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Towards Life Joyce’s lab turns their efforts now to tweaking the ABT system even more The goal is a system that undergoes self- sustained replication.

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Presentation on theme: "Towards Life Joyce’s lab turns their efforts now to tweaking the ABT system even more The goal is a system that undergoes self- sustained replication."— Presentation transcript:

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2 Towards Life Joyce’s lab turns their efforts now to tweaking the ABT system even more The goal is a system that undergoes self- sustained replication using the catalytic capabilities of the system (as we’ll see, this actually entails more than one ribozyme) The ABT system demonstrated limited exponential growth

3 Making ABT Cross-catalytic The ABT system was then modified for cross-catalysis rather than autocatalysis

4 Making ABT Cross-catalytic

5 The first attempt at a cross-catalytic system was to make the A and B sequences that base pair to an E that is not palindromic (as was T) Result was a ribozyme that would ligate substrates to form its complement, rather than itself (so E’ acts on A and B to form E, E acts on A’ and B’ to form E’, etc.)

6 Making ABT Cross-catalytic In order to do this, they changed the sequences at both ends of the ribozyme and in the central stem, and made extra nucleotides at the 5’ and 3’ ends of the E’ ribozyme (this would allow for distinguishing E and E’ when analyzing products since they are different lengths) This resulted in E and E’ that each had rate constants of about 0.03 min -1, with maximum extent of reaction (the “burst height” from the previous paper) no better than 10-20%

7 Making ABT Cross-catalytic These kinetic properties were determined for E and E’ separately (in separate reactions) When E, E’, A, B, A’, and B’ for these initial constructs were reacted all at the same time, even worse performance was obtained (about a 5-fold decrease in rate constants) Recall from the previous paper the issues associated with nonproductive complexes While in this system you won’t have the problem of forming EE, you will certainly have the problem of forming EE’, AB’, A’B (not to mention the ever- present nonspecific binding)

8 Improving on E and E’ An in vitro evolution approach was used to optimize sequences of E and E’ (and therefore A, A’, B, and B’) For example, they covalently attached E to B’ by adding in a loop, then randomized (12% chance of change per position) both the E-B’ sequences and the corresponding A’ sequences (in the catalytic core regions) The same was done for the E’ system, but E and E’ were run through the in vitro evolution process separately (along with the appropriate substrates)

9 Improving on E and E’ Round A-B-E´ A´-B´-E 1 2 h 2 h 2 1 min 5 min 3 15 s 30 s 4 15 s 15 s s 0.1 s s 0.01 s These rounds used mutagenic PCR to give greater diversity of sampled sequences Why were different times used?

10 Improving on E and E’ Each population was then cloned and sequences were determined for a number of clones of each A lot of sequence variability was observed, but every clone had the GU pairing near the ligation point

11 Improving on E and E’ So the “starting point” was an E sequence that was largely based on T from the previous paper The 3’ end was changed so it was no longer a palindrome, and the GU from the previous slide was introduced The E’ sequence was complementary to E, but the catalytic core was a little longer These sequences were not from the starting E and E’, but resulted from experiments described later

12 Improving on E and E’ Now E reacted with A’ and B’ with a rate constant of 1.3 min -1 and a maximum extent of 92% E’ reacted with A and B with a rate constant of 0.3 min -1 and a maximum extent of 88% Note that an extent of 100% would mean all of the substrates had been consumed, so for all practical purposes these reactions were probably not going to stop until everything was reacted

13 Improving on E and E’

14 To test whether these ribozymes really could keep going, and were only being limited by the supply of substrate, serial transfers of 4% of the first reactions were transferred to fresh substrate reaction mixtures and allowed to react some more, and this transfer process was repeated

15 Improving on E and E’ Over a total reaction time of 30 hrs, exponential growth of the product ribozyme was maintained, and at 30 hrs the total increase in product ribozyme was fold (presumably this increase is how much of E is produced per starting E’ or E’ produced per starting E)

16 Improving on E and E’ Even More It was assumed that changing the sequences at the 5’ and 3’ ends of the ribozymes would not substantially change the catalytic activity of the ribozymes In order to minimize complications, the 4 base pairs at each end that they changed were restricted to one GC and three AU base pairs

17 Improving on E and E’ Even More There were 32 possible sequence combinations that could be formed consisting of one GC and three AU base pairs, they selected 12 of them (inverting the 5’ sequence at the 3’ end to minimize complementarity of the ends) For each “end” sequence, they also made changes to the catalytic core So now they had 12 possible E and E’ sequences (the association of an end sequence with a catalytic core change was arbitrary), differing from each other at the 5’ and 3’ ends as well as the catalytic core

18 Improving on E and E’ Even More Note this is E1

19 Improving on E and E’ Even More Each E and corresponding E’ (and substrates A, A’, B, and B’) were analyzed to compare the effects of the changes made Relative replication efficiencies (rate of product production; all were exponential): E1>E10>E5>E4>E6>E3>E12>E7>E9>E8>E2>E11

20 Cross Catalysis Next a serial transfer experiment was done using a mixture of E1, E1’, E2, E2’, E3, E3’, E4, and E4’ and all 16 of their substrates 16 transfers (carrying over 5% of each round to the next) were done over 70 hrs Total of all of the E and E’ molecules

21 Cross Catalysis Products were cloned, and 25 were sequenced All 4 of the catalytic systems were found, so none of them dominated over the others 17 of the 25 clones showed “recombination” (cross catalysis) wherein, e.g., A1 was joined to B3

22 Cross Catalysis “Correct” reaction would be favored because the correct base pairing was such that it had several kcal/mol advantage in binding energy over incorrectly base-paired substrates (those in red are <3.5, meaning they are the more likely mismatches) Once a mismatch does occur, you’re stuck with it (although subsequent mismatching can result in a particular E/E’ reverting back to its original sequence)

23 Cross Catalysis Since some mismatches are more likely than others (based on the binding energies), there are predicted preferred pathways for mutation

24 Serious Cross Catalysis Another serial transfer experiment was done using all 12 of the E/E’ constructs and their 48 substrates (can get, in addition to normal replication of each E and E’, 132 possible pairs of recombinant replicators)

25 Serious Cross Catalysis 20 rounds of reaction were run over 100 hrs, total amplification of all ribozymes was fold

26 Serious Cross Catalysis After the last reaction, 50 E clones and 50 E’ clones were sequenced, only 7 were nonrecombinants (e.g., throughout the whole process E1 kept joining A1’ to B1’)

27 Serious Cross Catalysis There were preferred recombinants: A5B2, A5B3 (most common of all), and A5B4 represented 1/3 of all the products Since E1 was the best out of all the 12 original ribozymes, it was compared to A5B3

28 Serious Cross Catalysis E1 (circles) was better than A5B3 (squares) when each was only given its corresponding substrates (closed symbols) A5B3 was better when all 48 of the substrates were available (open symbols) A5B3 is relatively resistant to inhibition by presence of other substrates, may be why it dominates (also see subsequent slide for effects of preferred recombination pathways)

29 Serious Cross Catalysis A5B3 and its cross catalysis partner B5’A3’ was the most active system (and each had the same activity as the other) of all three predominating cross catalysis systems when incubated with the component substrates for all 3 systems (A5, B2, B3, B4, B5’, A2’, A3’, and A4’)

30 Serious Cross Catalysis Note that when A5B3 is “fed” A5, B2, B3, B4, B5’, A2’, A3’, and A4’ that it exhibits the best self-replication of the 3 major products of the original selection, and compare this to the preferred recombination pathways

31 Implications The ribozyme from previous work was made to self-replicate exponentially When variations were made in this exponential self-replicator, they could get new exponentially self-replicating ribozymes When these ribozymes (and all of their substrates) were reacted together, 3 self- replicators dominated, and all 3 were the result of recombination (i.e., mistakes) So mutations resulted in selection for the most fit ribozymes (Darwinian)

32 Implications This system represents 2 loci with 12 alleles per locus “An important challenge for an RNA-based genetic system is to support a broad range of encoded functions, well beyond replication itself” “Ultimately, the system should provide open- ended opportunities for discovering novel function, something that has probably not occurred on Earth since the time of the RNA world but presents an increasingly tangible research opportunity”


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