 Thank you very much for giving me this opportunity to talk about works. Today I will talk about one of the synthetic biology area by incorporating a natural component into DNA to expand the genetic alphabet. And also we talked about the application, so what we should do using this technology. So there are a lot of creatures living on Earth. Actually this is for a romance book. This is the next speaker and this is my pictures. So although these are very similar, but all creatures are very different from each other. About the same intellectual level. I think so. But these are all DNAs with the same chemical components of four base nucleotide, AGCT, as a genetic alphabet. This is very amazing because only four letters are ruling our lives. And my question is if we have more than four letters, so could we improve our functionality much more? So this is the simple question is also our objectives. So to do this we have to develop a third base pair. We call it a natural base pair. To this end, this natural base pair can function as a third base pair in replication, transcription and translation. And if we can make such a system, we can introduce new component into nucleic acid and proteins. Resulting in increasing their functionalities. So for example, this is a usual genetic code with four letters. But if you add two unnatural letters in this table, so you can increase the genetic code by 216. So you can add maybe 100 amino acids into proteins. So as for nucleic acid, nucleic acid has also versatile functionality. It's very amazing because they have only four very similar chemical compounds or bases. But the functionality might be less than proteins because protein has 20 standard amino acids. So this is nucleic acid with very four similar components. But if you add more different components into these tools, maybe you can make more functional nucleic acid. So this is our purpose. But until recently, such an experiment is just science fiction. This is my favorite picture of US TV X-Files. But this is very interesting because the researcher tried to examine the aliens DNA. And I don't know actually this is this aliens. But she said there is a gap on the sequencing band. So that is shows a equestrian basis in the DNA. This is just science fiction. But our sequencing method, including involving unnatural base pairs, is emerging from this science fiction data I will show you. So in our present, there are three unnatural base pairs of three groups or functions in PCR amplification or transcription. And one is Freud-Lobesberg of script. He is here and he's talking about this. So under our group and Steven Benner, there are three groups. So this time, I would like to focus our unnatural base pairs. So more than 18 years, we are struggling to create unnatural base pairs by improving step by step. And I will talk about the latest, our unnatural base pair, DSPX, that exhibit high fidelity and efficiency in PCR amplification. So this is the DSPX chemical structures. They have no hydrogen bonding interactions between pairing bases. And these shapes are different from those of natural bases. But we designed these unnatural base pairs by strictly refining the shape complementarity between parallel bases, faces. So the shape of DS, the DS base has a thiophane, a disposition. So the shape is very large for those of A or G. And in contrast, PX has a five-membered ring of the base part, two nitro pyloles. So T and C has a six-membered ring. So that's why the PX is smaller than T or C. And in this case, so if DS paired with T or DS pairs C, they are crushed each other statically. So A and PX, in this case, just shape is slightly fitted each other. So that's why we add the nitro group at this position. So electrostatically repair this nitrogen of A so that it cannot pair each other. Instead of A, DS has no nitrogen at this position. So that's why the shape of DS completely fit with the shape of PX. So that's why this DSPX pair can function as the base pair. First, we try to know the ability of the DSPX pair. We performed 100 cycles of PCR using these natural base pairs. We first made DNA containing DS by chemical synthesis and performed 100 cycles of PCR. But PCR is mostly after 20 cycles of PCR, the amplification is finished. So to maintain the exponential amplification, we first carried out 10 cycles of PCR, then diluted, then again we performed 10 cycles of dilution. We repeated 10 times these cycles to perform 100 cycles of PCR. So then we obtained a huge amount of DNA. So 10 to the 27th fold increased. It's okay. Which polymerase was used? In this case, we used deep bent DNA polymerase, which features a 3-primexon glass activity. So it depends on the polymerase. That's right. Next, how much unnatural base pairs included in the final amplified product? Then we performed DNA sequencing. This is a Sanger sequencing, but we added the unnatural base substrate in the sequencing, but we didn't add the oxyditaminate corresponding to the unnatural bases. So that's why the unnatural base position we can see as a gap like this. So after 100 cycles of PCR, the sequencing pattern is mostly the same. So after 100 cycles of PCR, the unnatural bases are still survived. And actually, this is X-Y, the sequencing like this. More detailed experiment, maybe, who I will talk about quantification to determine the selectivity and the efficiency. So that I will just omit more detailed experiment, but we just tell about the result. Finally, we found the selectivity of the unnatural base pairing is more than 99.90%. This is using a deep bent DNA polymerase. So another important thing is missing cooperation of unnatural base substrate opposite natural bases in template. In this case, the missing cooperation rate is less than 0.05% per replication per bases. This is a natural base missing cooperation rate. So that's why this unnatural base missing cooperation is mostly the same as natural base fidelity. So and PX has a very unique because we can attach to any functional group these positions like diodes, azide and ethanol. Azide and ethanol can be used for quick reactions to modify, to attach other functional groups. And also biotin can be attached to this position. So then and when we use these DS and modified PX bases, the PCR amplification is very high. So we now have such a third base pair. So that we are trying to several applications. Then today I just will talk about the DNA optoma applications here. So I just bit talk about DNA optomers and nucleic acid optomers single oligonucleotide fragments that specifically bind to target molecules. That's why nucleic acid are expected to alternative antibodies. So the unique point is optomers can be generated by in-viton selection system, repetitive selection and PCR amplification cycles by using nucleic acid libraries with random sequences. And the strong point is relative to antibodies that the optomers is high specificity and low immunogenicity and easy quality control and modifications for mass production because at first we determine the sequence by the selects. But after that we can chemically synthesized of the optomers. So that's why it's very easy to apply to diagnostic and therapeutic applications. But the weak point is the affinity is relatively low as compared to antibodies. This is because only four bases compared to 20 amino acids. That is a problem. So another one is low stability against nucleic acid. I explained first these problems. So this is another science fiction movie. This is also very interesting because some day simple bacteria came from the universe to Earth. And only few days they evolved to higher creatures. And the scientists determined the genetic materials. And they have 10 bases, 10 different bases. So this is just a science fiction. But I imagine this is very interesting because increased complexity by increasing letters. So if so rapid evolution is possible and also increased functionality. So that's why we try to evolutionary engineering method of selection by increasing the DNA letters in the libraries. So we performed selection procedures using the increased letter libraries. We first, we just add the DS bases into the library as a fifth base. There are two reasons. One is DS base is very hydrophobic. And nucleic acid is generally very hydrophilic. So it's just a bit difficult to bind to hydrophobic part of the proteins. So that's why we add the hydrophobic DS bases into the library like this. So another one is we didn't add the PX base in the library as a sixth base. Because DNA is always complementary paired to each other. So in this case DNA has just like a simple to form the single duplex. This is not good for optomers to increase structural diversity. And if you add the only DS base, DS base cannot pair as other bases. So that's why maybe the structure might be increased. So these are two reasons. Then we make this DNA library with five different bases and performed selection and after that we performed PCR amplification. In this case we add the DS and PX substrates and we make a double-stranded DNA. Then we separated only DS containing bases as an equestrand library. So first our demonstration we choose Begev 165. So this is very important for optomers researchers because only one modified RNA optomers named macogen was approved as treatment for age-related macular degeneration. Only one optomers approved. So that is a problem so that our first target is this. And this is actually so this is a macogen. So the KD value is relatively high, 5 to 100 picomoles. And this is RNA and another DNA optomer was generated by Ray-Gold group and in this case it's 300 picomoles. And it has two structures so that another one is much lower affinity. So then we performed in vitro selection targeting Begev 165. We performed seven rounds of selection like this. We also add the competitor of Ray-Gold optomers like this. And the important thing is we performed more than 150 cycles of PCR in total. So if the unnatural bases are removed during the PCR amplification that selection was maybe failed. But we most unnatural bases survived during the selection procedures. Then we got the DNA optomers. It has 47 DNA and only two unnatural bases existed here and here. So the KD value is less than one picomole. In the same conditions Ray-Gold's DNA optomers 100 picomoles. So that's why only two unnatural bases increased by more than 100 times higher affinity relative to the conventional DNA optomers. Then we replaced the DS base with A. Because the DS base is sometimes often replaced with A during PCR amplification. So that's why we also make a mutant. In this case the KD value is 300 picomole. So that's why only two unnatural bases effectively increased binding affinities. So this optomers also selectively bind to Begev 165. And it cannot bind to Begev 121 and other proteins. So that's why this optomers selectivity is also very high. So now we have several optomers by using this method as well under Begev optomers here and under Interferon gamma optomers and also under VWF optomers. 38 or 75 picomole which is much higher than the conventional DNA optomers. And recently Dr. Howard Young of the National Cancer Institute is interested in this Interferon gamma optomers. They have a system using a culture cells. Interferon gamma interact with the Interferon gamma receptor which stimulates stat 1 phosphorylation. And they have a system using a flow cytometry like this. So if we add our optomers into the system, if this optomers can inhibit the interaction so that we test it. 100 nanogram per liter of optomers completely. So you can see they completely inhibit the Interferon gamma and the receptor interaction like this. And as a control, we also tested using a conventional DNA optomers already reported. And in this case, more than 100 nanogram picomole it cannot inhibit the Interferon gamma activity at all. But another problem is even if we use this unnatural basis the DNA optomers cannot stable against nucleases. This is a general electrophoresis of optomers in the serum. After one day, the most DNA optomers are degraded in the serum. But we have another technology using, we call it Mini-Hairpin DNA. This is just natural DNA hairpin structures. If you add the 3 prime region of this Mini-Hairpin like this and also actually we found these two DS-paces are very important in this case. This is not so important. So that's why we replaced this region with Mini-Hairpin DNA. So if we modify like this story the optomers' stability is significantly increased. More than 80% survived after three days in the serum at 37 degrees. And also this optomers can show the in vitro activities. So I'll just be talking about Mini-Hairpin DNA structures because we still don't know the simple DNA sequences. So I also try to make a natural based incorporated DNA, artificial DNA. But during our experiment we still don't know the simple natural DNA. So I just be to introduce about this for new synthetic biology. So actually 25 years ago I accidentally found this unique DNA sequences. So this is a gel electrophoresis of this 21 chemical synthesized fragment. This is a complementary sequence. And a complementary sequence shows a usual gel mobility on denaturing gels. But this 21 more shows always faster mobility by one or two bases. This is 25 years ago so at that time even in chemical synthesis of DNA is also proceeding. So these are old methods. But if we use several methods, the purified DNA shows this position on the gel. At first we didn't know what happened with this DNA fragment. So that's why we made a shorter DNA fragment from the three prime timers of this 21 more. So in this case 1, 2, 3, 4, 5 more like this. And 6 more like this. And 7, 8, 9. So if you add make this fragment. So this fragment shows higher mobility. After that it glids like this. So that's why the 21 more shows lower mobility like this. And we also performed this experiment from the five prime region. Then we determined GCGA sequence shows this abnormal mobility. This is thermal stability. This fragment shows 10 values more than 82 degrees. And if you add the 7 more urea, even in the 7 more urea, the 10 values are 570 degrees. So that's why even in the denatured gel this fragment is still formed some structures. That's why the mobility is high. After that we determined the structure by NMR and also GCG NAGC. OR GCG NAGC shows a very stable structure. Then we call it a mini-hairpin structure like this. So this is NMR structures, tertiary structures by determined by NMR. Then two GC pairs and one shared type GA pair. And this is one A is stuck with neighboring bases. So the important thing is this thermal stable mini-hairpin DNA also resists against nucleases. These are very stable against nucleus P1 or nucleus S1. So that's why if we add the three prime terminals of the optimal, this structure blocked against three prime exonuclease activity. And also if you add the internal structure of the optimal, the optimal imparts higher increased structural stability. So then total structure is very stable even against nucleases. So last part I will just be talking about sense selects using our unnatural base pair selection systems. Optimal targets are not only proteins but also cells. So we performed in-bit selection against breast cancer cells. In this case, we just use cells as a target for the selection. After seventh round selection, we can see the gradually concentrates libraries like this. Then we obtained the DNA aftermaths that specifically bind to MCF7 like this. If we change DS base to A, this aftermath activity is completely lost. So this is a general imaging using these aftermaths. We also have aftermaths with the same selection so the cells can specifically die with these aftermaths. And by co-focal imaging, we also know that this aftermath was going to cells. Maybe this is one part. We also see that from the pictures. So that's why this aftermath can be used for diagnostics like petro something or it can be also used as a DDS. So finally, I just talk about myself. Actually, I cannot believe I'm in now Singapore. I was working in Japan at the D.K. Institute. But our group now moved to Singapore Institute of Bioengineering and Nanotechnology. And in this institute, we're focusing on the diagnostics of using these aftermaths and also to increase the studies of these genetic alphabet expansion systems. And we have also venture companies, diagnostics, biotechnology. It's still in Japan and they are focusing on therapeutic applications of these aftermaths. So now, to expand the genetic alphabet, we are expanding our research globally so that if you are interested in our research, please collaborate with each of us. And this study is also Matsunaga and Michiko are working with here. And I also thank Howard Young. Thank you very much. Thank you, Maria. It was a wonderful initial flavor of everything that Sheno biology can do for us. And we'll have various presentations of the meeting on that topic. All Young questions. Natural DNAs are well known for the so-called second chargups or parity-chargup rule. So what happens with this constraint in your synthetic artificial sequences? Does it break or complain? Actually, we don't know, but our natural base pair has no hydrogen bonding interactions. So that's why we cannot add a lot of this third pair into DNA. So maybe in this case, it cannot be work. Is it okay? I understand what you say, but it's not okay for me. I don't know if this applies for any number of base pairs. So A equals T in double X equals C and D equals P. Parity rule actually counts the oligomers over the same strand. It knows nothing about the opposite one. Trust me. I will know what I'm speaking about. In the same strand? Yeah, sure. Complimentary palindromes count over the same strand actually exhibit pretty close frequencies. Okay. Maybe, for a Romsberg, we made bacteria using a third base pair, so they will show the chargup rule as it's correct or not. That's exactly what I'm saying. To meet the constraint, you are to introduce not a single couple, but two couples of artificial places, we'll see. That's a trick. Okay, thank you so much again to the speaker.