2. Making R3C Autocatalytic
R3C sequence was modified to give a symmetrical “palindromic” sequence, maintains catalytic core of R3C
Not exactly palindromic (reads 5’-3’ on one strand the same as it does 5’-3’ on the complementary strand; catalytic core is not palindromic in this ribozyme)
Catalytic construct (“T” for template) joins pieces A and B to create another copy of T
A and B base pair to T through complementary sequences in the P1, P2, and P3 regions

3. Self-complementary R3C Derivative
R3C ribozyme
“Palindromic” R3C

4. General Reaction Scheme
Unproductive

5. Solving Problems
Certain issues had to be resolved to arrive at the optimal T
If P1 was too long, could get A•B•A•B complexes that would ligate to form T
If P1 was too short, would get steric hindrance of the catalytic cores

6. Solving Problems
Lengthening P3 would favor A•B•T over A•B•A•B (for long enough P3, trimolecular complex easier to form than tetramolecular?)
If P3 is too long, would favor T•T (bimolecular easier still?)

7. Solving Problems
Ligation reaction rates (i.e., formation of T from A and B) were measured for various combinations of lengths of P1 and P3
Reaction in absence of template indicates formation of A•B•A•B, in presence of template includes formation of both A•B•A•B and A•B•T
Optimal construct would minimize product (T) formation in absence of T, maximize in presence of T

8. Solving Problems

9. Optimizing Reaction Conditions
Order of addition matters
Preform A•B complexes and then add T: product T was formed (how would you distinguish between starting T and T formed from ligation of A and B?)
Preform A•T complexes and then add B: T was formed
Preform B•T complexes and then add A: T was formed faster than other two combinations
So for all ligation experiments, B and T were preincubated, and then the reaction was started by adding A

10. Binding Assays
Would like to know how tightly each of the parts are binding
Use electrophoretic gel mobility shift assays (EMSAs)
Incubate things that are binding, then run on a native (nondenaturing) gel
Things that bind will have their position in the gel shift vs. their position by themselves

11. Example of an Enzyme-DNA Binding EMSA

12. Binding Assays
EMSAs showed a “small amount” of A•A forms when [A]= mM
Another concern is formation of T•T, EMSA showed most T was not bound to another T for [T]= mM, small amounts of T•T and higher order complexes (T•T•T, etc.) did form
Modeling indicated that the monomeric T structure was stabilized by the opposite ends of the molecule base pairing (potentially a problem, since it wouldn’t bind A and B in this form)

13. Binding of Mixtures of A, B, and T
No way to tell difference
Note how little A•A forms
Note A•B is greatly favored, A•B•A favored over A•B•T (T forms intramolecular base pairs)
Note A and T don’t detectably bind

14. Ligation Reactions
Formation of T from A and B (at either 2 mM or 3 mM each) in the absence of starting T was minimal after 3 hrs
Much more T was formed by 3 hrs when A and B (both 2 mM) was reacted with T (1 mM; recall for this type of reaction B and T were preincubated)

15. Ligation Reactions
Are these results mathematically consistent with the model for a self- replication reaction? Note equation for the kinetics of this
Used 2 mM A and B (each) and varied [T] for kinetic analysis

16. Ligation Reactions
Figure 5 shows p (order of reaction)=1.0, ka (autocatalytic rate constant)=0.011 min-1, kb (rate in absence of starting T)=3.3X M•min-1
Similar results were observed for various other concentrations of A and B
Nonzero autocatalytic rate constant implies that T•T complexes do dissociate enough for autocatalysis

17. Ligation Reactions
Interestingly (and completely unmentioned by the authors), from Figure 4 it appears that the fraction of A and B that reacted (in the absence of T) at 3 hrs. was about 0.002
Calculate: (0.002)(2 mM)/180 min=2.22X10-11 M•min-1
Compare this to data from Figure 5: kb (rate in absence of starting T)=3.3X10-11 M•min-1
I would have pointed that out had I been them

18. Mechanisms for Forming T
By examining Figure 4, and doing additional experiments, can shed some light on how T is being formed
Reacting A, B, and T shows a burst of T formation (until about 30 min), followed by a slower rate
No such burst is observed for reacting A and B in the absence of T
They tried to increase the amplitude of the burst (which is about 5- 6% in Figure 4) by increasing starting concentrations of A and B (varied A and B from mM)
At A and B both at 16 mM, T formation in absence of starting T began to approach that for presence of starting T

19. Mechanisms for Forming T
If [B]>[A] then burst decreased, if [A]>[B] then burst increased
Maximum amplitude of the burst was obtained for [A]<16 mM and [B] at 2-3 fold<[A]
Remember, for these reactions, T is preincubated with B
Interpretation is that B and T form complex, when relatively large amount of A is added then it saturates B•T complexes and relatively low [B] minimizes A•B formation
For the reverse ([B]>[A]), lower [A] would generally give slower reaction (simple kinetics)

20. Mechanisms for Forming T
So they looked at kinetics for [A]=12 mM and [B]=4 mM in the absence and presence of 0.4 mM T
Initial burst in the presence of T (about 30 min) gave mM T product (this would be in addition to starting T; how would they know this?)
After leveling off, they let the reaction continue until 48 hrs
At about 17 hrs, the amount of T produced exceeded the amount of starting T (might consider this to be something like a complete cycle of replication)

21. Mechanisms for Forming T
Most interestingly, in the absence of starting T, there was no burst but the rate of the slow phase of the reaction was the same as that in the presence of starting T
Interpretation: the burst represents reaction of A•B•T, the slow phase is reaction of A•B (or actually A•B•A•B)
In other words, the end of the burst phase represents the exhaustion of the preformed B•T complexes, left with intramolecularly base- paired T and tightly bound A•B complexes
In light of this, the amount of T produced by A•B•T is less than the starting amount of T

22. Implications
So the formation of T was autocatalytic to a point, but it was not exponential and sustained
This type of self-replication (and other systems involving both nucleic acids and peptides that have been devised by other researchers) does not allow for Darwinian evolution
The Darwinian systems that have been thus far devised do not actually self-replicate (e.g., DNA-based systems require addition of a DNA polymerase enzyme)

23. Implications
They suggest the best approach may be a self-replicator that can work on an assortment of substrates (e.g., T can join A and B to make T, or C and D to make U, or E and F to make V, etc.)
In such a system, competition for the use of the substrates would allow for Darwinian evolution, but self-replication would be much easier than nucleotide-by-nucleotide copying of a long nucleic acid