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alright hi everyone um yeah so first of

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all thanks for having me um first time

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speaker so it's been going well so far

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and i work for the Shostak lab at

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harvard and we're interested in a

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probiotic RNA replication so I'm going

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to talk a little bit about non-enzymatic

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RNA replication and then how tides can

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yeah is this good great alright so we

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got a pretty nice introduction today to

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the RNA world by from the warm-up talk

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and so just briefly presidents

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present-day cells we believe that you

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know center dot central dogma holds you

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go from DNA to RNA to protein but

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previously you know in the evolution of

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life it's possible that RNA was the

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predominant molecule inside sales and

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because they store stores and passes

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down genetic information and it can

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catalyze chemical reactions and to that

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end multiple attempts have been

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undergone to kind of show whether

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prebiotic RNA synthesis and replication

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is possible and efficient and also

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research with with ribozymes

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specifically I'm really interested in

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non-enzymatic non ribose I matic RNA

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replication and you can imagine at some

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point it's probably likely that if you

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believe in the RNA world hypothesis

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first of all that um an RNA dependent

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RNA polymerase should have been

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necessary to efficiently replicate life

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so if we kind of go to this simplistic

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model here um you have a template and a

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primer templates and black primers in

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red and to get replication going you can

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extend the primer you form the

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full-length compliment you kind of break

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that up and then you repeat the process

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and this is what essentially PCR does on

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the larger and more exponential scale

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pcrs polymerase chain reaction can

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amplify DNA expect exponentially it's

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used in labs everywhere and just a kind

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of as a comparison so modern pcr and

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prebiotic what we believe could be

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prebiotic replication you don't have

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such a huge axis of primer requires more

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magnesium because the rate of reaction

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is slower we assume there's no

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polymerase enzyme or ribozyme and

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sometimes it's required that the amount

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of nucleotides that you use are

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activated kind of lower the energy

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barrier for this reaction and so if you

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go into a prebiotic kind of model once

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you get to this you know separated

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replicated strand this guy here if you

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want to kind of effect a second round of

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replication yes in primer but due to

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thermodynamic and kinetic issues what

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happens is the fully formed duplex RIA

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needles and that's because the full

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fully formed OOP flex full length is

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much more thermodynamically stable than

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the primer template complex and also you

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can undergo strand displacement and the

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longer strand will displace the shorter

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strands so if you get end up in kind of

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this regime you can't really replicate

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any more stops you're done cells dead or

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I mean it's not dead but can't grow you

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can't evolve and so all right my

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question then is like how can we inhibit

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this process how can we hit inhibit the

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rate of strain renewing and you know

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there's some things you can do in the

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only the second order process you can

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dilute it everything slows down but then

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you kind of it's pointless because all

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the reactions go slowed and might not be

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able to do anything you can in a

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laboratory setting increase the primer

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concentration but again in a prebiotic

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setting that might not have been totally

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plausible some people have tried to use

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complementary all goo nucleotide

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fragments of small fragments that bind

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to both strands that can physically

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block annealing but the fragments

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themselves if you like have them at high

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concentration they'll kind of self

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inhibit kind of be really strange and

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not really worked very well and we're

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currently also working on you know using

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some viscous environments that can also

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possibly you know physically slow down

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inhaling and so earlier in the first

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talk Nick was it he mentioned that there

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are a lot of RNA protein interactions

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that arm involve cationic peptides and

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cationic residues and it's been shown

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that ATP can interact with simple

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polypeptides made of lysine and arginine

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and they form these coacervate

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structures which is basically a second

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phase and it kind of separate and they

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can interact with RNA molecules RNA will

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preferentially separate into one of the

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two strands so we're like okay well it's

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possible that you know is it possible we

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can get some type of you know RNA

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binding event to these small

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polypeptides and kind of slow down the

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rate of annealing so basic polypeptides

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we studied specifically arginine but you

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know lysine could also work there also

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other issues with lysine I'm not going

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to talk about this in detail with a nice

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talk yesterday that kind of outline

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where all the plausible points of entry

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that peptides could have had in in the

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prebiotic earth you know all these

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different guys we can talk about this

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later if you're interested and most

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recently so some of you might know

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arginine is like pretty low in abundance

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in a lot of these experiments and in

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meteorites dust grains and reap most

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recently it's been shown that there is a

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prebiotic we plausible synthesis of

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arginine in cyanide rich environment

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that's reducing and contains a lot of

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hydrogen sulfide and this is work done

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by the Sutherland group in the UK and so

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here we go all right so can we use these

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peptides in a way to push the

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action in this way so that you can

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affect multiple rounds of polymerization

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and so we initially did this by testing

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the first rate of strand annealing in

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the presence and absence of peptides and

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we use the nucleic acid analog here's a

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to me no purine it's an editing analog

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and it you know bonds just as well to

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uracil and what's nice about this is

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it's fluorescent and it the fluorescence

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quenches when it's in a more ordered

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state so it's when when it's more

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stacked and when it's been a duplex the

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fluorescence will decrease and we can

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use this as a direct measure of the

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fraction of RNA and one or the other for

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in an assay and we use a stop folks

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spectrometer which you basically can

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inter inject both strands separately and

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measure kinetics immediately that's very

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unfortunate yeah alright well basically

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what this what this figure shows is that

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the y-axis is the fraction of RNA in a

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single-stranded state and the x axis is

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time and this is the annealing where the

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annealing of 2 15 more rnase in the

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presence of different peptides and the

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black is with no peptide and yellow is

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with a arginine 5m ER and these guys up

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here or longer our genes and we can see

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that the first curve here the black

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curve that without peptides the

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transition from single-stranded to

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double-stranded is very fast but once

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you add peptides in its starts to slow

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down is this like is it okay if I do it

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this way you guys all yeah all right

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because all the figures now are going to

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be like really crabby okay it's too bad

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so we by increasing the peptide

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concentration

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each of these colors is a different

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length of peptide and this is the

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annealing half-life of an annealing

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reaction and so by increasing the

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peptide concentration the time that it

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takes to neil increases so the nailing

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rate is inhibited somewhat and when you

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go from a 5m ER to a 7 to a 9 mer to

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attend mer the longer the arginine

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peptide that you're using the greater

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the inhibition of the annealing effect

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and so larger longer peptides a new

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cause a greater slowing of really you

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can increase the length of the RNA and

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the longer the RNA in the presence of

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peptide the longer takes 42 RNA to nail

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and this is great because of course the

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primer is much shorter than a replicated

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strand so we want in the presence of

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peptides to see that the primer a nails

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faster than this replicated strand

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otherwise this whole system is

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meaningless unfortunately we came across

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couple problems the traditional

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non-enzymatic replication machinery

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requires magnesium and magnesium at

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increasing concentrations decreases the

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annealing time of RNA so it kind of

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disrupts the peptide RNA interaction and

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you'll go back to a much faster in

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Newton time same with our activated

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monomer we use a modified G monomer if

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you're interested we can talk about this

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later and that also because it's charged

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it disrupts this interaction and

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decrease the effectiveness of the

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peptide in slowing the India trade so

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what do we do now so we took the we use

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this system here and it's a system where

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we can add up to 4G bases on to a

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fluorescently labeled primer we incubate

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this for many hours without enzymes

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anything and you can actually extend you

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can see extension over many hours

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so I think that's pretty cool and the

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rate of extension with and without

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peptides you know differs by about

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thirty percent but when you have peptide

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the annealing rate decreases or the

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annealing rate decreases by over three

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orders of magnitude so by sacrificing

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thirty percent in the right of the

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replication you can affect such a huge

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change in the annealing rate and so the

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kind of big experiment we did here is we

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started with a reverse complement and a

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template premio added peptide added

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primer heated it up cooled it down

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immediately due to some thermodynamic

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and kinetic considerations we have to

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cool down immediately otherwise the

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reaction doesn't work and we can see

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here that the third line is in the

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presence of reverse complement but

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without peptide and you can see that

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there's no primer extension there's no

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replication because presumably the the

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strand the blue strand is renewing

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faster but in the fourth line here you

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can see that in the presence of peptide

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we do get some form of primer extension

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we do get replication and I think this

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is one of the first times it's been

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shown that you can get multiple rounds

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of a non-enzymatic primer extension and

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so yeah we we hope to optimize this

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procedure to kind of push the

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equilibrium even further into this

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direction hopefully the ultimate goal is

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to kind of create this protocell that's

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self sufficient so thank you every one

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sorry about the figures I should have

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checked it I'm going to use my privilege

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as the first question so it looks like

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that was almost an exponential increases

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you were increasing the length of the

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peptide right have you gone beyond 10 is

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that does it turn over at some point and

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stop being as effective or um we haven't

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gone above 10 and the rationale there is

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just the shorter the peptide the easier

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it would have been ache I would guess

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that you could you would just keep going

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one of the things we tested was a

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cationic dextran polymer which is like a

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I think of polysaccharide and we see is

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the kneeling is barely visible it

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basically like completely stops it so

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cool other poly cations could also have

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the same effect any other questions so

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obviously this is sort of you started

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with the assumption of like an RNA world

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type scenario but then you've invoked

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peptides to sort of keep the RNA world

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going right and so this is something

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that seems to be coming up all over the

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place is that the RNA world was clearly

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not just an RNA world so I mean do you

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have anything that you think do you

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think RNA was playing specific roles or

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most roles or do you think that there

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was just sort of a lot of RNA but there

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was also a lot of other stuff going on

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yeah so I guess it's very possible that

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the peptides and RNA emerge kind of

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independently um weather at what point

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they kind of became interdependent I'm

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not so sure but I guess that would also

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depend on like in my opinion the length

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of both of the polymers that are made

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obviously the longer the polymer is the

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more like it's multivalent the more

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possibility for interaction so you know

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it's hard to say it'd be interesting to

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see you know some some more people work

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on this and I know a lot of people are

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kind of leaning towards a pet