1 00:00:10,920 --> 00:00:08,850 hi everyone I'm Raghav and from 2 00:00:13,709 --> 00:00:10,930 University of Missouri and I am 3 00:00:17,370 --> 00:00:13,719 interested in understanding rnase role 4 00:00:20,460 --> 00:00:17,380 in the origins of life and I'm I am 5 00:00:22,499 --> 00:00:20,470 basically concerned with how RNA 6 00:00:26,819 --> 00:00:22,509 catalyzes biologically relevant 7 00:00:28,979 --> 00:00:26,829 reactions I'm Thank You Kristen for the 8 00:00:30,960 --> 00:00:28,989 RNA world hypothesis intro that was 9 00:00:33,210 --> 00:00:30,970 really good so basically the RNA world 10 00:00:35,729 --> 00:00:33,220 hypothesis says that like during the 11 00:00:38,490 --> 00:00:35,739 early origins and evolution of life it 12 00:00:40,890 --> 00:00:38,500 was the RNA that was the workhorse both 13 00:00:43,710 --> 00:00:40,900 acting both as a catalyst and also as a 14 00:00:45,720 --> 00:00:43,720 genetic information carrier which is of 15 00:00:48,450 --> 00:00:45,730 course now done by the DNA and the 16 00:00:50,580 --> 00:00:48,460 protein although originally it was put 17 00:00:52,800 --> 00:00:50,590 forward to circumvent the chicken or egg 18 00:00:54,870 --> 00:00:52,810 paradox whether DNA came first or the 19 00:00:57,840 --> 00:00:54,880 army came first well recently we've 20 00:00:59,910 --> 00:00:57,850 actually found many catalytic rnase by 21 00:01:03,180 --> 00:00:59,920 in vitro evolution and it has really 22 00:01:06,090 --> 00:01:03,190 strengthened this hypothesis so I'm 23 00:01:08,789 --> 00:01:06,100 interested in phosphoryl transfer and 24 00:01:12,690 --> 00:01:08,799 and how RNA catalyzes phosphoryl 25 00:01:19,169 --> 00:01:12,700 transfer for example well in modern 26 00:01:23,940 --> 00:01:19,179 biology what we have ok so in modern 27 00:01:25,950 --> 00:01:23,950 biology we know that all these all the 28 00:01:30,749 --> 00:01:25,960 phosphorylation reactions are actually 29 00:01:32,399 --> 00:01:30,759 catalyzed by protein kinases and in case 30 00:01:34,709 --> 00:01:32,409 of the RNA well you do you think that 31 00:01:37,279 --> 00:01:34,719 there would be kinase ribozymes or RNA 32 00:01:41,580 --> 00:01:37,289 enzymes that can catalyze this reaction 33 00:01:44,129 --> 00:01:41,590 so how do you find catalytic RNA is that 34 00:01:46,109 --> 00:01:44,139 that have this activity to transfer 35 00:01:48,300 --> 00:01:46,119 phosphate group so we are actually 36 00:01:50,160 --> 00:01:48,310 selecting for our enzymes that can 37 00:01:53,519 --> 00:01:50,170 phosphorylate themselves so they take 38 00:01:55,739 --> 00:01:53,529 the phosphate off of a phosphoryl donor 39 00:01:59,999 --> 00:01:55,749 and then transfer onto themselves so 40 00:02:02,639 --> 00:02:00,009 that's what they would look like and the 41 00:02:04,349 --> 00:02:02,649 way you can find these active rnase well 42 00:02:06,620 --> 00:02:04,359 well the way you can partition the 43 00:02:09,059 --> 00:02:06,630 active rnase from the inactive rnase is 44 00:02:11,339 --> 00:02:09,069 so if you notice carefully there the 45 00:02:13,920 --> 00:02:11,349 phosphoryl donor actually has a sulfur 46 00:02:17,220 --> 00:02:13,930 instead of an oxygen on the gamma 47 00:02:19,470 --> 00:02:17,230 position of the triphosphate so any RNA 48 00:02:19,980 --> 00:02:19,480 that is active actually gets a sulfur 49 00:02:22,890 --> 00:02:19,990 along 50 00:02:25,620 --> 00:02:22,900 with the phosphate so if you separate 51 00:02:27,000 --> 00:02:25,630 these rnase out on a polyacrylamide gel 52 00:02:29,610 --> 00:02:27,010 where one of the layer in the middle 53 00:02:31,800 --> 00:02:29,620 contains mercury the sulfur interacts 54 00:02:34,110 --> 00:02:31,810 with the mercury and then all the rnase 55 00:02:37,110 --> 00:02:34,120 that have the sulfur get trapped in the 56 00:02:40,290 --> 00:02:37,120 mercury layer so if the RNA is active 57 00:02:42,900 --> 00:02:40,300 and if it is the activity of a guyanese 58 00:02:44,550 --> 00:02:42,910 ribozyme then it gets the sulfur along 59 00:02:46,080 --> 00:02:44,560 with the phosphate and gets trapped in 60 00:02:47,730 --> 00:02:46,090 the mercury later so you can you can 61 00:02:49,830 --> 00:02:47,740 basically cut out this band that is 62 00:02:52,530 --> 00:02:49,840 trapped in the mercury layer reverse 63 00:02:54,390 --> 00:02:52,540 transcribe it and then pcr-amplified do 64 00:02:57,990 --> 00:02:54,400 this process over and over again until 65 00:03:01,080 --> 00:02:58,000 you finally enrich your pool with active 66 00:03:03,330 --> 00:03:01,090 RNA so essentially you start out with 10 67 00:03:06,660 --> 00:03:03,340 to the 14th different sequences and you 68 00:03:10,500 --> 00:03:06,670 end up with a fraction of them that have 69 00:03:12,990 --> 00:03:10,510 the desired activity so this brings to 70 00:03:16,190 --> 00:03:13,000 part 1 of my talk which has to do with 71 00:03:19,170 --> 00:03:16,200 understanding how this ribozyme 72 00:03:21,030 --> 00:03:19,180 catalyzes phosphoryl transfer how ripe 73 00:03:25,620 --> 00:03:21,040 is not what kind of products can 74 00:03:27,480 --> 00:03:25,630 ribozymes form off of this reaction so 75 00:03:31,560 --> 00:03:27,490 this ribozyme was actually selected in 76 00:03:34,140 --> 00:03:31,570 my lab as so the GTP gamma s is the 77 00:03:37,260 --> 00:03:34,150 phosphate donor it uses magnesium and 78 00:03:42,330 --> 00:03:37,270 copper and phosphorylates at this second 79 00:03:44,550 --> 00:03:42,340 guanosine position and so that this is 80 00:03:46,920 --> 00:03:44,560 basically what a polyacrylamide gel 81 00:03:49,560 --> 00:03:46,930 would look like as the time increases 82 00:03:52,800 --> 00:03:49,570 you see more and more RNA getting 83 00:03:57,210 --> 00:03:52,810 phosphorylated and and gets trapped at 84 00:03:58,950 --> 00:03:57,220 the mercury layer okay so one of the 85 00:04:01,860 --> 00:03:58,960 first things I wanted to understand was 86 00:04:06,870 --> 00:04:01,870 how does the two prime hydroxyl in an 87 00:04:08,880 --> 00:04:06,880 RNA in this particular ribozyme play 88 00:04:13,290 --> 00:04:08,890 what can rule the two prime hydroxyl 89 00:04:15,150 --> 00:04:13,300 plays in this RNA structure so we know 90 00:04:16,920 --> 00:04:15,160 that two prime hydroxyl is involved in 91 00:04:19,070 --> 00:04:16,930 different things like a lot of different 92 00:04:23,550 --> 00:04:19,080 things for example here it's shown 93 00:04:26,580 --> 00:04:23,560 coordinating a metal ion and it is also 94 00:04:28,950 --> 00:04:26,590 involved in hydrogen bond as a hydrogen 95 00:04:32,850 --> 00:04:28,960 bond donor or hydrogen bond acceptor so 96 00:04:33,700 --> 00:04:32,860 so so it is very important to form the 97 00:04:37,470 --> 00:04:33,710 RNA straw 98 00:04:45,460 --> 00:04:37,480 the active RNA structures and ultimately 99 00:04:47,380 --> 00:04:45,470 give RNA the catalytic activity so to 100 00:04:49,780 --> 00:04:47,390 figure out whether the stew prime 101 00:04:52,570 --> 00:04:49,790 hydroxyls in this RNA is important what 102 00:04:55,630 --> 00:04:52,580 kind of two prime moriches are important 103 00:04:58,810 --> 00:04:55,640 in this ribozyme what we did was we we 104 00:05:00,730 --> 00:04:58,820 try to substitute 2 prime we try to 105 00:05:03,040 --> 00:05:00,740 substitute the nucleotides with two 106 00:05:05,440 --> 00:05:03,050 prime fluoro nucleotides so you 107 00:05:08,110 --> 00:05:05,450 basically transcribe this ribozyme with 108 00:05:10,630 --> 00:05:08,120 either ribonucleotides that have to 109 00:05:12,820 --> 00:05:10,640 prime hydroxyl or ribonucleotides that 110 00:05:14,710 --> 00:05:12,830 have to prime fluorine so we we either 111 00:05:17,710 --> 00:05:14,720 made the RNA with all two prime hydroxyl 112 00:05:19,660 --> 00:05:17,720 or substituted all geez with two prime 113 00:05:23,680 --> 00:05:19,670 fluoro or all a's with two prime photo 114 00:05:25,780 --> 00:05:23,690 all C's or all use one at a time so 115 00:05:29,260 --> 00:05:25,790 again the same idea to prime hydroxyl 116 00:05:30,930 --> 00:05:29,270 the RNA without the phosphate donor the 117 00:05:33,190 --> 00:05:30,940 RNA of course doesn't get phosphorylated 118 00:05:35,410 --> 00:05:33,200 once you have the phosphoryl donor it 119 00:05:38,740 --> 00:05:35,420 gets phosphorylated so get stuck at the 120 00:05:42,250 --> 00:05:38,750 mercury layer and we were really 121 00:05:44,680 --> 00:05:42,260 surprised that when you replace all 122 00:05:48,220 --> 00:05:44,690 these ribonucleotides with two prime 123 00:05:51,250 --> 00:05:48,230 floros they still get phosphorylated and 124 00:05:55,110 --> 00:05:51,260 the ribozyme is still active so we 125 00:05:57,490 --> 00:05:55,120 wanted to know more about about this 126 00:05:59,590 --> 00:05:57,500 this ribozyme because of what this tells 127 00:06:02,530 --> 00:05:59,600 us is that actually no two prime 128 00:06:04,570 --> 00:06:02,540 hydroxyl is important for this or no 129 00:06:07,240 --> 00:06:04,580 particular two prime hydroxyl is 130 00:06:09,730 --> 00:06:07,250 important for the activity because I 131 00:06:11,830 --> 00:06:09,740 also tried making the ribozyme as a 132 00:06:13,960 --> 00:06:11,840 complete deoxyribose I meant that is 133 00:06:15,880 --> 00:06:13,970 dead so of course like if you start 134 00:06:19,060 --> 00:06:15,890 replacing multiple to prime hydroxyls 135 00:06:25,810 --> 00:06:19,070 from multiple nucleotides you lose the 136 00:06:28,660 --> 00:06:25,820 activity so initially what we had found 137 00:06:32,200 --> 00:06:28,670 was that for the ribozyme that with the 138 00:06:34,840 --> 00:06:32,210 two prime hydroxyl the rate of the 139 00:06:37,120 --> 00:06:34,850 reaction increases as you increase the 140 00:06:39,370 --> 00:06:37,130 pH and one of the one obvious 141 00:06:41,500 --> 00:06:39,380 explanation to this would be as you 142 00:06:43,990 --> 00:06:41,510 increase the pH the two prime hydroxyl 143 00:06:47,290 --> 00:06:44,000 gets deprotonated and that somehow 144 00:06:49,589 --> 00:06:47,300 increases the rate of the reaction 145 00:06:52,779 --> 00:06:49,599 now that I had all the two prime 40 146 00:06:54,520 --> 00:06:52,789 ribozymes I I tested all these ribozymes 147 00:06:56,740 --> 00:06:54,530 with the two prime for in positions at 148 00:07:00,189 --> 00:06:56,750 different PHS as well and they all 149 00:07:03,129 --> 00:07:00,199 showed similar behavior and they all 150 00:07:05,439 --> 00:07:03,139 were being stimulated at higher pH as 151 00:07:07,270 --> 00:07:05,449 well now what this tells us is that it's 152 00:07:09,369 --> 00:07:07,280 not the two prime hydroxyl that is being 153 00:07:12,790 --> 00:07:09,379 deprotonated but there is something else 154 00:07:15,100 --> 00:07:12,800 going on and and and we would be really 155 00:07:17,200 --> 00:07:15,110 intrigued by this data because all this 156 00:07:19,450 --> 00:07:17,210 time we also thought that it was the two 157 00:07:21,760 --> 00:07:19,460 prime hydroxyl that was getting 158 00:07:24,490 --> 00:07:21,770 phosphorylated but it might not be the 159 00:07:26,740 --> 00:07:24,500 case anymore so to answer that the 160 00:07:30,249 --> 00:07:26,750 questions what we started to do was so 161 00:07:32,740 --> 00:07:30,259 this is the full ribozyme what I did was 162 00:07:34,719 --> 00:07:32,750 I basically designed a trans-acting 163 00:07:36,459 --> 00:07:34,729 version of the ribozyme where the 164 00:07:38,589 --> 00:07:36,469 phosphor the strength the part that gets 165 00:07:40,629 --> 00:07:38,599 phosphorylated is actually separated 166 00:07:42,459 --> 00:07:40,639 from the catalytic strand so you bring 167 00:07:44,499 --> 00:07:42,469 these two molecules together the 168 00:07:46,930 --> 00:07:44,509 catalytic strand base pairs with the 169 00:07:49,420 --> 00:07:46,940 substrate strand and phosphorylates at 170 00:07:52,420 --> 00:07:49,430 this guanosine position now what what 171 00:07:54,490 --> 00:07:52,430 this the design enabled us to do was we 172 00:07:56,830 --> 00:07:54,500 could actually test different substrates 173 00:07:59,860 --> 00:07:56,840 now and that's basically what I started 174 00:08:02,709 --> 00:07:59,870 out doing first of all I I used on all 175 00:08:04,420 --> 00:08:02,719 RNA backbone as a substrate and all DNA 176 00:08:08,939 --> 00:08:04,430 as the backbone as the substrate and 177 00:08:12,909 --> 00:08:08,949 then a DNA RNA hybrid substrate as well 178 00:08:14,200 --> 00:08:12,919 when to our surprise everything all of 179 00:08:16,390 --> 00:08:14,210 these three substrates got 180 00:08:18,969 --> 00:08:16,400 phosphorylated now what is really 181 00:08:22,379 --> 00:08:18,979 intriguing was that for the second 182 00:08:24,519 --> 00:08:22,389 guanosine the only like the only obvious 183 00:08:27,760 --> 00:08:24,529 phosphorylation site is the two prime 184 00:08:30,760 --> 00:08:27,770 hydroxyl and in a DNA we know from 185 00:08:34,209 --> 00:08:30,770 biochem or biology 101 that there is 186 00:08:37,029 --> 00:08:34,219 absolutely no two prime hydroxyl so this 187 00:08:40,000 --> 00:08:37,039 is actually first observation of direct 188 00:08:43,990 --> 00:08:40,010 nucleobase phosphorylation by a kinase 189 00:08:46,120 --> 00:08:44,000 ribozyme now why this is important is 190 00:08:49,360 --> 00:08:46,130 because once you start modifying 191 00:08:52,300 --> 00:08:49,370 nucleotides and early prebiotic earth 192 00:08:55,240 --> 00:08:52,310 would have more nucleotides to sample 193 00:08:57,699 --> 00:08:55,250 from it would have that's basically a 194 00:08:59,980 --> 00:08:57,709 way of generating different kinds of 195 00:09:01,180 --> 00:08:59,990 nucleotides so that nature can sample to 196 00:09:05,230 --> 00:09:01,190 see which one 197 00:09:06,700 --> 00:09:05,240 is optimum for life to use so if you 198 00:09:09,760 --> 00:09:06,710 actually go back to the structure of 199 00:09:11,770 --> 00:09:09,770 this complex everything from the 200 00:09:13,180 --> 00:09:11,780 substrate is actually base paired with 201 00:09:16,150 --> 00:09:13,190 the ribozyme so that's actually pretty 202 00:09:17,950 --> 00:09:16,160 boring it's just the DNA RNA helix 203 00:09:20,200 --> 00:09:17,960 there's not much going on but if you 204 00:09:23,800 --> 00:09:20,210 look at the business end here this GGA 205 00:09:26,020 --> 00:09:23,810 is this phi prime GG and a there it's 206 00:09:28,840 --> 00:09:26,030 free and it is free to do stuff it can 207 00:09:32,410 --> 00:09:28,850 actually interact with other parts of 208 00:09:35,170 --> 00:09:32,420 the ribozyme so i started out testing 209 00:09:37,810 --> 00:09:35,180 whether you can change this GG na so 210 00:09:41,830 --> 00:09:37,820 that's GGA to other nucleotides or not 211 00:09:44,470 --> 00:09:41,840 so if you mutated this G to a C or tea 212 00:09:46,390 --> 00:09:44,480 or a the ribozyme is dead so these are 213 00:09:49,060 --> 00:09:46,400 very soft mutations where you're only 214 00:09:51,160 --> 00:09:49,070 changing some atoms so in this case 215 00:09:54,220 --> 00:09:51,170 you're going from g2 and in a scene 216 00:09:58,210 --> 00:09:54,230 where you're only taking this amine out 217 00:10:00,910 --> 00:09:58,220 and for this g1 and g2 pretty much any 218 00:10:03,850 --> 00:10:00,920 soft mutation that you do the ribozyme 219 00:10:07,930 --> 00:10:03,860 is dead like it absolutely is very 220 00:10:09,220 --> 00:10:07,940 specific to this GG a sequence now this 221 00:10:11,320 --> 00:10:09,230 will this will be very important we'll 222 00:10:15,640 --> 00:10:11,330 come back to this again for the second 223 00:10:18,460 --> 00:10:15,650 part of my talk we basically then wanted 224 00:10:22,600 --> 00:10:18,470 to understand what kind of product is 225 00:10:25,180 --> 00:10:22,610 this ribozyme forming so do you answer 226 00:10:27,850 --> 00:10:25,190 that I basically took the substrate did 227 00:10:30,160 --> 00:10:27,860 not react with the ribozyme but added a 228 00:10:33,970 --> 00:10:30,170 phosphate on the very five prime end of 229 00:10:36,760 --> 00:10:33,980 the ribose sugar take this substrate 230 00:10:39,700 --> 00:10:36,770 incubated it at different ph is from 231 00:10:42,010 --> 00:10:39,710 nine to four point five and you can see 232 00:10:43,900 --> 00:10:42,020 that the Firefall now the product that 233 00:10:47,890 --> 00:10:43,910 has the phosphate the phosphate is still 234 00:10:50,350 --> 00:10:47,900 stable at all this pH range next what I 235 00:10:52,990 --> 00:10:50,360 did was I took the same substrate now 236 00:10:54,970 --> 00:10:53,000 this time added the phosphate using the 237 00:10:57,190 --> 00:10:54,980 ribozyme and and we know that the 238 00:10:59,050 --> 00:10:57,200 phosphate is on a nuclear base it's not 239 00:11:00,910 --> 00:10:59,060 on the two prime hydroxyl but we don't 240 00:11:03,880 --> 00:11:00,920 know what Adam the phosphate is attached 241 00:11:05,770 --> 00:11:03,890 to now you take this product formed by 242 00:11:07,990 --> 00:11:05,780 the ribosome and do the same experiment 243 00:11:09,970 --> 00:11:08,000 incubated at different pages the 244 00:11:13,450 --> 00:11:09,980 phosphate actually starts to come off so 245 00:11:14,889 --> 00:11:13,460 chemically speaking the ribozyme forms a 246 00:11:18,129 --> 00:11:14,899 different product they 247 00:11:21,369 --> 00:11:18,139 and what you have if it was just a 248 00:11:23,049 --> 00:11:21,379 ribose phosphorylation and then I 249 00:11:24,699 --> 00:11:23,059 started digging out literature to see 250 00:11:26,499 --> 00:11:24,709 like what people have done this is like 251 00:11:28,600 --> 00:11:26,509 I don't know I don't even know when this 252 00:11:30,460 --> 00:11:28,610 paper was probably uh give us from 2004 253 00:11:33,519 --> 00:11:30,470 but what what this paper essentially 254 00:11:37,359 --> 00:11:33,529 said was that in acidic conditions the 255 00:11:40,109 --> 00:11:37,369 pn bond they break faster than p 0 bond 256 00:11:42,730 --> 00:11:40,119 so what this suggests is that the 257 00:11:45,340 --> 00:11:42,740 phosphate might actually be linked in 258 00:11:46,989 --> 00:11:45,350 one of these two atoms so we still don't 259 00:11:50,230 --> 00:11:46,999 know where the phosphate is linked but 260 00:11:53,069 --> 00:11:50,240 it is pretty interesting that we are 261 00:11:57,189 --> 00:11:53,079 starting to see this novel targets for 262 00:11:59,679 --> 00:11:57,199 RNA catalyzed phosphorylation so that's 263 00:12:02,439 --> 00:11:59,689 the sort of summary of the first part of 264 00:12:04,329 --> 00:12:02,449 my talk I guess is that this ribozyme 265 00:12:08,619 --> 00:12:04,339 phosphorylates the nuclear base directly 266 00:12:10,090 --> 00:12:08,629 and the phosphoryl acceptor is likely an 267 00:12:12,910 --> 00:12:10,100 end we don't know if whether it's an end 268 00:12:14,919 --> 00:12:12,920 to or an n1 and obviously this is the 269 00:12:17,710 --> 00:12:14,929 first evidence of direct modification by 270 00:12:20,110 --> 00:12:17,720 of nuclear base by an re enzyme and as i 271 00:12:23,230 --> 00:12:20,120 said earlier nucleobase modification 272 00:12:25,299 --> 00:12:23,240 would have been a nice way to explore 273 00:12:30,220 --> 00:12:25,309 the chemical space four nucleotides in 274 00:12:33,129 --> 00:12:30,230 an early RNA work so this brings to sort 275 00:12:37,150 --> 00:12:33,139 of the second part of my talk and and 276 00:12:39,759 --> 00:12:37,160 and so you have all these ribosomes that 277 00:12:42,699 --> 00:12:39,769 people select and and they they say oh 278 00:12:44,710 --> 00:12:42,709 we found this ribozyme but they just end 279 00:12:47,379 --> 00:12:44,720 it at bat what I what are you going to 280 00:12:51,100 --> 00:12:47,389 do with it so I was looking for ideas to 281 00:12:54,790 --> 00:12:51,110 use this ribozyme to regulate other RNAs 282 00:12:59,199 --> 00:12:54,800 that are of biological or Astra 283 00:13:01,090 --> 00:12:59,209 biological significance I guess so if 284 00:13:03,639 --> 00:13:01,100 you look at the ribozyme structure with 285 00:13:05,350 --> 00:13:03,649 a substrate everything else as I pointed 286 00:13:07,239 --> 00:13:05,360 out earlier is base paired with a 287 00:13:09,669 --> 00:13:07,249 substrate which is pretty boring like in 288 00:13:12,220 --> 00:13:09,679 principle you should be able to change 289 00:13:14,650 --> 00:13:12,230 all these base pairs and the ribozyme 290 00:13:21,850 --> 00:13:14,660 should still be active as long as this 291 00:13:26,559 --> 00:13:21,860 part is kept constant and so that's 292 00:13:28,720 --> 00:13:26,569 basically what i was studying so one of 293 00:13:31,090 --> 00:13:28,730 the functional RNA is that i wanted 294 00:13:33,460 --> 00:13:31,100 target using this ribozyme was the ATP 295 00:13:37,240 --> 00:13:33,470 aptima so this RNA molecule actually 296 00:13:40,210 --> 00:13:37,250 binds to ATP and it was discovered by 297 00:13:43,120 --> 00:13:40,220 Jack szostak in and I think it was early 298 00:13:46,120 --> 00:13:43,130 90s but if you if you look at the 299 00:13:48,519 --> 00:13:46,130 sequence the sequence of the ATP aptima 300 00:13:50,470 --> 00:13:48,529 already has a GGA and and that's why I 301 00:13:53,980 --> 00:13:50,480 wanted you guys to keep in mind about 302 00:13:55,990 --> 00:13:53,990 that five prime GGA terminal because the 303 00:13:59,410 --> 00:13:56,000 ribozyme actually absolutely needs it 304 00:14:01,900 --> 00:13:59,420 and turns out that the second G that the 305 00:14:06,600 --> 00:14:01,910 ribozyme would modify it actually makes 306 00:14:09,490 --> 00:14:06,610 a direct contact with the ligand a MP 307 00:14:11,350 --> 00:14:09,500 acting as a hydrogen bond donor so we 308 00:14:13,810 --> 00:14:11,360 wanted to see if whether we could design 309 00:14:16,780 --> 00:14:13,820 a ribozyme that would phosphorylate ATP 310 00:14:20,230 --> 00:14:16,790 a primer and and and what would happen 311 00:14:22,720 --> 00:14:20,240 to the ATP after function so this is I 312 00:14:25,090 --> 00:14:22,730 guess the diagram kind of messed I'm 313 00:14:27,040 --> 00:14:25,100 sorry about that but but you can't 314 00:14:29,110 --> 00:14:27,050 basically say anything but anyway we 315 00:14:31,720 --> 00:14:29,120 designed the ribozyme and turns out that 316 00:14:34,960 --> 00:14:31,730 you can phosphorylate the ATP a primer 317 00:14:37,090 --> 00:14:34,970 and as you can see it the phosphorylated 318 00:14:40,750 --> 00:14:37,100 ATP aptima again is retain in that 319 00:14:43,780 --> 00:14:40,760 mercury layer so the next thing I want 320 00:14:46,090 --> 00:14:43,790 to study was what happens when this ATP 321 00:14:49,059 --> 00:14:46,100 a primer is phosphorylated is it still 322 00:14:53,889 --> 00:14:49,069 functional and and the way you can test 323 00:14:59,379 --> 00:14:53,899 that is you have a column with a gross 324 00:15:03,579 --> 00:14:59,389 that has ATP embedded in it and what you 325 00:15:06,400 --> 00:15:03,589 do is you add ATP epimer the the RNAs 326 00:15:08,379 --> 00:15:06,410 that bind to ATP bind to the column and 327 00:15:12,160 --> 00:15:08,389 the unbound RNA is basically flow 328 00:15:16,689 --> 00:15:12,170 through and you wash multiple times so 329 00:15:22,480 --> 00:15:16,699 all the unbound rnase is is alluded out 330 00:15:24,040 --> 00:15:22,490 and then you add a free ATP molecules so 331 00:15:27,189 --> 00:15:24,050 that the RNAs that are bound to the 332 00:15:30,069 --> 00:15:27,199 agarose now grab onto the free ATP 333 00:15:32,740 --> 00:15:30,079 molecules and then they allude out so 334 00:15:34,900 --> 00:15:32,750 this is basically what I did ATP aptima 335 00:15:36,970 --> 00:15:34,910 without the phosphate you add to the 336 00:15:41,439 --> 00:15:36,980 column then you start washing they come 337 00:15:42,249 --> 00:15:41,449 off and then well they come off and then 338 00:15:44,109 --> 00:15:42,259 the 339 00:15:45,969 --> 00:15:44,119 certain amount of ATP a primer that is 340 00:15:48,879 --> 00:15:45,979 still bound to the column and then when 341 00:15:51,729 --> 00:15:48,889 you wash it with ATP containing buffer 342 00:15:53,559 --> 00:15:51,739 then the eye primers bind to the 343 00:15:56,619 --> 00:15:53,569 free-floating ATP and then they allude 344 00:15:59,259 --> 00:15:56,629 out as well the story is different when 345 00:16:01,539 --> 00:15:59,269 the ATP a primer is phosphorylated the 346 00:16:03,489 --> 00:16:01,549 abbé de mer can no longer bind to the 347 00:16:07,269 --> 00:16:03,499 column so this could be a very 348 00:16:10,629 --> 00:16:07,279 interesting way of regulating functional 349 00:16:12,639 --> 00:16:10,639 rnase in an RNA world because because if 350 00:16:14,319 --> 00:16:12,649 there is an a functional area if there 351 00:16:17,019 --> 00:16:14,329 wasn't functional RNA molecule that 352 00:16:19,269 --> 00:16:17,029 bound to ntps and if there were 353 00:16:21,789 --> 00:16:19,279 ribozymes that started phosphorylating 354 00:16:24,279 --> 00:16:21,799 these these functional domains of these 355 00:16:27,969 --> 00:16:24,289 epimers then the ribozyme of the app 356 00:16:30,699 --> 00:16:27,979 members would no longer be functional so 357 00:16:32,439 --> 00:16:30,709 this brings to then I started looking at 358 00:16:34,419 --> 00:16:32,449 whether okay you can you can 359 00:16:38,199 --> 00:16:34,429 phosphorylate ATP a primers can you 360 00:16:41,229 --> 00:16:38,209 phosphorylate longer RNA chains and and 361 00:16:43,359 --> 00:16:41,239 this is sort of like a background of why 362 00:16:45,549 --> 00:16:43,369 we want to do that so this is just a 363 00:16:48,279 --> 00:16:45,559 simple primary extension a say that I'm 364 00:16:50,109 --> 00:16:48,289 showing here you have a template you 365 00:16:51,939 --> 00:16:50,119 have a primer the primer binds to the 366 00:16:54,429 --> 00:16:51,949 template and then you can add of DNA 367 00:16:58,299 --> 00:16:54,439 polymerase to extend it whereas if you 368 00:17:01,059 --> 00:16:58,309 have a phosphorylated template you add 369 00:17:02,769 --> 00:17:01,069 the primer anneal it extend it but the 370 00:17:05,860 --> 00:17:02,779 extension stops right where the 371 00:17:09,069 --> 00:17:05,870 phosphate is and this is basically what 372 00:17:11,470 --> 00:17:09,079 this just shows primer extended primer 373 00:17:13,569 --> 00:17:11,480 once you add the phosphate it stops 374 00:17:16,179 --> 00:17:13,579 right where the phosphate is you can 375 00:17:17,860 --> 00:17:16,189 make the template longer again you see 376 00:17:21,059 --> 00:17:17,870 extension but as soon as you add the 377 00:17:25,689 --> 00:17:21,069 phosphate it stops right there as well 378 00:17:27,669 --> 00:17:25,699 now what I was trying to get out was you 379 00:17:31,740 --> 00:17:27,679 have em RNA sequences that might also 380 00:17:34,869 --> 00:17:31,750 have GGA in them so what happens if you 381 00:17:36,909 --> 00:17:34,879 phosphorylated a G and and while the 382 00:17:39,430 --> 00:17:36,919 ribosome is trying to scan through this 383 00:17:43,649 --> 00:17:39,440 mRNA sequence what does it do when it 384 00:17:46,869 --> 00:17:43,659 encounters a modified nucleotide and and 385 00:17:49,360 --> 00:17:46,879 we know that the codon anticodon 386 00:17:51,519 --> 00:17:49,370 interactions are primarily governed by 387 00:17:53,799 --> 00:17:51,529 the Watson and Crick base pairing and 388 00:17:54,570 --> 00:17:53,809 when you're phosphorylating nuclear base 389 00:17:56,279 --> 00:17:54,580 and g 390 00:17:58,080 --> 00:17:56,289 you're essentially messing up with that 391 00:18:00,269 --> 00:17:58,090 watson-crick base pairing and and I 392 00:18:03,060 --> 00:18:00,279 think this might be a bird this might be 393 00:18:06,360 --> 00:18:03,070 a really good way to turn off genes as 394 00:18:09,389 --> 00:18:06,370 well so do you answer that question we 395 00:18:12,690 --> 00:18:09,399 started we started generating new 396 00:18:17,070 --> 00:18:12,700 ribozymes that would target sequences of 397 00:18:19,230 --> 00:18:17,080 a longer RNA chain so we this is an EG 398 00:18:21,870 --> 00:18:19,240 enhanced green fluorescent protein mRNA 399 00:18:25,769 --> 00:18:21,880 sequence and it turns out that it has 11 400 00:18:29,250 --> 00:18:25,779 sites that have a GGA sequence in them 401 00:18:30,990 --> 00:18:29,260 and i think everything has shifted so 402 00:18:33,509 --> 00:18:31,000 you can't really see there's one there's 403 00:18:36,269 --> 00:18:33,519 one there's one but but there are 11 404 00:18:38,370 --> 00:18:36,279 sites that have GGA in it so we 405 00:18:40,680 --> 00:18:38,380 basically generated ribozymes to target 406 00:18:42,629 --> 00:18:40,690 each of these 11 sites and turns out we 407 00:18:45,210 --> 00:18:42,639 can actually phosphorylate multiple 408 00:18:46,649 --> 00:18:45,220 sites in g enhance green fluorescent 409 00:18:47,879 --> 00:18:46,659 protein so what we're going to do next 410 00:18:50,220 --> 00:18:47,889 is we're going to take this 411 00:18:52,799 --> 00:18:50,230 phosphorylated mrna and do an in vitro 412 00:18:54,960 --> 00:18:52,809 translation experiment to see whether 413 00:18:58,220 --> 00:18:54,970 this phosphorylated mrnas are 414 00:19:01,830 --> 00:18:58,230 translatable or not if they are well if 415 00:19:04,710 --> 00:19:01,840 what we hope is that the translation is 416 00:19:07,860 --> 00:19:04,720 shut off after phosphorylation and this 417 00:19:10,730 --> 00:19:07,870 would mean that phosphorylation by 418 00:19:13,110 --> 00:19:10,740 ribosomes could be a new avenue for 419 00:19:14,909 --> 00:19:13,120 artificial genetic regulation and 420 00:19:17,490 --> 00:19:14,919 possibly could be used in synthetic 421 00:19:20,700 --> 00:19:17,500 biology as well we're also working on to 422 00:19:22,680 --> 00:19:20,710 reverse the phosphor alysha the first 423 00:19:25,649 --> 00:19:22,690 part of my talk I showed that at low PH 424 00:19:27,629 --> 00:19:25,659 you start losing the phosphate so we're 425 00:19:30,060 --> 00:19:27,639 also trying to explore what happens once 426 00:19:32,190 --> 00:19:30,070 the phosphate is lost can you regain the 427 00:19:35,840 --> 00:19:32,200 active functional activity that you 428 00:19:38,070 --> 00:19:35,850 initially had as well so with that well 429 00:19:41,850 --> 00:19:38,080 this is a summary of the second part 430 00:19:44,789 --> 00:19:41,860 well functional rnase can be targeted 431 00:19:46,799 --> 00:19:44,799 for ribozyme mediated regulation of 432 00:19:50,850 --> 00:19:46,809 other functional RNA molecules so you're 433 00:19:52,860 --> 00:19:50,860 you're basically so this is literally 434 00:19:55,830 --> 00:19:52,870 making like an RNA world because you are 435 00:19:59,659 --> 00:19:55,840 controlling activity of one functional 436 00:20:03,360 --> 00:19:59,669 RNA molecules with another ribozyme and 437 00:20:06,000 --> 00:20:03,370 mRNA molecules mrna phosphorylation may 438 00:20:08,130 --> 00:20:06,010 potentially be useful for artificially 439 00:20:11,100 --> 00:20:08,140 regulating gene expression 440 00:20:14,310 --> 00:20:11,110 and this basically means that metabolic 441 00:20:16,980 --> 00:20:14,320 networks in an RNA world could be 442 00:20:18,930 --> 00:20:16,990 mediated by ribosomes and with that I 443 00:20:21,810 --> 00:20:18,940 think that's it yeah I'd like to thank 444 00:20:30,100 --> 00:20:21,820 my lab and of course of you all for your 445 00:20:42,109 --> 00:20:39,680 any questions thanks for that it was an 446 00:20:44,659 --> 00:20:42,119 interesting talk now you mentioned a 447 00:20:46,460 --> 00:20:44,669 little bit before the relationship 448 00:20:48,830 --> 00:20:46,470 between that proteins and and sort of 449 00:20:51,019 --> 00:20:48,840 RNA enzymes and when I think of a 450 00:20:54,200 --> 00:20:51,029 protein you know the active site usually 451 00:20:56,960 --> 00:20:54,210 has a metal there right right so how 452 00:20:59,570 --> 00:20:56,970 does this work with RNA enzymes beat DJ 453 00:21:02,810 --> 00:20:59,580 is it so you incorporate a metal at some 454 00:21:05,539 --> 00:21:02,820 point well some are enzymes they utilize 455 00:21:07,039 --> 00:21:05,549 may require metals absolutely but there 456 00:21:09,859 --> 00:21:07,049 have been some that are metal 457 00:21:11,930 --> 00:21:09,869 independent as well this one this one 458 00:21:14,419 --> 00:21:11,940 particular actually uses magnesium and 459 00:21:16,129 --> 00:21:14,429 and copper for some reason but yeah just 460 00:21:17,749 --> 00:21:16,139 a quick follow-up do you know how those 461 00:21:20,060 --> 00:21:17,759 metals are incorporated I mean today 462 00:21:21,889 --> 00:21:20,070 mostly are they kind of dynamic like do 463 00:21:23,869 --> 00:21:21,899 they come in and go out or are they 464 00:21:25,310 --> 00:21:23,879 fixed like when you have this kind of 465 00:21:27,830 --> 00:21:25,320 structure that's almost like a protein 466 00:21:29,419 --> 00:21:27,840 it's pretty much fixed just like 467 00:21:31,669 --> 00:21:29,429 proteins there's specific metal binding 468 00:21:33,169 --> 00:21:31,679 sites metal ion binding sites and and 469 00:21:35,180 --> 00:21:33,179 these metal ions usually interact with 470 00:21:36,710 --> 00:21:35,190 the two prime hydroxyls as well and so 471 00:21:44,509 --> 00:21:36,720 it's really important for the RNA 472 00:21:47,210 --> 00:21:44,519 folding Thanks a quick question about 473 00:21:50,979 --> 00:21:47,220 your first part of your talk so you are 474 00:21:55,070 --> 00:21:50,989 showing the the function of your 475 00:21:57,919 --> 00:21:55,080 ribozyme against pH right I noticed the 476 00:21:59,869 --> 00:21:57,929 the Ranger you did was a little bit 477 00:22:02,180 --> 00:21:59,879 tight did you try pushing it very far 478 00:22:04,190 --> 00:22:02,190 out of here actually yes so that work 479 00:22:06,229 --> 00:22:04,200 had already been done before and I was 480 00:22:07,999 --> 00:22:06,239 basically only testing it at a small 481 00:22:10,340 --> 00:22:08,009 range and and yes it is evident in our 482 00:22:13,220 --> 00:22:10,350 larger range as well I think we have 483 00:22:15,229 --> 00:22:13,230 gone from five to eight point five or 484 00:22:18,019 --> 00:22:15,239 something like that Thank you Thank 485 00:22:24,229 --> 00:22:18,029 other questions maybe some from say