1 00:00:00,790 --> 00:00:07,559 [Music] 2 00:00:13,020 --> 00:00:12,390 I thanks to the organizers for letting 3 00:00:15,750 --> 00:00:13,030 me be here 4 00:00:17,760 --> 00:00:15,760 the original tiled title was alternative 5 00:00:19,620 --> 00:00:17,770 biopolymers in the early evolution but I 6 00:00:22,769 --> 00:00:19,630 figured I should be a little more 7 00:00:25,079 --> 00:00:22,779 specific about what it is that I want to 8 00:00:27,150 --> 00:00:25,089 present today and so I chose this 9 00:00:29,790 --> 00:00:27,160 subtitle assessing the evolutionary 10 00:00:32,429 --> 00:00:29,800 potential of nucleic acid libraries with 11 00:00:34,740 --> 00:00:32,439 increasing increased information density 12 00:00:36,930 --> 00:00:34,750 and what I would like to show you today 13 00:00:41,189 --> 00:00:36,940 is a set of experiments that we started 14 00:00:45,330 --> 00:00:41,199 in the attempt to understand what is the 15 00:00:48,029 --> 00:00:45,340 real information density that is needed 16 00:00:50,750 --> 00:00:48,039 for a library to achieve evolution while 17 00:00:54,569 --> 00:00:50,760 still maintaining the least possible 18 00:00:57,829 --> 00:00:54,579 amount of well while still using the 19 00:01:01,740 --> 00:00:57,839 least possible amount of energy to do so 20 00:01:05,760 --> 00:01:01,750 so why would we want alternative 21 00:01:07,650 --> 00:01:05,770 biopolymers to to achieve that one of 22 00:01:10,020 --> 00:01:07,660 the reasons is of course that our innate 23 00:01:11,910 --> 00:01:10,030 prebiotic chemistry is hard and only 24 00:01:13,560 --> 00:01:11,920 works on under very specific sets of 25 00:01:16,020 --> 00:01:13,570 conditions and we heard about it all 26 00:01:16,740 --> 00:01:16,030 through this conference actually this 27 00:01:20,249 --> 00:01:16,750 morning 28 00:01:21,480 --> 00:01:20,259 Tyler Rush made a few good points about 29 00:01:24,420 --> 00:01:21,490 that 30 00:01:26,700 --> 00:01:24,430 also this one I should have raised it 31 00:01:28,590 --> 00:01:26,710 after this it's a fast talk this was 32 00:01:32,160 --> 00:01:28,600 very good actually 33 00:01:35,190 --> 00:01:32,170 so I think that a replicase is still our 34 00:01:38,219 --> 00:01:35,200 work in progress but people as we just 35 00:01:40,050 --> 00:01:38,229 saw in the Arab and in Holly girls love 36 00:01:42,709 --> 00:01:40,060 and enjoys lab actually they're doing 37 00:01:45,270 --> 00:01:42,719 great things with them but yet 38 00:01:47,670 --> 00:01:45,280 replicators are a key play player in a 39 00:01:50,340 --> 00:01:47,680 hypothetical RNA world and are still a 40 00:01:54,270 --> 00:01:50,350 little bit of a challenge that needs to 41 00:01:56,520 --> 00:01:54,280 be overcome another point is that alien 42 00:01:57,870 --> 00:01:56,530 life arising independently of life on 43 00:02:00,029 --> 00:01:57,880 Earth might have had a different 44 00:02:02,190 --> 00:02:00,039 molecular biology than what we see or 45 00:02:03,870 --> 00:02:02,200 was experienced here in the past and 46 00:02:06,420 --> 00:02:03,880 this is also something that people have 47 00:02:09,510 --> 00:02:06,430 been talking about quite a bit this past 48 00:02:11,970 --> 00:02:09,520 few days but even if the biopolymer of 49 00:02:13,290 --> 00:02:11,980 choice has been had been the same alien 50 00:02:14,820 --> 00:02:13,300 life might have chosen a different 51 00:02:18,360 --> 00:02:14,830 evolutionary pathway 52 00:02:19,880 --> 00:02:18,370 no matter what and even without having 53 00:02:24,110 --> 00:02:19,890 to go to all the planets 54 00:02:26,120 --> 00:02:24,120 or or before or after the evolution of 55 00:02:28,400 --> 00:02:26,130 life today's natural RNA 56 00:02:31,520 --> 00:02:28,410 post-transcriptional modifications are 57 00:02:33,440 --> 00:02:31,530 seen by many as remnants of an RNA like 58 00:02:36,980 --> 00:02:33,450 world and they might be considered as 59 00:02:39,500 --> 00:02:36,990 hints that in prebiotic times these 60 00:02:41,330 --> 00:02:39,510 modifications could have arise and 61 00:02:43,610 --> 00:02:41,340 during rough rather than after the 62 00:02:45,470 --> 00:02:43,620 establishment of the biopolymer so in 63 00:02:48,380 --> 00:02:45,480 other words these features that we call 64 00:02:49,670 --> 00:02:48,390 modifications or alternative now and 65 00:02:52,340 --> 00:02:49,680 here might have been the main 66 00:02:55,010 --> 00:02:52,350 evolutionary players in other times and 67 00:02:57,620 --> 00:02:55,020 players and in planets I did like these 68 00:02:59,780 --> 00:02:57,630 to add this quote from yesterday 69 00:03:02,720 --> 00:02:59,790 morning's talk from Christine Keating 70 00:03:04,250 --> 00:03:02,730 that I think submarines well what a lot 71 00:03:07,009 --> 00:03:04,260 of people have been saying these days 72 00:03:08,990 --> 00:03:07,019 what happens so there's this dichotomy 73 00:03:11,780 --> 00:03:09,000 in which what happens what happened who 74 00:03:14,509 --> 00:03:11,790 has versus what could happen or could 75 00:03:16,160 --> 00:03:14,519 have happened in this dichotomy the 76 00:03:19,190 --> 00:03:16,170 second is a much larger group of 77 00:03:23,240 --> 00:03:19,200 reactions so we need to start to 78 00:03:26,750 --> 00:03:23,250 investigate this I'll go back for a 79 00:03:28,580 --> 00:03:26,760 second so what as I said what we said 80 00:03:30,580 --> 00:03:28,590 what we decided to do is to set up a 81 00:03:34,340 --> 00:03:30,590 series of experiments a couple of 82 00:03:36,560 --> 00:03:34,350 initial in vitro selections starting 83 00:03:38,990 --> 00:03:36,570 with library so the the two selections 84 00:03:41,120 --> 00:03:39,000 that we want to do in parallel would go 85 00:03:43,160 --> 00:03:41,130 towards the same function but starting 86 00:03:46,090 --> 00:03:43,170 with two libraries that are the same 87 00:03:49,850 --> 00:03:46,100 length but carry different information 88 00:03:53,500 --> 00:03:49,860 contents so how do we achieve this these 89 00:03:55,699 --> 00:03:53,510 enrichment or depletion of information 90 00:03:58,039 --> 00:03:55,709 nothing would make me more happy than 91 00:04:02,360 --> 00:03:58,049 being able to do it with the naked 92 00:04:04,759 --> 00:04:02,370 systems or the neutral sites in the 93 00:04:06,770 --> 00:04:04,769 Taylor showed this morning but we are 94 00:04:10,280 --> 00:04:06,780 still lacking a molecular biology for 95 00:04:13,910 --> 00:04:10,290 those systems so what do we do have in 96 00:04:15,440 --> 00:04:13,920 the lab actually at home at Fame is this 97 00:04:17,449 --> 00:04:15,450 artificially expanded genetic 98 00:04:19,819 --> 00:04:17,459 information system that was developed in 99 00:04:21,979 --> 00:04:19,829 Boehner's lab in the last 20 years and 100 00:04:24,110 --> 00:04:21,989 for this system we do have a very 101 00:04:27,500 --> 00:04:24,120 well-established molecular biology 102 00:04:29,570 --> 00:04:27,510 structural biology synthetic biology and 103 00:04:31,430 --> 00:04:29,580 we have shown in the past years that we 104 00:04:32,910 --> 00:04:31,440 can actually use this system for 105 00:04:36,090 --> 00:04:32,920 evolutionary 106 00:04:38,280 --> 00:04:36,100 easily and effectively so the idea 107 00:04:40,620 --> 00:04:38,290 behind ages is to complete the 108 00:04:42,570 --> 00:04:40,630 watson-crick pairing concept as it's 109 00:04:44,400 --> 00:04:42,580 shown in this scheme by shuffling 110 00:04:46,860 --> 00:04:44,410 hydrogen bond donors and acceptors 111 00:04:49,950 --> 00:04:46,870 groups forming additional orthogonal 112 00:04:51,930 --> 00:04:49,960 nuclear base pairs they resemble net 113 00:04:53,520 --> 00:04:51,940 natural nucleotides in size shape and 114 00:04:55,530 --> 00:04:53,530 pairing geometries they are 115 00:04:57,390 --> 00:04:55,540 independently replicable do not 116 00:04:59,610 --> 00:04:57,400 interfere with the RNA folding or the 117 00:05:01,470 --> 00:04:59,620 DNA double helix structures they 118 00:05:03,120 --> 00:05:01,480 increase the information density and 119 00:05:05,130 --> 00:05:03,130 they have the potential to increase 120 00:05:08,130 --> 00:05:05,140 functionality and that's what we want to 121 00:05:10,800 --> 00:05:08,140 test so what we are going what we have 122 00:05:13,920 --> 00:05:10,810 done in this preliminary studies is we 123 00:05:16,650 --> 00:05:13,930 started out by using these pair Z's in 124 00:05:21,000 --> 00:05:16,660 peace where he has an extra nitrogen 125 00:05:25,560 --> 00:05:21,010 group and five while and also is has an 126 00:05:29,010 --> 00:05:25,570 extra hydrogen here at n 3 and P is 127 00:05:32,580 --> 00:05:29,020 lacking hydrogen right here and has an 128 00:05:34,290 --> 00:05:32,590 extra nitrogen in and v 2 so we pick 129 00:05:35,910 --> 00:05:34,300 this couple at first because it's the 130 00:05:37,470 --> 00:05:35,920 one that we have studied more and we 131 00:05:39,630 --> 00:05:37,480 know it works and it's a first step 132 00:05:44,100 --> 00:05:39,640 toward increasing the diversity and the 133 00:05:46,230 --> 00:05:44,110 information inside the library now the 134 00:05:47,940 --> 00:05:46,240 next step is to try to understand what 135 00:05:50,850 --> 00:05:47,950 is the sequence space coverage that we 136 00:05:53,480 --> 00:05:50,860 are trying to to achieve as I said we 137 00:05:56,520 --> 00:05:53,490 are going to start with same length 138 00:05:58,140 --> 00:05:56,530 libraries but one will have six letters 139 00:06:00,180 --> 00:05:58,150 DNA and the other one will have four 140 00:06:03,330 --> 00:06:00,190 letters DNA if you look at this table 141 00:06:04,830 --> 00:06:03,340 well signal space is defined for us at 142 00:06:07,830 --> 00:06:04,840 least his define is the number of 143 00:06:10,230 --> 00:06:07,840 library window block blocks to the N 144 00:06:12,660 --> 00:06:10,240 where n is the length of the library so 145 00:06:15,390 --> 00:06:12,670 if we have 19 nucleotides and six letter 146 00:06:17,880 --> 00:06:15,400 DNA we will achieve 100 percent coverage 147 00:06:20,310 --> 00:06:17,890 when we use one nano mole of material 148 00:06:22,140 --> 00:06:20,320 which is what you usually are able to 149 00:06:25,110 --> 00:06:22,150 handle in a in a laboratory experiment 150 00:06:27,720 --> 00:06:25,120 while if we have four letters DNA for 151 00:06:30,510 --> 00:06:27,730 the same amount you will actually have 152 00:06:33,240 --> 00:06:30,520 it about a thousand molecules a thousand 153 00:06:35,100 --> 00:06:33,250 copies for each sequence that you have 154 00:06:37,190 --> 00:06:35,110 in your library so obviously our for 155 00:06:39,570 --> 00:06:37,200 light of DNA always has an advantage 156 00:06:41,610 --> 00:06:39,580 compared to a six letter DNA when you're 157 00:06:43,439 --> 00:06:41,620 trying to do wet lab experiments 158 00:06:45,570 --> 00:06:43,449 evolutionary experiment at 159 00:06:47,519 --> 00:06:45,580 at least so if you look at the table and 160 00:06:49,649 --> 00:06:47,529 you keep going through the numbers you 161 00:06:52,640 --> 00:06:49,659 will actually see I'll do the math for 162 00:06:57,390 --> 00:06:52,650 you you will actually see that for every 163 00:06:58,980 --> 00:06:57,400 there is a the column space coverage 164 00:07:02,100 --> 00:06:58,990 decreases of one order of magnitude 165 00:07:03,869 --> 00:07:02,110 every five nucleotide lengths increase 166 00:07:06,899 --> 00:07:03,879 in the library for two bits of 167 00:07:09,299 --> 00:07:06,909 information difference so the longer you 168 00:07:12,929 --> 00:07:09,309 go the more you actually have the four 169 00:07:16,070 --> 00:07:12,939 letters DNA have an advantage versus the 170 00:07:19,170 --> 00:07:16,080 six letters the six letter DNA that is 171 00:07:21,200 --> 00:07:19,180 unless the functionality and the extra 172 00:07:24,809 --> 00:07:21,210 nucleotides that you are adding are 173 00:07:27,989 --> 00:07:24,819 actually more powerful for more 174 00:07:32,040 --> 00:07:27,999 effective for the type of phenomenon 175 00:07:33,899 --> 00:07:32,050 that you're trying to evolve down here 176 00:07:36,119 --> 00:07:33,909 we actually have the the actual 177 00:07:37,950 --> 00:07:36,129 libraries that we have used for these 178 00:07:40,769 --> 00:07:37,960 preliminary studies we started with an n 179 00:07:43,889 --> 00:07:40,779 25 for the full nucleotides we started 180 00:07:47,070 --> 00:07:43,899 with 0.5 nanomoles just this was a first 181 00:07:50,550 --> 00:07:47,080 run so we covered 25% of sequence page 182 00:07:52,969 --> 00:07:50,560 space for the standard DNA library while 183 00:07:57,899 --> 00:07:52,979 for the ages library we actually covered 184 00:08:00,749 --> 00:07:57,909 0.00 11% of the sequence space so we did 185 00:08:02,429 --> 00:08:00,759 the next step would be which reaction 186 00:08:04,409 --> 00:08:02,439 are we gonna pick as a proof of 187 00:08:08,070 --> 00:08:04,419 principle probably one of the easiest 188 00:08:10,589 --> 00:08:08,080 reaction to pick is selecting for DNA 189 00:08:12,629 --> 00:08:10,599 RNA cleaving DN enzymes raghava talked 190 00:08:16,050 --> 00:08:12,639 about it a little bit this morning it's 191 00:08:18,029 --> 00:08:16,060 a very well-known type of molecule it's 192 00:08:20,100 --> 00:08:18,039 very well studied so we have a lot of 193 00:08:22,139 --> 00:08:20,110 literature to compare to once we get 194 00:08:25,139 --> 00:08:22,149 some results and people have actually 195 00:08:27,959 --> 00:08:25,149 been done selection on the n enzymes for 196 00:08:32,189 --> 00:08:27,969 the past maybe decade and they were of 197 00:08:35,309 --> 00:08:32,199 course first selected by and in the 198 00:08:38,159 --> 00:08:35,319 Joyce lab Santora enjoys 1997 this is 199 00:08:40,800 --> 00:08:38,169 Craig Craig jerem did all of it is he's 200 00:08:42,930 --> 00:08:40,810 a technician in in at Fame he's a very 201 00:08:45,809 --> 00:08:42,940 skillet guy but he needs still a little 202 00:08:47,879 --> 00:08:45,819 training this is the schematic of the 203 00:08:50,550 --> 00:08:47,889 selection so we start with an RNA target 204 00:08:52,740 --> 00:08:50,560 that is here in blue that is attached to 205 00:08:54,990 --> 00:08:52,750 a primer that will amplify a library and 206 00:08:56,090 --> 00:08:55,000 at the Phi Prime and actually has a 207 00:09:01,069 --> 00:08:56,100 biotin 208 00:09:03,680 --> 00:09:01,079 that you see after the you got the 209 00:09:05,900 --> 00:09:03,690 double-stranded DNA you can remove the 210 00:09:07,550 --> 00:09:05,910 the one of the strands and everything 211 00:09:10,069 --> 00:09:07,560 all your system will be attached to it 212 00:09:13,309 --> 00:09:10,079 back to the biotin this portion which is 213 00:09:15,710 --> 00:09:13,319 your evolving ribozyme can fold over the 214 00:09:18,230 --> 00:09:15,720 target cleave it and you will actually 215 00:09:21,769 --> 00:09:18,240 have in the supernatant your evolving 216 00:09:23,540 --> 00:09:21,779 library that you can amplify and this is 217 00:09:26,059 --> 00:09:23,550 just a slightly modified from the 218 00:09:27,949 --> 00:09:26,069 original protocol to make it a little 219 00:09:30,199 --> 00:09:27,959 more effective and remove a few steps 220 00:09:31,340 --> 00:09:30,209 these are the conditions that we used is 221 00:09:34,579 --> 00:09:31,350 to millimolar magnesium because 222 00:09:39,290 --> 00:09:34,589 everybody cares about magnesium and what 223 00:09:41,329 --> 00:09:39,300 we care about is that we use pH 7.8 for 224 00:09:44,900 --> 00:09:41,339 a very specific reason that I will tell 225 00:09:47,180 --> 00:09:44,910 that I will say in a moment and okay so 226 00:09:49,610 --> 00:09:47,190 this is the the big deal we did the 227 00:09:53,150 --> 00:09:49,620 parallel in-vitro selection and what we 228 00:09:55,970 --> 00:09:53,160 could see is that while the standard DNA 229 00:09:59,150 --> 00:09:55,980 had a really hard time growing at least 230 00:10:01,340 --> 00:09:59,160 in our conditions at 25 nucleotide long 231 00:10:03,710 --> 00:10:01,350 library library and with only two 232 00:10:08,120 --> 00:10:03,720 millimolar magnesium and instead the 233 00:10:12,110 --> 00:10:08,130 ages they the known standard library 234 00:10:15,170 --> 00:10:12,120 grew very well and and and was happy so 235 00:10:17,509 --> 00:10:15,180 these by itself is already a surprising 236 00:10:20,960 --> 00:10:17,519 result because while we did expect that 237 00:10:23,449 --> 00:10:20,970 ages might have some some advantages 238 00:10:26,960 --> 00:10:23,459 because of the extra groups that we had 239 00:10:29,780 --> 00:10:26,970 we didn't expect that we would have such 240 00:10:31,490 --> 00:10:29,790 a such a big diverse difference also I'm 241 00:10:32,960 --> 00:10:31,500 a little disappointed we did these two 242 00:10:35,090 --> 00:10:32,970 or three times I think three times in 243 00:10:37,160 --> 00:10:35,100 total we really can't grow this DN 244 00:10:39,470 --> 00:10:37,170 enzymes with standard nucleotides it 245 00:10:41,210 --> 00:10:39,480 really looks like only four it's not 246 00:10:42,829 --> 00:10:41,220 enough for two millimolar magnesium 247 00:10:49,429 --> 00:10:42,839 thank you 248 00:10:53,360 --> 00:10:49,439 and for 25 nucleotides long sequence one 249 00:10:56,420 --> 00:10:53,370 reason might be in the Z nucleotide 250 00:10:58,429 --> 00:10:56,430 which I reported here again which has a 251 00:11:01,069 --> 00:10:58,439 pKa of seven point eight which is 252 00:11:04,759 --> 00:11:01,079 exactly the pKa then the pH that we use 253 00:11:05,850 --> 00:11:04,769 during selection so ideally you can see 254 00:11:08,370 --> 00:11:05,860 here the 255 00:11:10,650 --> 00:11:08,380 the output of the deep sequencing that 256 00:11:14,610 --> 00:11:10,660 we did so we actually were able to we 257 00:11:17,670 --> 00:11:14,620 have a technique to transform our ages 258 00:11:19,949 --> 00:11:17,680 DNA into standard DNA submitted to deep 259 00:11:22,410 --> 00:11:19,959 sequencing and then keep track of where 260 00:11:23,550 --> 00:11:22,420 the original season peas were so we 261 00:11:25,740 --> 00:11:23,560 applied that technique to these 262 00:11:28,970 --> 00:11:25,750 libraries and we actually sequenced each 263 00:11:32,190 --> 00:11:28,980 cycle of the selection and what was 264 00:11:34,350 --> 00:11:32,200 surprising surprising but maybe not is 265 00:11:36,660 --> 00:11:34,360 that there was a preponderance with the 266 00:11:39,240 --> 00:11:36,670 up Z's in all the major clusters that 267 00:11:41,250 --> 00:11:39,250 were that survived the selection here 268 00:11:44,100 --> 00:11:41,260 there's just a graphic representation of 269 00:11:45,780 --> 00:11:44,110 how the various different clusters 270 00:11:48,630 --> 00:11:45,790 appear and disappear during the 271 00:11:51,180 --> 00:11:48,640 selection this is a table that shows you 272 00:11:54,030 --> 00:11:51,190 at which psychology cluster will will 273 00:11:55,920 --> 00:11:54,040 appear but back to the Z's so our main 274 00:11:59,220 --> 00:11:55,930 sequence the cluster that actually 275 00:12:01,440 --> 00:11:59,230 showed most prominently in the deep 276 00:12:04,530 --> 00:12:01,450 sequencing analysis actually carries two 277 00:12:09,660 --> 00:12:04,540 for Z's and it has two sides of cleavage 278 00:12:12,540 --> 00:12:09,670 so in an ideal in in the hypothesis that 279 00:12:14,490 --> 00:12:12,550 the pKa of Z is actually very important 280 00:12:16,590 --> 00:12:14,500 here you can see how half of the 281 00:12:18,660 --> 00:12:16,600 molecules will be protonated half of the 282 00:12:21,690 --> 00:12:18,670 molecules will be deprotonated and they 283 00:12:24,870 --> 00:12:21,700 will act each of them every couple 284 00:12:30,180 --> 00:12:24,880 around one of the size of cleavage to to 285 00:12:34,230 --> 00:12:30,190 achieve cleavage so this is one example 286 00:12:36,360 --> 00:12:34,240 of all the of all this and she's acting 287 00:12:39,389 --> 00:12:36,370 clusters that we found we got three 288 00:12:42,150 --> 00:12:39,399 families of Ages DN enzymes when one as 289 00:12:44,130 --> 00:12:42,160 I just said is cuts at you nine and you 290 00:12:46,769 --> 00:12:44,140 thirteen see one two six and seven 291 00:12:49,230 --> 00:12:46,779 cluster one two six and seven then we 292 00:12:54,150 --> 00:12:49,240 have cleavage at a sixteen and then we 293 00:12:55,949 --> 00:12:54,160 have creases at a in a a ten sorry so we 294 00:12:58,010 --> 00:12:55,959 we got these three families and we 295 00:13:00,660 --> 00:12:58,020 decided to go ahead and and try to 296 00:13:02,819 --> 00:13:00,670 characterize exactly what was happening 297 00:13:04,530 --> 00:13:02,829 in each of these clusters we took a 298 00:13:06,990 --> 00:13:04,540 cluster one to begin with 299 00:13:09,180 --> 00:13:07,000 so in sis it will just have a you know a 300 00:13:12,180 --> 00:13:09,190 decent activity but this is a really big 301 00:13:13,980 --> 00:13:12,190 molecule you do see that the evolving 302 00:13:16,769 --> 00:13:13,990 portion of the library actually does 303 00:13:17,230 --> 00:13:16,779 best pairs very nicely with the RNA and 304 00:13:19,389 --> 00:13:17,240 then 305 00:13:21,010 --> 00:13:19,399 you have the Z's over here so I'm what I 306 00:13:23,079 --> 00:13:21,020 think is happening is actually that 307 00:13:25,300 --> 00:13:23,089 we're getting a very big 3d 308 00:13:27,430 --> 00:13:25,310 restructuring around the double 309 00:13:29,769 --> 00:13:27,440 partially double stranded area this is 310 00:13:31,150 --> 00:13:29,779 actually there's a GU bubble and here 311 00:13:34,870 --> 00:13:31,160 it's probably open because there's this 312 00:13:38,290 --> 00:13:34,880 huge structure area but we then so we 313 00:13:39,970 --> 00:13:38,300 checked that see one with no Z would not 314 00:13:42,070 --> 00:13:39,980 actually have the same activity and 315 00:13:43,720 --> 00:13:42,080 that's what is shown here in two really 316 00:13:45,970 --> 00:13:43,730 different representations so here you're 317 00:13:47,769 --> 00:13:45,980 monitoring the appearance of the bands 318 00:13:50,740 --> 00:13:47,779 that will show you cleavage here you're 319 00:13:53,019 --> 00:13:50,750 monitoring the disappearance of of the 320 00:13:55,000 --> 00:13:53,029 full-length product and the red one is 321 00:13:57,040 --> 00:13:55,010 the one that does not contain Z and the 322 00:13:58,930 --> 00:13:57,050 black one is the one that does contain 323 00:14:00,960 --> 00:13:58,940 see I'm running out of time so I'm just 324 00:14:04,150 --> 00:14:00,970 gonna go very fast 325 00:14:06,910 --> 00:14:04,160 we did find yeah this is like it's a 326 00:14:09,970 --> 00:14:06,920 very dense slide that basically we did a 327 00:14:12,730 --> 00:14:09,980 bunch of biochemistry assays in which we 328 00:14:15,519 --> 00:14:12,740 separated it two strands and we finally 329 00:14:17,980 --> 00:14:15,529 could see that we got a cake out of 10 330 00:14:20,139 --> 00:14:17,990 to the nine we get a km of 10 to the 5 331 00:14:22,840 --> 00:14:20,149 and a catalytic efficiency of about 10 332 00:14:26,139 --> 00:14:22,850 to the fifth per mole per minute that is 333 00:14:28,210 --> 00:14:26,149 very very comparable to I don't know if 334 00:14:31,930 --> 00:14:28,220 you guys know the work of David Perrin 335 00:14:34,269 --> 00:14:31,940 he works with heavily modified DN 336 00:14:37,120 --> 00:14:34,279 enzymes and he got exactly the same type 337 00:14:39,130 --> 00:14:37,130 well but we got similar catalytic 338 00:14:41,380 --> 00:14:39,140 efficiencies just with one extra 339 00:14:43,870 --> 00:14:41,390 nucleotide so that's that's very 340 00:14:47,050 --> 00:14:43,880 interesting conclusion conclusions and 341 00:14:50,500 --> 00:14:47,060 perspectives this is just a summary what 342 00:14:53,560 --> 00:14:50,510 we would like to do after is actually 343 00:14:56,380 --> 00:14:53,570 start to perform multiple parallel 344 00:14:58,540 --> 00:14:56,390 strand ages standard ages selections 345 00:15:00,280 --> 00:14:58,550 with increased and decreased lens number 346 00:15:02,889 --> 00:15:00,290 of nucleotides and extra nuclear base 347 00:15:07,290 --> 00:15:02,899 modifications so two four six seven 348 00:15:10,150 --> 00:15:07,300 eight and and whatnot I am going to 349 00:15:12,699 --> 00:15:10,160 thank of course Craig Jerome who is the 350 00:15:14,740 --> 00:15:12,709 one who did all of these all our funding 351 00:15:18,310 --> 00:15:14,750 agencies although this specific project 352 00:15:20,290 --> 00:15:18,320 is not yet funded and everybody at fame 353 00:15:24,180 --> 00:15:20,300 and firebird thank you I'll take any 354 00:15:29,230 --> 00:15:27,790 if there's time for one question okay 355 00:15:48,520 --> 00:15:29,240 all right 356 00:15:58,330 --> 00:15:48,530 we're gonna go to the back yes can you 357 00:16:00,640 --> 00:15:58,340 repeat that I'm sorry well see in our 358 00:16:03,210 --> 00:16:00,650 life studies actually we did have we did 359 00:16:06,580 --> 00:16:03,220 found that when these MPs and other 360 00:16:09,600 --> 00:16:06,590 pairs in the ages system are not best 361 00:16:12,370 --> 00:16:09,610 paired they actually contribute into 362 00:16:14,140 --> 00:16:12,380 into secondary and tertiary structures 363 00:16:17,530 --> 00:16:14,150 that we are still investigating but seem 364 00:16:20,620 --> 00:16:17,540 to be extremely interesting and not seen 365 00:16:23,500 --> 00:16:20,630 before in DNA and RNA what what I meant 366 00:16:25,000 --> 00:16:23,510 with that point was that we have also a 367 00:16:26,800 --> 00:16:25,010 lot of data that shows that when you 368 00:16:28,780 --> 00:16:26,810 substitute C and P in like 369 00:16:32,020 --> 00:16:28,790 double-stranded structures you actually 370 00:16:34,240 --> 00:16:32,030 do get the same the same foldings that 371 00:16:35,980 --> 00:16:34,250 you expect from known molecules you 372 00:16:37,960 --> 00:16:35,990 might have seen you know they hatch much 373 00:16:41,020 --> 00:16:37,970 in DNA paper with the spinach septum ER 374 00:16:43,600 --> 00:16:41,030 and some ribose which that we actually 375 00:16:45,250 --> 00:16:43,610 substituted these in piece for but yes 376 00:16:48,400 --> 00:16:45,260 it's true some of these nucleotides will 377 00:16:50,230 --> 00:16:48,410 actually provide I think we'll we don't 378 00:16:51,430 --> 00:16:50,240 have confirmation yet but I really think 379 00:16:56,050 --> 00:16:51,440 we'll have actually provided new 380 00:16:58,060 --> 00:16:56,060 structures for DNA and RNA molecules yep 381 00:16:59,380 --> 00:16:58,070 all right great let's thank ELISA again