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[Music]

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[Applause]

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so most of you are familiar with the

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ironing world hypothesis which proposes

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that early in the evolution of life RNA

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serve both genetic and catalytic roles

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roles which have been taken over mostly

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by DNA and enzymatic proteins so one

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important tool for the RNA world would

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be having an RNA polymerase ribozyme not

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only able of self replication but also a

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more general transcription of an RNA

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template so a number of labs are looking

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into this and it all started with the in

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vitro selected class one ligase by

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Bartel and Shaw stack and it can

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catalyze the 3 prime to 5 prime

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phosphodiester bond formation of two RNA

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molecules with that is a starting point

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a second functional domain was in vitro

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selected and that donate domain was

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called an accessory domain and this

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transformed it from ligase to a

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polymerase and what this polymerase can

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do now is extend an RNA primer that's

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been hybridized on an RNA template in a

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template dependent manner however it

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does have a preference for gc-rich

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templates so the example shown here is B

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six point six one it was a polymerase

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ribozyme evolved later in our lab and

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what it can do is extend the primer by

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about twenty nucleotides in twenty-four

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hours so a major problem with these

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polymerases is a lack of processivity so

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they tend to dissociate from the primer

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template complex in between nucleotide

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additions so one solution that has been

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found and used to increase processivity

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by a number of labs such as the

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Hollinger and the Joyce lab was to

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directly hybridize that the polymerase

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or the ribozyme straight to the template

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that way you can dissociate away and

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this has resulted in extensions of up to

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200 nucleotides over seven days and this

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again is on GC rich templates with

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multiple repeats this is the latest

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Joyce ribozyme 24.3 and it was able to

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transcribe small functional RNAs

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but even thither it has there's a

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trade-off between template complexity

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that it can transcribe and process

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tivity so we can see that the

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full-length product is not in a

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and there's a number of stalling that

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happens during transcription so at this

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point we thought would it be possible

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and it would be interesting to make a

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polymerase ribozyme that has high

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processivity without that feathering and

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also that has more biological features

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so what I mean by biological features is

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looking at bacterial DNA dependent RNA

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polymerases and one thing that they have

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is specificity factors so they use Sigma

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factors which recognize promoter region

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on the double-stranded DNA template and

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it localizes the polymerase to this

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promoter region so similarly we're

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thinking that we can do the same with an

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RNA specificity factor termed here in

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RNA primer and this primer can recognize

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a promoter region on an RNA template by

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via hybridization and again localize

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this polymerase to the template another

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thing that the DNA dependent RNA

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polymerase is have this process Savini

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and the way they get this is either by

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clamping onto the DNA template so they

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don't associate or by in training the

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RNA product so again we think we can do

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this by selecting - in vitro selection a

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third functional domain a clamped domain

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and this domain would be in an open clam

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conformation when bound to the

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specificity factor the RNA primer and

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once the primer is displaced onto the

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promoter region of the template this

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open clamp domain can close and circling

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the template and being able to slide

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back and forth on the template in an

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untethered fashion so to select for such

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a polymerase ribozyme I started with B

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six point six one as my progenitor to

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make the pool and it has the ligase

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course only and in blue

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the accessory domain in green and this

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is a little cartoon signifying that so

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first I designed a five prime primer

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binding site shown here in orange and

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this is partially complementary to this

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RNA primer or the specificity factor and

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this serves as the first arm of the

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clamp domain for the second arm of the

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clamp domain this is where the in vitro

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selection comes in handy and I've

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appended random sequence so the three

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prime end of

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the accessory domain and this gave me an

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RNA pool with a diversity about 10 to

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the 13th this is the general schematic

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of the in vitro selection where I

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started with a B six point six one DNA

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pool that has been fused with random

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nucleotides it's being transcribed to

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make an RNA pool then multiple selective

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steps are undertaken to remove inactive

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RNAs while selecting for sliding and

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active RNA clamp polymerases which are

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then reverse transcribed to make a cDNA

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library and then PCR amplified to feed

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back into the cycle so to go over in

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more details about the Selective steps

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I've used the first elliptic selective

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step looked for clamping domains alone

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so primer was given to the pool to

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create this open clam conformation and

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then it was incubated on circular

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template that has been immobilized on

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streptavidin magnetic beads so ribozymes

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with functioning clamps would be

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directed to the promoter region by the

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specificity factor or the RNA primer

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where the primer would be displaced and

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the clamping domain would go from an

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open conformation to a closed

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conformation being trapped on the

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circular template and now all non

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functioning claps would be able to be

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washed away at this point to recover

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this functioning active ribozymes fresh

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primer is added to ask the the clamp to

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open back up and this allows collection

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of them off the circular template and

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we're able to monitor this process by

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radio leaving the radio label in the

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pool and then the whole process can be

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carried out multiple times to increase

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the stringency so this is what we did so

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by monitoring the pool that comes off

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with fresh primer off the beads we're

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able to look these are the last two

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rounds that used this selection protocol

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and the Selective steps hasn't been used

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every single round that's why I round

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twenty is missing so while we see that

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primer recovery is higher than just

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buffer recovery and this is the first on

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and off event the second and then in

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between rounds this amount increases the

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second selections scheme looked

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both clamping and Templi dependent

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extension of a primer so again an open

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clamp confirmation was it was done by

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adding primer to the pool and then

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incubated on a circular template with an

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TPS but instead of using a regular CTP

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we used an analog upaya tune latest CTP

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this way an active polymerase or an

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active ribozyme would be able to

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incorporate this by 2 in latest CTP into

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the extended primer this allows

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purification of active by molecular

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complexes on script Avenue marinated

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beets anything that isn't active is

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washed away and again we can monitor the

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active guys by recovering them with a

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primer focusing on this opening the

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clamp one more time so these are the

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last three rounds that we did with this

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selective step so you can see that the

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amount of pool recovered with primer

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increases in between each round so this

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is the final round of selection that

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that we've done so far and we looked at

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the activity of this round 27 so first

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we looked at at its processivity on the

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template that it was selected so I

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mentioned that be six point six one was

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able to extend 20 nucleotides on a GC

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rich template however we used an 8 you

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reach template and B six point six one

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can only extend about three to four

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nucleotides wide around 27 pool is able

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we then switched the system from a red

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primer red templates to a secondary

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green primer green template with a

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different promoter and we were able to

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see extension over 24 hours quite

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precisely and we see about an extension

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about 3035 nucleotides on the left side

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on template 1 and about 20 to 25 on the

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right side on templates 2 and again both

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these templates are au rich so then we

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wanted to see if a specificity factor is

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actually working and we are doing

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driving template specific extension so

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we took the pool and incubated them with

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either the red primer or the green

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primer and gave them both templates at

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the same time and based on the extension

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pattern we can see that with the red

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primer there's a preference for the red

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template with a green primer there's a

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preference for the green template so

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I've showed so far that red primer red

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templates being extended green primer

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green templates being extended we wanted

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to see if we're able to make the pool

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switch in between templates so once

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given into a complex we're able to want

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it to see if we can add fresh primer to

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open up the clamp give the primer of the

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different color and then make a switch

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to a secondary template this would allow

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for multiple rounds of transcription if

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it works properly

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now one activity we did not want was the

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same complex but if given a primer

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that's already hybridized to the

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template the clamp domain should remain

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closed and stay on the primary template

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so the stable at the top just is exactly

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like the images from the previous slide

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so red primer red template we see

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extension green primer green template we

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see extension we also see that by

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switching primers and templates we're

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able to see extensions on the secondary

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templates were able to switch templates

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however the pool doesn't perfectly stop

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the undesired behavior of switching to a

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template that's already been primed I'll

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be at a lesser extent then when properly

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primed and switched over the last thing

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we wanted to test since we're selecting

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for a sliding clamp domain we

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hypothesized that they should see more

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processivity with the pool on a circular

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template over a linear template as on a

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circular template clamped ribozyme would

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be able to slide back and forth and not

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dissociate from the template where on a

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linear it would be able to slide off the

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ends and it would lose its primer

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template remarkably that's exactly what

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we see or there's more processivity on

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the circular template in the linear

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we're able to see this with the second

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green primer and green template and we

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also tested five other templates for the

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same behavior with different lengths and

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we see the same behavior so it's

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important to note at this point that all

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the data has been done with the

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selection pool so what we see is an

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average of activities of the different

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individuals and just last week we cloned

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in sequenced

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45 clones of the pool and we have to

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further characterized the individuals

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inside the pool to look for the optimal

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activity so just to conclude comparing

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our in-vitro selected polymerase

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ribozyme with the DNA dependent RNA

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polymerase although RNA might never be

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able to get the proximity of a protein

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we're able to increase the profit eekum

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pair to the progenitor be six point six

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one on a you rich templates we're also

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able to show promoter recognition using

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an RNA specificity factor and we were

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partially able to show a switching of

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templates which can lead to multiple

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replication events and we believe that

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the process civet e and the switching of

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templates are linked and then we have to

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characterize the pool to find

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individuals that are better at doing

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both these processes and the last

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question that remains unanswered even in

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the field is how do we do strand

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displacement perhaps with a fourth

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fourth domain but yes so once RNA is

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transcribed of an RNA template how do we

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get it to come off without he donation

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with that I would like to thank my

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supervisor dr. Ron Rao my lab member is

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time for one question maybe two yeah so

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your list of features on protein enzymes

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I think there's something really missing

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there in that they are motors they they

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take chemical energy and they do work

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with it in a really directed way and I

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and I think on your list where you you

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know you're just not you're not doing

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that being possessive by locking it onto

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the template is not the same thing as

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making it into a directed motor yes so

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there's been multiple publications where

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even the protein polymerases have had

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their clamps removed and their proximity

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goes way lower by thousandfold so

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clamping does play a major role in being

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processive but they're they're

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definitely more complex and can go much

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further distances than an RNA polymerase

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would ever be able to I'm allowed to

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have one question so first I think he

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does have some form of a motor because

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the hydrolysis of each ntp drives it

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forward as long as you bind to this

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interface so you that could be an

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analogy to motor my question or

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suggestion is in our hands high

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throughput sequencing has been

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incredibly powerful in analyzing these

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rounds and that not only allows you to

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cluster sequences but also to find

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sequences that are getting enriched

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within one cluster and Irene Chen has

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been teaching us how to do that are you

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planning to do that yes so we're

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planning to do high throughput

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sequencing so again this was just the

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data from last week I was analyzing them

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in in the hotel room but yes and we

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might carry out a couple more selective

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schemes to get them a little further and

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then we do high throughput sequencing

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all right great let's thank Rosalyn

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again next time