Hello, welcome to the Sativa webinar. We want to talk today about our work observing the viral RNA polymerase complex of SAS Coronavirus at work using the Amersham branta typhoon via biomolecular imager. We are we work at the laboratory, our sheet actually function, the macromolecule logic in my safe hands. This laboratory belongs to the CNRS into X Massey University. And we are working in the group of via replicators. We are actually Shannon, a postdoc in our lab and berbahasa Disco. I'm a research associate. And the work that we are talking about was published recently in Nature Communications. So if you need more details, you're very welcome to Reno publication.
Okay, so this is the protein complex that we've been interested in our lab and more specifically, it's the SARS Coronavirus, RNA polymerase complex. And this is formed by three proteins and SP 12, and SP seven and NSPA. And so the major player here is in SP 12, which contains a viral RNA dependent RNA polymerase. And so here's just a depiction of the structure here. And the polymerase is shown in three colors here. But in order for it to be active, it requires interaction with two viral cofactors. And these are NSP seven and NSPA. And so NSP seven is shown here in blue and NSPA, is shown here in green. And so together, these three proteins make up the minimal functional RNA polymerase complex, which is required for viral RNA synthesis.
And so to look at the activity of this complex, we often use these annealed primer template RNA pairs. So here we have a 20 mer or a 20 nucleotide, template RNA, and we anneal this with a primer RNA, which importantly, has a sci fi fluorescent dye at its five prime end. And so then we find the complex by NSP 12, in SP seven and NSPA. And so here, we're using a covalently linked version of NSP seven and eight, which we refer to as NSP seven link date, and we add supplementary NSPA into the reaction as well. And then we incubate this complex with this primary template RNA pair. And we start the reaction via the addition of NTPs.
And so here in this reaction, we've just added three out of four of the NTPs. And so the polymerase complex binds here in this double stranded RNA region, and it starts synthesis in a complementary fashion against this template RNA. And so it stalls or stops here with this templating G, because there's no CTP in the reaction. And so we stop this reaction at different time points in the EDTA. Mix. And then we analyze the samples through these denaturing page urea sequencing gels.
So this separates the different sizes of RNA through the gel, and then we scan this gel through the Amersham tyffyn imager, looking for the fluorescence of this cipher dye. So here's just an example of one of the gels or what we'd expect. And down the bottom here we see the P 10. RNA. This is just off the time zero. And then as the polymerase complex reacts with this as a time post reaction here, we see that this grows into this plus seven product here. And so we use this system to optimize firstly, the polymerase complex. And so what we had here was different ratios of NSP 12, seven, linked eight and eight to get the optimal ratio. So this is the ratios that we use shown here. And again, this is just a time series with the primer, the P 10. Primer here, and then we see extension over time up into this full length product here. And so importantly, in these reactions, we use one micromolar of the protein complex and point to micro molar of this sci fi p 1020. RNA pair here. And then what we actually run in each individual Lane only contains point one, seven, pick a mole of this sci fi die.
So the Amersham typhoon imager is very sensitive, it's able to detect even these very low concentrations of this primer. And so we then analyze these product bands using the image quant software, which comes with the with the typhoon imager. And so we can analyze each of these bands individually to get the optimal rates. And so what this experiment showed with this optimal condition of this complex is a one to three to three ratio. And so similarly, this is another experiment we did to look at the actual speed of the complex.
So this was done with pre steady state, rapid quench experiments. And so here we stopped the reaction at very short time points. So the lowest time point here is 10 milli seconds and we got to one second. And so what we see here is very clearly the gradual extension from the primer into the plus one product the plus two plus three up to this plus seven product too. And so what we can analyze from this with the image quant software is the production of each of these bands individually. So here we see the plus one product which was produced, and then it's consumed into the plus two. And so then we see the extension up into this. And so that's what's depicted here. So as an example, in green, here, we have the plus one product which is rapidly produced and then consumed into the next product, which is shown here in blue.
And so we can also analyze an average rate for this by looking just at the consumption of the of the primer here, into the full length product. So that's what's shown here with the primer shown in red, which is consumed, and then the full length being produced here. And so what we get when we analyze this at multiple NTP concentrations is an average rate of polymerization. And so what we calculated here was a rate of 90 nucleotides per second at 25 degrees Celsius. So just to put this in perspective, when we compare this with other polymerases, like for example, Dengue or polio, these elongate at around five to 20 nucleotides per second. So depending on the RNA template that we gave the polymerase complex, we found that it was up to five to one up to five times faster than these other polymerase complexes. So that makes this complex the fastest polymerase or viral polymerase complex known so far.
Yes, and then the following series of experiments, we wanted to see if this pole very fast polymerase complex is able to incorporate nucleotide analogues that then can be used as antiviral drugs. For this first serious we used to nucleotide analogs t 705. And t 1105. ribose triphosphates. And T 705 comes from protrack, which was approved for influenza virus, it's a broad spectrum antiviral which is called Favi PR view. And then in the cell, this base is a ribose is added in the triphosphate is added to this space and this is the nucleotide analogue that we use in our assays. These molecules are considered as a GTP analog mainly, and this comes from specially from this carboxy I made group which is connected to two pyrazine rain.
And so in this series of experiments, we wanted to see if they are incorporated instead of GTP. And we tested also ATP. For this purpose, we use the again, the primer template combination, which is specific to SAS with Psy five fluorescent primer, and we use, we use the nucleotides that are able to have to form a finished product. And again, when we analyze the our reactions on the gel, we used only point 33 pico moles in each lane. You have two series of experiments and then one without GTP and one without ATP. On the left kenetic here you'll see what happens when you're when we didn't use nucleotide analogs.
In this case, the problem is stops after forming you a lot dunk. elongating the primer until this position, you can see that here. It's just before the G and when we give the analogs, it's the Primus really finished, which indicates that they are very efficiently incorporated here. Instead of GTP. Your if you compare 10 micromolar of each analog you'll see clearly that 1105 is incorporated more efficiently than 705. What happened when we didn't use ATP so here you have normally it should be a UAE so there is no a available and what the polymerase complex does is one missile cooperation of a G and then it's it stops. And here again, when we use the nucleotide analogs. It produces a finished product especially in the case of 1105 with a 705. Again, it has problems to come to a finished product, but this indicates that it's also incorporated instead of ATP here at this positions with the red point.
And clearly it's more efficiently as an HTTP analog, then, as an ATP analog we did, then a test in cooperation with a different kind of substrate in this case it was happen to happen is at the same time template, which is here, and then prime which stops here. And then if we give the four nucleotides, the primer will be elongated until the end. And this is a long finished hairpin. The difference to our primer template was also that we use another fluorophore. This time, it's six fam, that can be read then with a blue laser. And here on this gel, you'll see again, that for both analogs, they are incorporated in the hairpin substrate instead of GTP.
And again, 705 is less efficiently incorporated than 1105. In the next experiments, we use the these two substrate at the same time, so you can use the one labeled with six fam and another labeled with sy five, and then you do just two scans. So one with a red laser, and one with a blue laser. And you can see both substrate elongations at the same time. And what we did use here, it's, as I said, they happen and then the primer was a new 240 nucleotide template. So here you have the finished product and the 14 nucleotides. In this part, you'll see the elongation of the happen substrate so when we don't use use the, the analog about us or nucleotides, you'll see the finished product here and then when we use the analogs in absence of the ATP or of the absence of GTP, we see the finished product here the difference this difference in migration can be explained by the different nature of secondary structure that remains in this long double stranded substrates, we cannot denature them 100%
And the rest of secondary structure depends on the frequency and the nature of the analogues that are incorporated. Here on this part of the gel, you'll see that there is some complete stop of elongation when several nucleotides incorporated nucleotide analogues, sorry, incorporated at one after the other in a consecutive way. And so, in this case, we see to chain termination on the other hand, this is not very likely to happen to happen in vivo. And we also saw that the polymerase gets slower just here before the incorporation of the nucleotide this can be seen by the accumulation of the band just before the incorporation and it can also get slower. After the incorporation of the nucleotides, yes, you will see the accumulation of the band which contains the nucleotide analogue. So to summarize our observations and results, we could see that the sauce RNA polymerase complex is able to rapidly elongate RNA primer or hairpin substrates.
We saw that our nucleotide analogues, I incorporated instead of G and E, and that they mainly act by stalling polymerization and incorporation and their incorporation leads to the generation of mutants, and this was corroborated in infected cells that were treated with a pro drug for VPR via t 705. Console concerning the imager so we saw that the Amercian typhoon is really can visualize the RNA elongation of fluorescent primers or hairpin substrings in a very efficient way on this denaturing resolutive sequencing gels, and this is perfectly identical to the conditions that we used before for using radioactive labeled RNA substrates, if you want to compare directly by yourself, you can refer to our publication antiviral research where we present similar experiments done with a radioactively labeled substrate. Finally, I want to thank all the people involved in this work, especially the people that organize or contributed to the to buy the machine typhoon for all laboratory. That was if born our director in one Liga, and
Bruno Kona, Kana or team leaders that obtain the financing and then people from our group who contributed with experiments into writing of the article, We are Nick Francois noon, caffeine Etn, and a very important collaborator who was on sabbatical here from Colorado State University or Pearson. Then the chemists from the university handbook that synthesize the nucleotide analogues you are now walking and Chris Maya and the people from a pH N. Opito DiMasi. Who did the work with infectious virus and in cell lines. And we will also want to thank our support staff from sativa. We hope it was clear and you found it interesting to our work. If you have any questions, don't hesitate to contact us. We put our email addresses in the first slide. And thank you very much for your attention. Thank you for listening.
