and there was some sort of DNA-pol impurity in the sample or whatsoever. Anyhow, S641 apparently has no influence on rNTP/dNTP discrimination.

In the following years considerable effort was made to generate polymerases that could produce nucleic acids containing non-canonical NTPs for uses in the clinic or as biosensors (e.g. as described in the paper Khalid provided).  These approaches, however, always aimed at engineering polymerases to tolerate bulkier substituents at the ribose's C2 position instead of tuning specificity towards dNTPs.

Perhaps one should try to use a directed evolution method to find appropriate enzyme variants or maybe it would be enough to combine the Y639F mutation with a deliberately introduced steric gate since T7RNAP only has a glycine at the respective position.

Please let me know your thoughts or if you know whether somebody ever tried to generate this double mutant.

If you are interested in this topic especially because you are fascinated by the notion of primerless DNA-synthesis then please read:

https://www.ncbi.nlm.nih.gov/pubmed/28265063

PS: You can find a list with all the relevant papers I could discover attached to this post.

Dear Julian,

You raised an interesting subject indeed. I have also checked the literature when I came across your question. I could not find any.

I guess the challenge will be engineering of RNA polymerase that can synthesize a nucleic acid (RNA or DNA) from only dNTPs with pre-defined sequences. How it could be possible without using primers and templates? It looks tough!

Sorry guys, I should have been more specific about the question.

In short: I wonder if somebody ever tried to engineer for example T7 RNA polymerase to accept dNTPs instead of rNTPs so that it "transcribes" DNA instead of RNA with the advantage that you could generate DNA from RNA without priming which is needed by all DNA polymerases (also reverse transcriptases).

There is some research about the underlying physicochemical principles of differentiation between dNTPs and rNTPs like https://www.ncbi.nlm.nih.gov/pubmed/27480935 and also there are reverse transcriptases that can use "protein priming" like those employed by hepadnaviruses. But this still is not what I am looking for.

I can not think of a really obvious reason why this should not work, though, but perhaps nobody ever tried.

Physically I think there is no reason RNA pol can't use dNTPs for synthesis.

The recognition of binding site (such as TATA sequence) requires sigma factor to begin synthesis.

So, if you can engineer RNA pol having sigma factor domain attached or separate addition of sigma factor in addition to RNA pol, it is interesting to see whether that's enough for RNA pol synthesizes DNA.

I agree with the previous comment that physically there is no reason why RNA polymerase could not use dNTPs for synthesis. However, would there be an issue with recognition when converting RNA to DNA, in that uracil replaces thymine? I would imagine so. 

Dear all,

thanks for all your comments and Khalid for your article! Finally, I found some time to take a closer look at the topic and this is what I can tell you so far:

Most monomeric RNAPs (like T7 RNAP) as well as DNAPs are members of a structural superfamily resembling an "half open right hand" sharing strong similarities in active site architecture and reaction mechanism. Substrate (i.e. r/dNTP) discrimination for DNAPs is accomplished by a "steric gate" (i.e. a bulky amino acid residue that would clash with the 2'OH-group of incoming rNTPs; e.g. E615 in Taq-pol., E480 in T7DNAP or E710 in Klenow Fragment) while RNAPs positively select for the 2'OH via hydrogen bonding (e.g. with Y639 in T7RNAP).

Accordingly, T7RNAP Y639F readily incorporates dNTPs into RNA transcripts but shows significantly decreased activity in vitro when only the 4 dNTPs are present (Sousa et al 1995). Likewise in 1995, Kostyuk et al reported that S641 would act as a "substrate specificity switch" and exchanging it with Ala would turn the T7 RNAP into an efficient DNAP. However, this was not reproduced by Huang and coworkers as stated in their 1997 paper and also the Moscow group around Kochetkov did not mention these earlier results about the S641A single mutant in their later works. So, I guess, unfortunately this would have been too convenient for us to be true and there was some sort of DNA-pol impurity in the sample or whatsoever. Anyhow, S641 apparently has no influence on rNTP/dNTP discrimination.

In the following years considerable effort was made to generate polymerases that could produce nucleic acids containing non-canonical NTPs for uses in the clinic or as biosensors (e.g. as described in the paper Khalid provided).  These approaches, however, always aimed at engineering polymerases to tolerate bulkier substituents at the ribose's C2 position instead of tuning specificity towards dNTPs.

Perhaps one should try to use a directed evolution method to find appropriate enzyme variants or maybe it would be enough to combine the Y639F mutation with a deliberately introduced steric gate since T7RNAP only has a glycine at the respective position.

Please let me know your thoughts or if you know whether somebody ever tried to generate this double mutant.

If you are interested in this topic especially because you are fascinated by the notion of primerless DNA-synthesis then please read:

https://www.ncbi.nlm.nih.gov/pubmed/28265063

PS: You can find a list with all the relevant papers I could discover attached to this post.