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[Music]

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[Applause]

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so we've been interested in complex

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class rates as model prebiotic

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compartments because they are very easy

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to assemble and they can accumulate

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different biomolecules that are very

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relevant to provide a chemistry so a

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little bit of background on how these

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guys form is through assembly of

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polyelectrolytes so I'm showing here

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poly anion and poly cation they can

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assemble together to form these dense

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phase and dilute phase so the dense

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phase actually contains a lot of all the

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electrolytes and and charged molecules

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since and since RNA is highly charged

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they can also be expected to accumulate

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within these droplets so before I joined

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Phil's lab graduates in the lab Erica

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Frankel she had done a lot of nice work

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with the system of poly allele amine

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hydrochloride and ATP where obviously

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the poly a lil amine is the poly cation

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on the ATP is the poly anion and when

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you add these things together you form

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these really nice droplets that

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accumulate I'm not gonna go into too

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much detail but what she showed was you

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could really accumulate magnesium

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nucleotide and RNA for example when she

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used ADP and poly ethyl amine to form

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these Kumasi rates and only added about

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5 milli molar magnesium bulk the

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concentration of magnesium in the

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coacervate swear upwards of molar so

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that's tremendous enhancements of

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magnesium levels which might be very

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relevant for chemistry's like non

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enzymatic RNA polymerize ation or even

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ribozyme catalyze RNA polymerize ation

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as well and obviously what she also

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found was that RNA different rnase were

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also highly enriched in these Kumasi

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rates so what we started to do was we

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started exploring different poly cations

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and poly anions to form these class

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rates and ask whether they can

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accumulate rnase and whether they can

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participate in chemistry's that we care

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about in terms of RNA world hypothesis

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so you can see that all the go lysine

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poly dialy o dimethyl

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for IP DAC all the arginine they all

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formed these nice class roommates when

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we add all ego RNA to the solution and

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what I didn't tell you was that we

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actually added a size five or site we

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labeled RNA in these class arrays and

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you can see that the RNA really

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accumulates in these droplets and we're

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going to use the same RNA for template

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directed RNA polymerize ation that i'm

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gonna talk to you guys here so again

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these are the different classifications

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that we're using we're using poly a RNA

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to form the to have the poly anionic

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part and we're using pollyali Lamine

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PTAC illegal I seen illegal arginine as

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the poly cationic part and what we do is

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we add this RNA primer template complex

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and you can see that the template has a

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stretch of C's where this activated

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nucleotide that is aji can come in and

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the primer gets extended and what we do

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is that we add everything together and

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then we separate the two phases so that

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the coacervate phase is separate from

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the dilute phase and we monitor the

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reaction in these two phases separately

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so what happens when you don't have any

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class rates of course you see that the

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RNA starts to the primer starts to get

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longer because this monomer gets

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incorporated and what was really

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interesting was that in the case of the

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dilute phase which is the supernatant

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phase you don't really see that much RNA

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which is expected because a lot of the

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RNA ends up in the condensed phase

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inside the coacervate but while striking

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was that in the condensed phase only the

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B tack only the class of rates that

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formed that were formed from the B DAC

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and all ego RNA they supported the non

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enzymatic polymerization and you can see

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that all the ones that don't support non

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enzymatic polymerization with PAH oligo

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arginine and all ego lysine they can not

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only engage in short charged

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interactions but they also have this

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ability to hydrogen bonds so we think

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that this super high concentration of

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hydrogen bond donors acceptors and and

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charged groups within the co acid rates

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they might be somehow inhibiting this

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reaction potentially miss folding or

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unfolding the primer temper

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complex since we knew that some of the

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crafts are eight other crafts of eight

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systems in increase the magnesium

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concentrations within the condensed

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phase we thought it might be interesting

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to look at non enzymatic polymerization

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in different magnesium concentrations in

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the presence of colossal rates so you

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can see that without any class of rates

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in buffer as you decrease the magnesium

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level you do see you do take a hit in

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how long the RNA is formed by this

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reaction and what was really interesting

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was when we did the same thing but in

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the presence of P deck and oligo RNA

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Kassovitz you deuce take a little bit of

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hit in the beginning but then even when

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we didn't add any magnesium you stills

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formed like decent amount of plus one

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and you even see some amount of plus two

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of band form from non enzymatic

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polymerization and and that's quantified

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here and so to really get into more

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detail of what was going on we decided

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to add an excess of EDTA to the reaction

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so when we added ATT a very little

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background reaction that was that was

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happening even in the absence of

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magnesium that goes away as well as

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expected because there might be some

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background dye valence that the ETA is

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chelating and what was really surprising

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was that when we added the PTAC which is

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the cationic polymers that we were using

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to form the coacervate we actually

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rescue the the the the reaction back so

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what this was telling us was that not

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only that Kosar rates can act as this

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passive compartments that are just

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taking up RNA molecules but they might

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be actually engaging in in the chemistry

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as well so that was really interesting

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so having looked at non enzymatic

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polymerization we decided to now you

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know go study a little more complex

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system and the the sequel to this you've

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already heard yesterday from phil where

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we wanted to look at ribozyme catalysis

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within within complex krasin rate system

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and the model we use is the hammerhead

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ribozyme which is shown in shown here so

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the black strand is the enzyme strand

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which base pairs with a substrate strand

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which is shown in green and then cuts

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the substrate right where this red

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triangle is

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and so the idea was you know if in an

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early Earth the the enzymes were

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probably not very abundant they're

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probably very scarce and so how do you

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and further insult for this particular

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reaction to happen of course the enzyme

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needs to find the substrate so if you

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are and by the way this is just a native

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gel that shows the association between

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the enzyme and the substrate so if you

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have a lot of enzyme present then you

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start to associate with the substrate so

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if the molecule becomes larger so it

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moves up but if you don't have enough

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enzyme you're below the dissociation

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constant then you don't see much

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association and and and so the substrate

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is not really interacting with the

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enzyme and presumably when the

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functional molecules were very scarce in

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the early Earth you are probably but we

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were probably dealing with this sort of

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regime SOCAN complex Craster rates

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activate these kind of chemistry's when

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you have a very limited functional RNA

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molecules so let's see what happens so

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again we tested these sort of ribozyme

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reactions with PTAC and an oligarchic

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acid casa rates so the way we do these

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experiments is we first add the poly

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cation and the pollyanna and to form the

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coacervate and then we add the ribozyme

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and the substrate separately and and and

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and monitor the reaction so to our

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surprise indeed well in case a buffer

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where we don't have any co acid rates

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you don't see a lot of reaction which is

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the cleavage of the substrate because of

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course since there's only 5 nano mol or

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enzyme and the KD i think you at 1

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millimolar we estimated it to be about

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250 nanomolar so there's not a lot of

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association between the ribozyme and the

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substrate so you don't see a lot of

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reaction whereas when we have the p deck

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and the d-10 class of rates you you see

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robust enhancement of the ribozyme

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chemistry so not only about what we

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think is going on is that the cursor

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rates are bringing these RNA molecules

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together and promoting RNA RNA

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interactions that are not possible in

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dilute solutions we tested several

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different class rate compositions for

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example PTAC and illegal aspartic acid

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obviously they activate the ribozyme

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illegal lysine and all the grass partic

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acid they

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these classmates also tended to activate

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a ribozyme a legal arginine on the other

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hand we saw inhibition of the reaction

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and we do the same and I think based on

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Irene's work

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we know that arginine is highly

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interactive with RNA and then I think

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the Aligarh journey is probably

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misfolding the ribozyme when it gets

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inside the complex

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Craster rates and finally what we

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decided to do was we decided to test

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whether this sort of system is

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widespread can other enzymes also be

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activated using these this mechanism and

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so this is the 10:23 DNA's I'm which was

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which is from Gerry Joyce's lab and it

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also catalyzed a similar chemistry the

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DNA strand base pairs with a substrate

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which is shown in blue and cuts where

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the red triangle is and here you can see

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that with high ends the DNA is I'm

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concentration you can see nice cleavage

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of the substrate but when you reduce the

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DNA's I'm to five nano molar you don't

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see a lot of chemistry being happening

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and when we do the same reaction now in

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the presence of complex class of eight

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so you can see that the DNA's I'm also

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gets turned on and we've done this with

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also hairpin ribozyme which I didn't

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have time to talk about in that case the

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enzyme actually gets activated by

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concentration of sperm in which is not

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really in our neighbor a small molecule

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so there there there multiple ways that

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coacervate seem to be enhancing

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different nucleic acid enzymes and

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that's just the quantification from

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different magnesium levels so in summary

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what I want to talk about is that you

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know we've shown that specific complex

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class rates can not only partition RNA

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molecules but they can also enhance

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template directed polymerization under

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limiting magnesium concentrations and

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concentration of RNA and other Co

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solutes can lead to enhancements of RNA

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catalysis and finally we we've shown

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that complex crossbred mediated

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enhancements seem to be general as in

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different different composition of cras

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rates can activate different nucleic

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acid enzymes so we think that this might

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be a very useful thing to study for

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diverse nucleic acid enzymes and

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with that I would like to thank Phil's

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lab and and Chris's lab who's been like

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really instrumental in and we have had

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an awesome collaboration in all those

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liquid liquid phase separated systems

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and we're starting to look at liquid

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liquid phase separation in cells with

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Geraldine City at Johns Hopkins we're

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starting to do some ribozyme catalyzed

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RNA polymerize ation with Gerry Joyce

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and we're also looking nan non-enzymatic

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oligonucleotide phosphorylation with Ron

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Krishnamurthy lab so a lot of cool