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[Music]

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[Applause]

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thank you thanks very much for the

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opportunity to speak so today I'd like

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to tell you about our work and looking

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at ribozyme catalysis inside of complex

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Kosar baits and I'm going to describe

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how this is assisted by anions and so we

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just heard a little bit about protocells

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and how those could come about from

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complex coacervate and we've been

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studying this in the lab for for several

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years now this is a collaboration with

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the Keating lab and some of the first

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work that we published looks at the

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coacervate in a poly cation poly a lil

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amine and a oligo anion ATP and when

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these come together by associative face

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operation to make a liquid liquid phase

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separation and these form just got cut

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off a little bit here there should be a

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picture here of coacervate sand then

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these are enriched with certain species

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that are favorable for for ribosome

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reactions such as enhanced

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concentrations of magnesium ions

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nucleotides and RNA oligomers and I'm

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going to show you a story today which we

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recently published just a few weeks ago

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came out an ACS chemical biology in

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which we have coacervate sand and in

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these are ribosomes our RNA enzymes

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which are shown in green and they go

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from this Mis folded state where they're

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associated with the poly cation and that

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we can rescue their activity by adding

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oligo anions that can displace these and

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so condensates are formed by liquid

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liquid phase separation in they're

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common in modern cells and so here's an

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image from cliff brain wines lab showing

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phase separation and this is with the

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the unstructured region of the laughs

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helicase and these come together to form

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these these condensates which are on the

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the micron size scale and we can make

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these in the lab as well so these are

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association in this case this is between

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spermine

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and a poly you as well as we can have

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them in which they're in which the poly

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cation is is the polymer and these give

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these give compartments that are similar

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on their size scale and the microns

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scale to the ones that are found in vivo

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and in order to form these condensates

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some of the features that are important

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are polyelectrolytes unstructured

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peptide domains Crowder's and co solutes

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as well as it depends on the pH ions and

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temperature and recently we published a

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review on this in biochemistry in which

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we see to see a number of processes that

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are important that could emerge for RNA

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function in an RNA world scenario and

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those are the concentration of

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nucleotides that can happen and then

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function in terms of non enzymatic these

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are templated polymerization reactions

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as well as ribosome reactions and then

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we also get the concentration not only

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are the nucleotides but also of the

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longer rnas which are enriched and

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encapsulated within the proto cell and

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these shorter ones which are less less

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so in that in that sense and I just want

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to point out that a lot of the work much

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of the work I'm going to talk about

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today is to work from a postdoc in our

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lab dr. Raghava Dial and he will be

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speaking on Thursday on template

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directed RNA polymerize ation and

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enhanced ribozyme catalysis inside of

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membrane compartments formed by

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coacervate so we're going to use

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ribosomes in order to look at function

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inside of side of complex class debates

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as models for proto cells and one of the

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reasons to look at ribozymes is we're

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interested in whether or not RNA can

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fold into a functional form inside of

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these coacervate sand ribosomes are

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ideal for that because they reveal their

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folding through the self cleavage and

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their enzymatic activity plus RNA

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activity and reactions are of an

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intrinsic interest to the RNA world

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scenario so this begins to show the

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process of an in a mechanistic way in

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the absence of coacervate that are going

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to help us understand in some way about

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how coacervate s-- themselves promote

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the reaction and it's a fairly simple

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reaction which we can understand through

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kind of a Michaelis Menten types of a

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formula that's shown here these are

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under single turnover conditions meaning

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that the substrate is in limiting

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amounts in this particular example it's

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radio labeled and it can associate with

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the enzyme to make a complex that we can

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look at on a native page this is a

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particular case where the reaction is

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prevented from advancing because at the

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cleavage site here for the hammerhead

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ribozyme there's actually a deoxy which

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removes the nucleophile in the reaction

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and we can see that as we increase the

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enzyme concentration that it shifts the

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limiting amount of the limiting reagent

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of the substrate up to this higher

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mobility species which is the enzyme

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substrate complex and we can plot this

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the fraction of the substrates that's

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bound as a function of enzyme

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concentration and we can see that in

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order to get most of the substrate bound

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it takes an increasing amount of enzyme

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concentration as its we're all familiar

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with and the question is then how does a

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similar type of reaction happen inside

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of coacervate so these are coacervate

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that are made from a quaternary amine

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polymer poly daya will dimethyl ammonium

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chloride which is a 53 mer and we're

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going to bring this together with some

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algo aspartic acids there are varying

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lengths from 10 out to a hundred

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monomeric units and here you can see in

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the ddic image that these coacervate s--

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are forming and that they're on the

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micron scale and that these also

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encapsulate the ribosome and so if the

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enzyme strand is labeled with a

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fluorescing you can see that it's

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enriched in the same droplets here again

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on the micron scale and what's important

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is that if we take this and we make a

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calibration curve and solution we can

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see that the concentration from the

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dilute or bulk phase into the coacervate

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s-- is quite large it's increased about

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five thousand times so if we add only 10

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ten animal or of the enzyme its enriched

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up to 44 micromolar and so so now we'll

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go in and we'll look at the effect of

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different lengths of the Ala go aspartic

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acid from ten to thirty fifty and a

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hundred MERS and in this particular

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assay with the poly cation apollyon i

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and are allowed to come together a

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charge matched conditions of one to one

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and then the first the enzyme is

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introduced and then the substrate is

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introduced and we'll look at the

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reaction that happens and this is again

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with a radio labeled substrate and so

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when reaction happens it goes from this

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higher mobility species down to the

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product here from the unreacted to the

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cleaves so these are now denaturing page

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gels and what we can see that is if the

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reaction is done in buffer and these are

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done under conditions under so-called

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k-kat over km conditions though so that

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even though the enzyme is in excess it's

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far below the the the KD for the

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reaction so there's very little reaction

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out to 30 minutes but that as we bring

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in the the coacervate we begin to see

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more and more cleaved product form and

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then we can look at and plot the amount

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of product that's formed at 30 minutes

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as a function of the length of the anion

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in these coacervate sand we can see that

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we get an increase in that there there's

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a certain range of the length of the

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polygon ion that's optimal or what I'm

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calling a so-called Goldilocks effect so

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if it's too short the reactions not as

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good and if it's too long it's also not

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as good so we wanted to delve into what

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could be the mechanistic basis for this

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and so the first thing we did was to

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look at the encapsulation of the bribe

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design in this case we're looking at the

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encapsulation of the substrate into

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coacervate that come from the different

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lengths of the poly anions and you can

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see somewhat paradoxically that the

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shorter poly anion of the 10 mer has

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greater encapsulation than the longer

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one does even though this is one of the

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shorter reaction rates and so if we plot

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this out these these colors and in all

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the slides are color matched and you can

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see indeed that there's less substrate

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less substrate encapsulated

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inside of the inside of the coacervate

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and so what seems to be happening is

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that the larger polygon ions are

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preventing uptake of the ribozyme

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because the larger polygon ions are

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strongly associated with the cations but

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on the other hand here in the shorter

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poly anions the ribosome goes in more

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more readily but it associates so

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strongly with with the poly cations that

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it's probably not in the correct fold so

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having made this observation we then

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came up with the idea that we perhaps

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could stimulate that reaction by adding

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even more poly on ions and so here's the

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effect of adding excess on poly anions

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and you'll see that excess short poly

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anions actually enhance catalysis and so

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so you can see here this is now in the

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case of the of the d-10 if we go away

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from charge match conditions to a 2 fold

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up to 5 fold excess of the all ago anion

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you can see that the rate of the

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reaction is enhanced up to 2 fold more

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and now this is a chart of the the

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various encapsulation of the enzyme

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under these various conditions and the

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two most optimal conditions for

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catalysis are with the excess charge on

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the d-10 as well as then with the charge

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matched on the d50 and you can see that

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they have similar uptake kind of modest

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uptake in which it kind of finds that

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sort of magic zone between being up

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takes some what the the RNA being up to

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taken up into the coacervate somewhat

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but not I'm so strongly that it's miss

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folded so then we wanted to see whether

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these effects are general which would

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make them kind of more robust in an

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early Earth scenario so the next thing

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we did was move away from biogenic to a

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biogenic on carboxylates

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at going from 30 to 45 and we can see

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that the reaction in 45 is also much

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enhanced over the buffer if we go a

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little bit shorter in the 25 mirror we

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can see the rate it's not as high and

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the charge matched but again if we add

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excess poly an i and just like we saw in

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the other case that the rate is enhanced

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but if we add too much that the rate

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starts to come back down to further test

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generality of this mechanism we then

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looked at these Pollyanna and assisted

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catalysis and asked whether other poly

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anions that that might be present and to

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test the robustness of this without

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whether these could also stimulate the

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reactions so we test this with sulfates

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and phosphates and the first thing is if

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we look at phosphates by adding the

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excess anion as RNA and the phosphate

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you can see that the rate is stimulated

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this is by adding half of an equivalent

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excess of the of the poly an i and it

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also works with sulfate and here this is

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heparin and this also enhances the

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reaction so then in the last data slide

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that I have we're going to test whether

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excess pollyannas poly anions can rescue

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RNA catalysis in otherwise incompatible

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complex coacervate and so we start here

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and rather rather than starting with the

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the allgäu aspartic acid interacting

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weakly with a quaternary amine we let it

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interact strongly with with al ago

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arginine in which there's very little

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reaction and you can see that as you add

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increasing amounts of the ala go

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arginine that the reaction increases and

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so the first thing again is as we move

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from the quaternary amine to the illegal

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arginine the rate drops tenfold but then

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as we add in up to threefold excess of

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the ala goal arginine the rate is r is

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rescued and it increases back up 12 fold

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so so quite strikingly these that this

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mechanism is general and works in in

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cases where there's otherwise

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incompatible coacervate so just to wrap

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up and what i just told you so up here

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is is the mechanistic diagram for what's

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happening and here's a little reaction

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i'm a chemist so i'd like to think about

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things in terms of simple reactions so

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in some ways you can think of this as a

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single displacement reaction and with

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little version so little a minus little

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algo anions come in and displace the

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larger pollyana and the RNA and allow it

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to go from a miss folded to a folded

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state as shown here and there's certain

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conditions that are special and they

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have little gold stars to indicate that

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they work really well and those can be

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short all ago anions where we add large

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amounts of them or for the longer ones

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where they're charged matched and so

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there's three conclusions or three

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takeaways first is that the complex

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coacervate spar tition ribozymes very

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strongly about 5,000 fold the maximal

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rate of catalysis at the charge match

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conditions occur at an intermediate poly

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anion length which is a type of

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Goldilocks effect and the mechanistic

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basis for this seems to be a balance of

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strong RNA sequestration which in the

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sense that when you have short poly on

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ions that are any a strongly sequestered

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in a way that's good because it's inside

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the bioreactor

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but it's kind of bad in the sense that

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the interactions so strong that the RNA

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might not be able to fold very well and

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the final conclusion is that if you add

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excess polygon ions you can enhance

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catalysis and complex coacervate and

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it's very general so short poly anions

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work the best they can be any kind of

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poly anion carboxylates phosphates

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sulfates they work well can be any kind

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of poly cation P DAC and I'll go

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arginine work perfectly fine the the

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anions and cations can be biogenic or a

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biogenic and it works in any ribozyme a

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release in two ribozymes i showed you

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the data on the Hammerhead and we also

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have data on the hairpin ribozyme that

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he didn't have time to to show you and

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then finally the excess short poly

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anions can rescue RNA catalysis and

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otherwise incompatible protocells

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and so this comes from a collaboration

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of my lab and these are the people have

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contributed the most and in particular

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the the work that I show today is

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primarily that of raghava dial and drew

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Venus who this is done lab that's mine

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okay I don't know how to stop in lab

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retreat photo and here's raga hiding

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right there and there's drew and then

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collaboration with with with the Keating

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lab and this work has been funded by

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NASA as well as rogue ops supporters

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come from the Simons Foundation I'd be

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we have time for one short question so

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if your question is short please keep

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your hand up hey fascinating work bill

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but my question is about the coercive

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eighths themselves is there a minimum

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length for the the polymers that make

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the coacervate in order to get a

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coercive in other words prebiotic ly how

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long could would you need these polymers

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to get in order to make a coacervate yes

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so that's ongoing work and and see human

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sitting right next to you it's been

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investigating that and and I think you

379
00:16:04,160 --> 00:16:01,020
can get a little bit shorter than than

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00:16:07,910 --> 00:16:04,170
ten but certainly with with the ala go

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00:16:10,519 --> 00:16:07,920
on D 10 and you get the coacervate but

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00:16:12,590 --> 00:16:10,529
as I said the ribosome is inactive but

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00:16:15,110 --> 00:16:12,600
then you can rescue that activity by

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00:16:17,150 --> 00:16:15,120
adding more and more of that and so so I

385
00:16:18,380 --> 00:16:17,160
don't mean to sort of stand up here and

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00:16:20,240 --> 00:16:18,390
say that this is the way that it

387
00:16:22,610 --> 00:16:20,250
happened that the coacervate were

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00:16:24,590 --> 00:16:22,620
necessarily made out of this anion or

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00:16:26,600 --> 00:16:24,600
this cation in some ways it doesn't

390
00:16:28,850 --> 00:16:26,610
matter right so you can see lots of

391
00:16:33,440 --> 00:16:28,860
different an ion cation combinations can

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00:16:35,920 --> 00:16:33,450
work and basically if the if the RNA in

393
00:16:38,510 --> 00:16:35,930
this case but I'm sure could be any any

394
00:16:40,190 --> 00:16:38,520
informational polymer goes into there if

395
00:16:42,110 --> 00:16:40,200
it goes in too strongly and interacts

396
00:16:43,460 --> 00:16:42,120
too strongly it gets unfolded and so

397
00:16:45,860 --> 00:16:43,470
then you kind of need to loosen that up

398
00:16:48,170 --> 00:16:45,870
so so I think you see here a specific

399
00:16:49,760 --> 00:16:48,180
example but really the take-home is that

400
00:16:51,470 --> 00:16:49,770
sort of the general principles for

401
00:16:53,750 --> 00:16:51,480
drawing it in but not drawing it in too

402
00:16:55,940 --> 00:16:53,760
strongly to allow it to fold and

403
00:16:59,500 --> 00:16:55,950
hopefully and robustly and I would I

404
00:17:02,990 --> 00:16:59,510
think many many different systems

405
00:17:04,270 --> 00:17:03,000
alright lets thankful again