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[Music]

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[Applause]

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good afternoon everybody

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this is going to be a kind of a tale of

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a set of reactions based on this DCC

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system okay so a little bit of a

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preamble or a prologue to the story

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there are various kinds of hypothesis as

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to how prebiotic life could have emerged

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into the last Universal common ancestor

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but assuming that there was a

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hypothetical RNA world the question

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remains as to how the our own

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hypothetical on a wall got to the Lucas

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stage if we look at life from a top-down

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approach one eventually goes down to the

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ribosome which I strongly believe based

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on the evidence it's the largest white

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elephant in the room of prebiotic life

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and also the Luca if you look at the

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ribosome you go deep down to the

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ribosome you end up with the PTC but

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it's almost like at the end of the

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ladder when you look down what do you

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see what do you see is a prebiotic wall

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assuming that there was a hypothetical

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on a wall how did we get from the

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hypothetical on a wall to the locust

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stage so the the current hypothesis is

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that the hypothetical in a world was

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probably terminated by a proto Ramazan

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wall an ancient peptide making machine

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however there's a small problem the

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absence of a demonstrable replicase

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therefore the absence of this replicase

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we have heard a lot about the dry cycles

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in a similar fashion we have this

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alternate system which was developed in

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a lab it's called the dynamic

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combinatorial chemistry system which

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uses cycles of synthesis and degradation

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degradation not much different for the

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wet/dry cycles there's one big caveat

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though the the enzymes that we use are

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not particularly very prebiotic not at

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all however that's not the point whether

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we use enzymes or whether it was any the

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system of degradation and ligation the

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RNA sequence accretion pathways are

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likely to have been along similar lines

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that's the central idea now what did we

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do we use two enzymes one of them is the

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nucleus

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nucleic acids while having absolutely

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zero proteolytic activity and when it

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does the degradation it creates a five

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prime phosphate N and three prime

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evasion in the degraded product which is

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perfect as a substrate for the following

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enzyme which is a t4 RNA ligase one

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which catalyzes the ligation of these

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two products if the help of ATP so what

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do we do we put them together in a

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buffer system which we establish along

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with an RNA ala go and while one enzyme

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cuts the other our game enzyme puts it

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puts it together and this cycle goes on

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or a period of time in day and the going

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assumption is that this will help in

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increase in buildup of the RNA size if

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not complexity I'm I'm very nervous

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about using the word complexity after

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today's morning session yeah so this is

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exactly what happens shuffling of cards

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but with one small difference

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assuming that instead of just one deck

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of cards there are several decks of

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cards each deck of cards refers to one

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oligo in the entire mix so every time

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you shuffle them there is a new

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combination and hopefully build up of

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size not complexity we do not know yet

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okay so what we did we chose the seed

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Allah go which is a reasonably humble

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oligo it has no secondary structure so

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convenient and it has just one small

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little repeat of a followed by three C's

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but absolutely no secondary structure so

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you take the seed Allah go combine it

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with the benzene is and ligase along

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with ATP run it for 180 minutes take

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wood samples every 30 minutes sequence

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the same and also run gels of each of

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these samples together and what do we

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see when we run the gel we immediately

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see a forty mer which we suspect to be

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the only cue ligated with itself and we

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also see bands that gradually increase

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in intensity but past the 120 minute

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point they start decreasing because the

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ATP gets used up okay when we sequence

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these reaction pools we make DNA

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libraries

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which had its own set of problems are

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not to be talking about them but when

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you look at the sequences the blue bar

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here is the seed oligo which decreases

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over time which I which one would think

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is intuitively expected but what is

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interesting is the increase in the

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number of unique sequences which is

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unique to each time point at 30 minute

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time point there is a set of unique

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sequences that are a comparative

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compared to the seed or liqueur here at

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this level and then they keep on

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increasing in percentage

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this shows the percentage and at the 120

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and 150 minute points they are almost

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more than 85% of the total RNA pol but

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as we saw on the gel past the 120 150

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minute point 80 baguettes used up so the

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total oral oral RNA pool starts getting

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degraded because the benzene here takes

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all it's almost like a catch me if you

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can experiment so what did we learned

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from this particular explained is that

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the DCC system definitely produces

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products larger than the scene and point

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mutations are very very rare in this

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system and most of the changes involve

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insertion and deletion of small blocks

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akin to recombination events however if

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only we could manage to figure out a way

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to keep on adding ATP we'll have more

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room for extra RNA sequence space

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exploration past one 180 minutes okay

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now it is not realistic that in the

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ancient are on a wall if there was one

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that they'll just be 1 L ago what if

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there are multiple early goes so we use

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multiple seeds what we what we chose was

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this mini helix alanine our tRNA which

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has we just split it into four different

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parts and put them all together hoping

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to see what happens and what happened

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after 15 minutes distinctly this

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particular section with the CCA end

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completely disappeared you cannot see

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rain right here but after 15 minutes you

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still see the other other 3 on top but

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after 180 minutes

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you find that this three these three

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have been ligated together - the fourth

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part and there this one was nowhere in

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the picture in the initial RNA pool they

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have not occupied the amongst the first

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top ten now the original seed from which

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the four roll egos were derived had just

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one copy number that kind of reverses

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back to the stiffened golden paradox as

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I call it if you had to start life with

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pieces of the ribosomal RNA with

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something like this or even wet/dry

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cycles it's highly unlikely we will get

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something like the pre PTC so that's a

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huge challenge we have to keep in mind

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okay now it is also a reasonably fair

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assumption based on all the talks we

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have listened to that the RNA world was

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not just an RNA wall it must have had

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inputs of peptides or amino acids so how

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do the presence of peptides or amino

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acids could have impacted this sequence

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space exploration to do that we chose

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two peptides one a peptide of ancient

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origin which is highly conserved

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extension of the ribosomal protein l2

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and as a control we choose a second

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peptide which is of much more recent

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origin which is FM LP which plays an

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important role in the human immune

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system it's a short peptide and we did

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the same by putting each of these

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separately into this DCC system with the

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two enzymes along along with the oligo

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what do we see we see that while l2

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inhibits formation of the larger product

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FML b appears to favor the ligated

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product we do not know why but this is a

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very good sample to just show how the

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presence of peptides or amino acids

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could impact sequence space exploration

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when they went without them now looking

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ahead we are thinking of using the DC

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system we with rnas as well as prebiotic

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little-o and amino acids these are amino

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acids which have been catalogued very

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well by Paul Higgs and as well as

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attentions group and I'm still issuing

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using combination of these amino acids

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and short peptides again I have to make

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this caveat that's P P odd prebiotic

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systems did not have these enzymes we

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are only interested

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the dynamics of the RNA sequence space

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exploration right now here is their blog

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it's a seminal paper from Col who's in

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an atom to determine what sorts of

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prebiotic interactions between poly

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nucleotides and poly mean acids or their

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derivatives of possible while doing so

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we hope that this knowledge will in turn

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lead to the development of a concept of

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what a workable primitive form of

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translation could have been like so

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towards this visionary goal of the late

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Carlos I think it is important for us to

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trace the roots of the ribosome through

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various systems including the DCC that's

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all at thank you are there any questions

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out in the audience if anyone's

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gathering up the courage for actually

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have a question these sequences that you

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do see that are showing up are they

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correlated with any particular property

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that causes them to show up because it

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seems like in terms of sequence base

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there are so many possibilities so is

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there any commonality for what keeps

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showing up you Moore's life that they

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removed because I might be running out

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of time which is a very good habit of

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mine so benzene is the enzyme is not

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particularly agnostic it's very we found

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that from the from the statistics it

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cuts at anywhere after or before a C so

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that could be the reason why they are

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appearing however if we also tried a

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different system by using alkaline

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hydrolysis combined with another laggies

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which combines three prime phosphate

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with F with Phi prime OS but that enzyme

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number one it forms cyclical products it

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doesn't push the reaction forward number

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two it's ridiculously expensive all

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right dr. Thirumalai thank you very much