 What I would like to do today is, too, because I think that we are speaking mostly for a biological audience, to talk about the principle of orthogonal nucleic acids. What do we mean with that from the point of view of a chemist? And to try to explain how we analyze things and also how difficult it is to design really something orthogonal that will work in a vivo situation. Because what we would like to hand at the end is really an orthogonal plasmid, or a complete genome, or a complete chromosome that's completely different as an information system. That is an artificial information system. And the question is, how do we look at that from a chemical point of view? I do these presentations as well from our group in the ESSB, because we were closing together to reach that goal. Now, why we do that? I think to summarize what we think about synthetic biology, because most things you get, the first question, you do why you do these things, what are the applications? Now, in this whole game of designing new genomes, which we like to introduce in microorganisms, is that what we do metabolically? And then I don't speak only about the genetic system, where we like to change information, but we also like to change metabolism. We also like to change catalytic function. And the application is in therapeutics, energy, food, and environment from a four-level of microorganism. Of course, when you do something here, when you look after new genetic systems, for example, or new metabolism, this can be also implemented in vivo directly on therapeutics. And you saw one example of Shiro, who looked for a new information system and applied it directly in synthetic optomers, which is an in vitro system. So synthetic biology might also deliver you new drugs without going to evolved microorganisms, which is looking from another point of view. Now, how do we look as a chemistry to the field? Most of the people look from synthetic biology, from the kind of view of biologists. We use natural biology and re-engineer this biology. So what I do, they re-engineer biologicals and they ask themselves questions about circuit designs, bioinformatics systems, biology, standardization, whatever. Now, if you do chemistry, you look from a chemical point of view on it. We call that, indeed, which is already introduced, xenobiology, but we look after synthetic chemicals, evolution or enzymology, cellular implementation. So I think that the xenobiology is, in fact, a chemical approach to the biologist and not to re-engineer natural biological systems. So we look, in fact, at this branch of synthetic biology from a chemical point of view. So this is how it is defined. And Floyd already gave an example of new base pair systems. But we would like construct cells that would store genetic information in alternative informational polymers consisting of XNA, different base pairs, non-canonical amino acids. You've also heard examples today. Alternate genetic code systems, which is generally called by Schmidt as a genetic firewall. Now, we use for that two abbreviations, which were introduced. One abbreviation is this XNA, xenonical acids. The other abbreviation is this. But the difference I will talk about both today. XNA are completely orthogonal systems. This is not really orthogonal in start, but could become orthogonal after the vivo evolution process. So they are two different ways of looking at the systems. So let's first talk about an orthogonal information system, which is an information system that are synthetically and functionally isolated from the cell and do not code potential with the natural nucleic acid. So they do not interfere with the natural nucleic acid. They don't recognize that they have its own genetic system, which do not correspond, which do not talk to the natural system. So it's really a synthetic nucleic acid. It's really a synthetic information system. We do not recognize the natural system. Now, the first thing to think about that, orthogonality, is that you could think orthogonality in a biological sense or in a chemical sense. If you think about biological orthogonality, this means that your molecule is not recognized in the natural system. As a biological entity, it does not show up toxicity, it's not conferred to triphosphates. It's a completely entity, which is completely strange to a metabolic, to any kind of metabolism in the cell. When you talk about chemical and always most of, I will talk about the chemical orthogonality, is that once you use this monomers to construct alternative nucleic acids, alternative genetic systems, that they do not hybridize with DNA and RNA, that they do not hybridize at all, that they do recognize, they don't form duplexes at all. They may form duplexes in itself or whatever structure, but not with DNA and RNA. An example is these 2-prime fluoride nucleic acids, which is neither biological nor chemical orthogonal. Exit on the nucleic acids is chemical orthogonal, not orthogonal, but it's biological orthogonal. It's quite inert if you use it in a biological system. You will not see an effect on the monomeric level. They are not phosphorylated. But I pretty appreciate this minus, because we only test it in a human system, not in a bacterial system, so they might still interfere with the metabolic process. But you are chemical, not inert, if you make it, only going to clotide from that. They will recognize in the cell RNA, and they will block RNA functions. So once they will polymerize into the cell, they will block cellular functions. And then it's not an orthogonal system, because it will interfere in the biological system, not at the level of the metabolism, but at the level of the nucleic acids itself. This is silent nucleic acids, and I explain to you why. These are so biologically chemically inert, which makes, of course, evolution a very difficult problem. The second question is, if we go for orthogonal information systems, do we need one or do we need two? Which means the natural system works with DNA with RNA. If we go for Nixon's system, an orthogonal information system, do we also need a couple? When I talk about the sugar now, I only talk about sugar modifications. Do we all need the deoxy-analog and the ribo-analog to have a DNA, as well as non-naming, or can we do it with one? It's a question. Now, we look structurally at this pair, and compare this pair and this pair, to try to explain why this is chemical orthogonal to a natural system. So the difference is not big, so one carbon atom will change from conformation, they are epimeres. That's the only difference. Now, if you speak about chemical orthogonality, then you go to analyze its structure. What happens if you make an information system from these chemical modifications? We go back to the natural system, because we look after also pairings, so the deoxy-analog and ribo-analog. This is the classical Watson-Trick systems. You know, the A-type, nucleic acids, helical structures, and the B-type, which come back to the structure of the sugar. The sugar conformation determines if you have a B-type and an A-type. A is not flexible, it's RNA only take one structures, DNA can take A and B-type, mostly it's presented as a B-type, because it's a preferential conformation of the sugar. There is a sugar conformation that determines your orthogonality, which means that it determines your structure of the DNA and RNA, it's not a base. So, which means that to go to a complete orthogonality, it's easier to work on sugar modifications than base modifications. Well, let's look in reality, if we keep this in mind, at this helical DNA structures, what happens if we go for deoxy-nucleic acid structure? This is deoxy-xylone nucleic acid structure. You see this structure with determined binomore, and they're not at all helical anymore. This is inside in the mining groove, this is inside the mining groove, and they have just a very, very, very small term, and they still have Watson-Cake base pair systems. So, you move in fact to a nucleic acid structure, which is completely different from DNA. They do not hybridize DNA anymore, it's an orthogonal structure. If you go from the ribo, I mean the xylone nucleic acid, so with the two-prime hydroxy, the RNA mimic, and we took also in a more structure of that, you see the same in the mining groove, in the major groove, a little bit twist, but not much. And you see also that this structure is stabilized by interstrand stacking. The stability of a duplex of nucleic acid duplex is determined primarily by interstrand stackings. And this interstrand stacking, because you see that the base of one strand is stacking above the base of the other strand, and that forms a very stable structure. This is of course due to this structure here, because it's not helical structure anymore, it's quite a straight structure. It's different from DNA from RNA. Now, the easiest way to analyze nucleic acid structures is not go to all the synthesis, but not going to an amorphic structure. It's simple to do a CD experiment. Now, if you take this deoxyxilium nucleic acid, and you take a CD experiment in water, you see this structure is a temperature dependent. Of course, it's a default when you increase the temperature and your curve is going down. But this is in water. If you put zero point or 100 micromolar sodium chloride on it, you see you have a completely other structure. It's go from that structure to that structure. Which means that the deoxyxilium nucleic acid is quite a flexible structure also. Also in DNA, DNA as a helical structure can take BNA type, which is the same here, but it's much more weird. If you analyze the structure here, you see that for the deoxyxilium nucleic acid, they can take this kind of structure. This is DNA structure, the Watson-Take structure. These are all the different conformations you could have as a conformational cycle. And if you go to a straight forward, so this is a structure we have with xilium nucleic acid, but the deoxyxilium nucleic acid can also move in that direction and give a completely left handed dependent on the circumstances. But that's the orthogonal. You see the overlap and how easy it is to analyze it by circular dichromism. You have deoxyxilium nucleic acid, you have xilium nucleic acid, and here you have ARNA and ZDNA and BDNA. See, it's completely chemical orthogonal. They don't recognize DNA anymore. They are a system on itself, comparable with the DNA RNA structure. See that also on the helical parameters, for those who are interested in helical parameters, the helical parameters give you the structures. Twist inclination is how forward is inclined to right or left. See here, minus five, minus 52. The most easy parameter to have the right inclination is the twist value here. Twist value give you how many bases you have in one turn. If you have a turn of DNA, 360 degrees. If you have for every base 10 degrees or 36 degrees, we have 10 base pairs in one helical turn. So 36 is what you see here, and if you go to Skylo, you have here values of 2.7 and 10. If you have only 2.7 degrees, which means that in one turn you have 60 base pairs. No, you have 90 base pairs for one turn. So it's completely different structures. And that's what we mean with chemical orthogonality, and that's what in fact we are looking for when we want to have orthogonal information systems. You could say yes, but okay, you put a burden on the generation of the polymerases that want to polymerize the nucleic acids, which is quite difficult. We had a collaboration also with Andreas Marx, who looks about the oxyxyl in the nucleic acids and several polymerases, and he could show that this oxyxyl adenine, the oxyxyl timidine is not much, but you already could incorporate a couple of this very weird, by his polymerases, his mutant polymerases, which he has a library from. He could always show that you have two bases which are already introduced. And if you have two bases, you have a start to do an individual evolution system to generate the polymerases that go to this, that recognizes the oxyxyl on the nucleic acids. So to summarize this first third of my talk, that I would say that small changes in the constitution of DNA and RNA, just an immunization reaction, has a large effect on this 3D structure and can easily lead chemical to a new orthogonal information system. Then the second part, which I would talk is a little bit about base pairs. But not about hydrophobic bases, which have been covered, and we never worked on that, but a little bit about the old systems which have been used and first introduced a long time ago by Alexander Richen, which have been tested of a student very intensively by Stephen Benner. So this is the ISOC, ISOG base pair. Now normally, the G base pairs have brought on donors and acceptors and acceptors, and this is the opposite and acceptor donor system. If you go to the ISOC, ISO metal base pair, you change this position from a donor to an acceptor and you also change this position. So it looks like an orthogonal systems, but he already showed a long time ago that the problem is that this ISOG system also recovers nice T, because you can have different tautomeric forms. You see here a ketone function, you see in the null functions. And this of course complicated the field which is already mentioned by Stephen Benner a long time ago, where he put out when he do PCR, this base pair systems show only 96% fidelity in amplifications which leads to indirect loss of information during amplification. So it's not a good base pair system to introduce in vivo system. Now the question is, is the base pair system not good or is the context in which we use the base pair systems? Not good. So the other questions now, if you combine that, if you use now a classical base pair system, it's not so good, but we change according to sugar, because in an ideal situation, we will not only have in an orthogonal system and modified backbone, but we would like also to have modified sugar, modified base pair systems on the modified backbone and what happens when you start to combine them? So here you see the ISOC picked it again and the spare is the ISOC material and here we put a six-membered ring which we call the HNA, which is in fact on itself, so both as not a base, as a sugar or really completely orthogonal. Now you see already that by doing a simple experiment, the situation already changes. This is the ISOC base pair in an oligonucleotide and we compare just the problem which you might get in a vivo situation is that your base system is also recognized by ATGC and that you lose your information, yeah? So we looked about melting temperature of the ISOC, exit all D and exit all ISOC, so there's all the different backbones against ATCG. What we expected, what is already shown before, is that the ISOC also form mismatches with T, it also pairs with T, less with the other ones. But if you go to an exit all system, it goes to G. So you have a change just by taking a TM or selectivity if you change the backbone. Of course it also forms base pairs a little bit with A and T, but it's moved in fact from T to base pairing with G and with C. So by introducing sugar modifications, you can influence the base pairing. If you go by the ISOC, it's recognized by G as a mispair, but if you change it to exit all, it's also just recognized G. Now, the next step was of course to see what happens in vivo. And if that correlates with what we see in vitro, because in vitro taking a TM is an easy experiment to see how it behaves in a bacterial environment is a little bit more complicated. This is a test which has been used as a test which has been developed already 20 years ago, by Sylvie Posse and Philippe Marlier, which is now used by Valerie Pezzo in the group in ESB and E3. So to put it in simple, in the active side of timid late synthetase, the artificial, this is the green one, is a symbol of the artificial sequence. Fine, not the artificial sequence, the artificial nucleic acids which we introduce here and this is then transformed and then she looks if they are timid late synthetase positive transformants on a medium without time. So that means when there is growing, you see that the information is read. So this is the same base pair in vivo, you see that the oxy iso G is in vivo, in fact behaving as an A, which recognized by T and in a second place also by gene vivo. The difference is not big. But if you go to Higgs et al, Iso C, it's in vivo, still recognized by A, although it was more in G in vitro experiment, a little bit in vivo, but you see that the quantity is going much more down. So you see that if you combine a modified base and modified sugar, it's almost not recognized anymore. So the intensity in which is recognized is very much reduced. You see the same with Iso C, Iso C is recognized in vivo as a C, but also a little bit as a T. There's not much more difference. But if you put a Higgs et al on it, you see that the number is going down. You have average ratio, you have not much clones anymore. And if you put two in a row, you don't have information anymore. So in fact, if you only put two Higgs et al with an Iso C metal, it's not recognized anymore in the natural environment, it's not recognized by the bacteria anymore. So a combination of, if you go to different base pairs and different backbone, the combination of them might in fact easily lead to orthogonality. So a combination of modified base and modified sugars made it easier to orthogonality than you use single modifications, only a sugar or only a base pair. Now we extended this study to some other base pair systems. This is CG and this is Iso C, Iso G. Now a base pair which has been described by Frank Zehler, at least in vitro experiments, was Iso C metal 8AZG. So we asked the question, could we turn this base pair also to something useful for us? And could we generate here a base pair which where the sugar is connected to the 8 position? And the other one is a normal one, which we could recognize each other by Hoogstein base pairing. The question is, is Hoogstein base pairing also possible in an in vivo situation and do we have to account on that to have to think of that problem and generating new artificial systems? So you see that this is a simple model where 8 amino hypoxantin. Of course we face here a lot of problems not to understand what we do and that's why we made also some other analogs. This is an 8 position. This is the 8 amino deoxynosin. But it could be theoretically recognized from this side in vivo by a C. That's why we put here a metal, the one metal over here, which avoid recognition in the Watson-Krieg base precision and also only in the Hoogstein systems. You have a DDA donor acceptor with acceptor-acceptor-acceptor donor. We also made the 8-oxo analog. We turned it to an acceptor donor acceptor which should not form base pairs anymore. So what will happen in vivo? Does it recognize them in vitro? And these are also the 8-oxo-diamino, which is the opposite from this one. So this should not be recognized at all. So here you see some of the melting studies. This is the ISOC, it is a DG base pair. With the 8-amino, you have 45 degrees, a little bit down. But you see if you put an end-metallic, it's the same. If you put 8-oxo, it's the same. 8-oxo-diamino is the same. So they also form base pairs in vitro situation just by taking the TM. 8-oxo-diamino, that's two, and it's not recognized at all. If you look after the recognition against ATG, the N8AZ is also recognized as Gs for the question is it's also so in vivo. What do we see in vivo? The 8-amino is recognized by C, the 8-metallic 8-amino, it's recognized by G even if you block the Watson-Trick port. 8-oxo-di is recognized by A, in vitro at least, and those is recognized by C and T. So this means if you see this data and you see the previous data, that the problem is in fact, and that was Floyd was mentioned, that when you start to do it from the rational point of view, that the orthogonal base pair design is not always a logical design because you have to take care that you can have also wobble base pairs. You have to take care that also the Watson-Trick system is not only the Watson-Trick system, but the Hoogsen systems is recognized. So these are recognizing each other in a wobble base pair system and also these can be recognized each other at least in vitro in a wobble base pair system. So when we look at that in vivo situation, this is the test which is done, so we look after the incorporation and look if GC or T or A is incorporated and then the next sequence. You see that in fact, in vitro hybridizations to this, not correspond always with an in vivo recognition system. And A is at the G, in vitro behaves as a C, but in vivo behaves as an A. And until 8-minutes the I in vitro behaves as a C, but in vivo behaves as a G. Hexatol, iso-G in vitro behaves as a CG, but in vivo it behaves as an A. That's what is recognized by the biological system. Which makes of course in design just based on in vitro experiments just the M-studies very difficult. And even if you look after incorporations to this by polymerase it's even worse. Because the data you generate it just depends on the polymerase you use. Sometimes it recognizes A and sometimes G depending just on the polymerase you select. And it could be indeed at Hoagstein base pairing in vivo is probably possible because 8-oxo-DI is recognized by DI as well in vitro as in vivo situation. So we analyze all the systems. We don't have an ideal base pair at least when these all don't be DNA. At least when we look after this base pair systems with a DNA backbone because the N-metal 8-amino is still somewhat recognized by C and the HA is at G is still somewhat recognized by C in vivo situation. This does not mean if you would put another sugar on that that you come in D to an orthogonal information system. DNA is not a sugar and a base and a backbone it's all together. And when you look after new information systems you have to analyze the whole system and not partially the system. Now the last part of my talk is searching for a new set of four nuclear bases that can be used for faithfully and both information in vivo. This also has to do with when we change the four bases in DNA can that be changed by other four bases in DNA and still be recognized in a biological system? I don't talk about replication I just talk about getting recognized and be converted in DNA getting read by the natural system. Just the first step. Why we do that because this is a system which could easily lead to orthogonality if you go the next step to in vivo evolution. You can start by small modification of the natural system and come to a orthogonality by evolution or by small chemical changes. Now if you go back to the literature some people have analyzed that if you see this paper in 1957 these people have studied the effect of five halogenated uracin on the growth of Escherichia coli and their bacterophage and they saw that in the bacteria in the virus there is replacing of timidine residues and they also talk about quantitatively about this five-holigonated stuff and they use indeed timidine oxotrophic but in this case by using sulfonylamides. But it means that these five halogenated is in fact a system which maybe can also be used by a natural system. But this is a test which has been long, long time ago but for another purpose of course for looking at the toxicity. In another paper in 1956 they use Bacillus Sirius and 8ase agonin and they see that's for 40% incorporated in ribonucleic acid but only less than 1% in deoxy nucleic acid in DNA. Which gives already an idea that this might be not a very good system not very good base in the DNA context but maybe in the RNA context. So we did a lot of modifications. Five chloro, U, 70ase A, 8ase, 8ase, 70ase. Some of them were here. We replaced metal C and 5luro C and 70ase G and 8ase G and we also question which other four set of nucleobases that can be used in vivo situation and transformed to DNA. Now the setup is in fact not so difficult. You synthesize modified bases. With PCR, you synthesize a gene. This is gene for dihalophilate reductase and then you transform that in vivo and you see if this gene can still be converted to DNA. It looks obvious but it's not like that because you have to PCR multiply which is with the thermophilic polymerases which is a completely other situation as in the vivo situation where you have mesophilic and other polymerases. It's not because you can do it in PCR. You can do PCR a lot because there are a lot of different and they accept a lot of modifications but most of the things you do in PCR by multiplying, modified nucleic acid for example for haptamer design they do not work in bacteria. It's not because you can make a gene in PCR that it also is recognized by the polymerases from the bacteria which are mesophilic polymerases and have another subsets specificity. You can maybe do that with small species but we looked at the whole gene. Now the first experiment which we do is a simple TM studies. There is always a simple physical chemical experiment you can do. We had a sequence and we look always at the coding system. The information unit for a chemist is one nucleotide. The information unit for a biologist is a coding system. So we always have to look after three. So we put here three modified. This is A. When you have three As and three Ts you have TM is 48.5 degrees. You see here if you start to modify with other bases, the T is still okay, 48.2 degrees but if you go from other pyrimidine bases you get from two U-chloroen until U you see that the stability is going down also certainly with the other base modifications. But the only combination that stands even in a simple TM experiment is the two experiments are done with Chloro-Russell, 7-DESA-A and N8, 7-DESA-A. They are the same TM as an AT. So there is something in this kind of molecules in that Chloro which makes in fact the thermodynamic stability the same as a natural base pair. If you look at the same base pair it's the same. You have G, 7-DESA, N8, and Asa, C-fluorose, CF-metal temperature 47.1 degrees. It goes up here with metal C. But if you go to the fluorose C, 7-DESA-G combination, it's about the same TM as you see at least in one codon system of the GC. The other are up, some are down but most are different. So only with this combination you have in fact the same thermo-stability as a codon in a codon system as a natural system. This is the 57-Mayer template PCR with vent polymerases and you see here that the combination of A and T, 7-DESA-chloro-U is similar. 8-ASA-5-chloro-U is less well done. So this combination seems not only in the TM, but also when you use it these of course together with G and C quite well accepted in a 57-Mayer template as a modified basis. So if you decide about 7-DESA-5-chloro-U as one base pair replacing A and T you can do the combinations of the changes of G and C. You see these are the number of cycles it goes to 40 PCR cycles and you see here that if you take this combination still give good results also this combination give good results also this combination give good results but this is in a short template. This is the combination of the four bases but on a 525 base pair this is already allowed G. So does it work PCR amplifications to make a 525 base pair system, umplical system? You see here that and these are the different combinations on the different bases but it's always here 7-DESA-A that looks good this is with 5-chloro-U still okay. This is by Ventes, by TAC a combination with 7-DESA-G 8-ASA-G, 8-ASA does not work anymore it works when you make a 60 mer but once you go to a 500 mer it's not useful anymore so it's not because you can incorporate 20-30 you have a good genomic system it fails when you go to 500 but it stands still for these combinations of 7-DESA-A5-chloro-U and if you see also that the gene you make is fully stable against restriction and on nucleases while normal DNA in AGCT DNA is hydrolyzed by restriction of nucleases so the next step was indeed to analyze the PCR product from the dihydrofolate reductase gene which is resistance to 3-metroprim you see here the AGCT and this is the gene because there is also still AG and CNT because we use it in the primers now we don't use it anymore but these are the first experiments so you could show that the gene is okay and then it is brought in a vector which is then transformed and you look for if this this whole dihydrofolate reductase gene is recognized and you see here the number of clones we are not going to into the details these are unmodified DNA these are with 5-chloro-U system and even if you combine that with 7-DASIA it is still marvelous and this is of course the fully modified you don't have so many clones anymore but it still is completely still converted in vivo in DNA enough to survive and to have several clones I will not go into these are the very few mutations you see there this gives most of the time even the same amino acids and this is the growth of the fully modified versus the natural and then versus some negative references which means that in vivo this is the for nuclear-based cassette which is used in the if there is a mistake here it should be not a double bond the AGCT is used by nature Steven Bender always called it God's mistake so it can be at least in DNA to multiply by another DNA we didn't do replication for the moment it changes these four bases which could be another mistake but at least you can have a gene which is with four different bases they are all four changed in the natural system they are still recognized by the natural system and I have a lot of couple of slides because of this molecule which has been used by Philip Malier to evolve to a completely orthogonal biological system in vivo we did some physical chemical studies in that how remarkable it is if you look after the TM again after one codon system and this is what I show you you have the 49.6 degrees which is similar to the metal analog but it's quite remarkable because if you look after the PK of this molecule the PK of time is 9.7 the PK of uracil is 9.3 that's why nature used uracil in RNA and time in DNA it's because of the difference in PK if you look after chloralute 7.9 so it should be partially ionized in vivo because then we work at the PK of 9 if you look after the delta PK value which is the PK between the pyrimine and pyrimine bases it should be as large as possible to have a stable hydrogen base pair so in fact based on this delta PK value which is the difference between the PK of an acid and a base and the largest is different in PK is a stronger hydrogen bonding it's very low so if you do a simple physical chemical thinking it would never work despite of that it worked so we do together with Martin Aigli some x-ray structures to see how the chloralute is recognized by an adenine and here it's recognized by a guanosine which is by wobble based pairing so at least an x-ray structure and you see also mistakes which is made in vivo between A between chloralute and G but if you look after the same principle and the principle was already mentioned by an introduced by Eric Hall and mentioned by Floyd here it has a practical perfect shape of the entire T between an A T base pair and a chloralute base pair if you overlap that you don't see many differences and you also see that in the wobble base pair system this chlorine is hysterically in fact the perfect shaping meep of a metallic group and that's why it's so interesting to do all this in vivo work because chloralute is always substrate in present biochemistry it's not found in nature the carbon-chlorine bond is alien to biochemistry it gives a stable genome it gives some biggest base pairing for the evolution and it respect the principle of safe recognition in biology and we hope of course that with this no four base pair systems we can continue doing this kind of in vivo evolution process to become to an artificial not only an after field chromosome but I think also that this might be the way to an x-ray system because at least it's recognized by the nature system and by substituting them and evolving polymerases you can easily come to an x-ray system out of a d-z-ray system which you still can sequence because you still have the Watson-Krieg port to sequence which is always the problem in natural base pairs you don't know what you have because you cannot sequence them so it could be that in this way we come easily to orthogonality than going to more advanced technologies I would like to thank these people we do that together with Philippe this whole thinking and setting up the experiments for the in vivo work has been done by Valery Pezzo and Aaron Braddock in every synthesis done in Leuven by Omprekash Bande, Mikhailov Bramov and his shapers the whole structure biology work is done in our laboratory by a PhD student Moetosh Maitey and the whole work with d-z-ray is also done by a PhD student which is called Reina Eremaev and I hope I had convinced you a little bit more about how we think about orthogonality and how we think based on chemical orthogonality I can reach sooner or later biological orthogonal systems in vivo and I thank you for your attention