towards life joyce’s lab turns their efforts now to tweaking the abt system even more the goal is...

Towards Life

• Joyce’s lab turns their efforts now to tweaking the A•B•T 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 A•B•T system demonstrated limited exponential growth

Making A•B•T Cross-catalytic

• The A•B•T system was then modified for cross-catalysis rather than autocatalysis


• 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.)


• 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%


• 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 E•E, you will certainly have the problem of forming E•E’, A•B’, A’•B (not to mention the ever-present nonspecific binding)

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)


• Round A-B-E´ A´-B´-E1 2 h 2 h2 1 min 5 min3 15 s 30 s4 15 s 15 s5 0.1 s 0.1 s6 0.01 s 0.01 s

These rounds used mutagenic PCR to give greater diversity of sampled sequences

Why were different times used?


• 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 G•U pairing near the ligation point


• 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 G•U 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


• 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


• 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


• 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 108-fold (presumably this increase is how much of E is produced per starting E’ or E’ produced per starting E)

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 G•C and three A•U base pairs


• There were 32 possible sequence combinations that could be formed consisting of one G•C and three A•U 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


Note this is E1


• 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

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

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

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)

Cross Catalysis

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

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)


• 20 rounds of reaction were run over 100 hrs, total amplification of all ribozymes was 1025-fold


• 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’)


• 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


• 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)


• 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’)


• 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

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)

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”

towards life joyce’s lab turns their efforts now to tweaking the abt system even more the goal is...

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e system

sequences of e

e ribozyme

starting e

e acts

eb sequences

enow e

distinguishing e