Session 1: Origins of Life: Protocells and Non-Enzymatic Template-Directed RNA Synthesis

00:00:00.00 My name is Jack Szostak,
00:00:01.25 I'm a Professor of Genetics at Harvard Medical School,
00:00:05.22 I'm an Investigator at Massachusetts General Hospital, where my labs are,
00:00:09.18 and I'm also an Investigator of the Howard Hughes Medical Institute.
00:00:14.17 In this lecture, what I'd like to tell you about is recent advances
00:00:21.06 in work from my lab on the origin of cellular life on the early Earth.
00:00:26.22 But before I get into those experiments,
00:00:29.08 I'd like to step back from the origin of life per se,
00:00:33.08 and talk a little bit about some insights from modern biology
00:00:39.00 that bear on this question,
00:00:41.01 in particular why the question has attracted so much interest and attention recently.
00:00:46.16 So, this is one of the iconic images of hydrothermal deep sea vents.
00:00:56.02 This is an environment characterized by very high temperature and pressure,
00:01:01.21 and of course the surrounding area is just teeming with life.
00:01:07.04 Here's another example: an image from Norm Pace.
00:01:12.04 You can see a layer of green cells growing inside the rock.
00:01:18.07 These are photosynthetic cyanobacteria,
00:01:21.29 and they're living in the pores of the rock at very low pH.
00:01:28.23 This is one of the famous hot springs in Yellowstone National Park.
00:01:34.26 Again, a very high-temperature environment; again, full of life.
00:01:43.29 And here's yet another distinct kind of extreme environment,
00:01:48.21 another very low pH environment.
00:01:50.14 This is the Rio Tinto in Spain.
00:01:53.27 Very acidic water, but again teeming with life:
00:01:59.00 microbial, eukaryotic life.
00:02:04.17 There are even more extreme examples of this kind of environment in acid mine drainage sites,
00:02:12.00 where the water that's flowing out is basically sulfuric acid at a pH close to zero.
00:02:18.24 And again there is microbial life.
00:02:21.11 So with all of these examples,
00:02:22.22 what it's telling us is just the remarkable extent which our planet
00:02:27.21 has been colonized by life.
00:02:31.17 And even environments that we would've considered incredibly hostile and extreme
00:02:38.20 are apparently easily adapted to by life.
00:02:43.10 And of course, this is a consequence of the power of Darwinian evolution,
00:02:48.10 to lead to adaptations to diverse environments.
00:02:53.16 So, if you put this together with recent observations
00:03:00.01 from our astronomy colleagues, in terms of the discovery of extrasolar planets,
00:03:06.02 it really puts into focus the question of whether there is life out there,
00:03:13.13 apart from our planet.
00:03:16.07 So this is an image of the Milky Way, of course.
00:03:22.20 Up to a couple of years ago,
00:03:24.21 astronomers had discovered on the order of 500 extrasolar planets,
00:03:29.04 planets orbiting other stars.
00:03:32.08 But more recently, as a result of the Kepler mission,
00:03:36.00 a space telescope that is just pointed continuously at a very dense starfield,
00:03:45.22 a large number of additional planets have been found,
00:03:48.24 about 1200 candidates at the last count.
00:03:52.28 And these are detected as the planets orbit around their star,
00:03:57.29 and if they eclipse the star, if they transit in front of it,
00:04:00.27 they block out some of the light, and you can detect that little dip in the intensity of the light.
00:04:06.23 So this has given us a big enough sample to actually make extrapolations,
00:04:12.02 and what I've heard from scientists associated with the Kepler mission
00:04:17.17 is that those extrapolations suggest that there could be roughly on the order of
00:04:23.06 500 million, perhaps even a billion, Earth-like planets
00:04:27.00 orbiting sun-like stars out there in our galaxy.
00:04:32.13 And so, if you put that together with the fact that we know,
00:04:37.03 on our planet, that at least microbial life can live in incredibly harsh and diverse environments,
00:04:44.28 it's pretty clear that there will environments out there on
00:04:49.12 these other planets that could support life.
00:04:52.03 So the question is, and the thing we all really want to know is:
00:04:56.01 Is there life out there?
00:04:58.01 Are we alone, or is the universe, is our galaxy, full of life?
00:05:03.15 So this really comes down to the question you see here.
00:05:07.25 Is it easy or hard for life to emerge from the chemistry of early planets?
00:05:14.13 And, unfortunately, it's going to be a long time before we can answer that question
00:05:18.28 in the most satisfying way, by direct observation.
00:05:23.16 Even to get indirect evidence from spectroscopy of planetary atmospheres
00:05:30.27 may take 10, 20, 50 years, to look at Earth-like planets.
00:05:38.28 So what can we do in the meantime to try to get some clues to answer this question?
00:05:46.27 So, what we've been doing, and other people have been doing,
00:05:49.29 is to go into the lab and do simple, chemical experiments
00:05:54.29 and try to work out a complete, step-by-step, plausible pathway,
00:06:02.17 all the way from simple chemistry to more complex chemistry to simple biology.
00:06:08.02 And if we can actually show that there's a continuous pathway
00:06:13.26 with no super-hard steps along the way,
00:06:17.20 then I think we can conclude that it's likely that there is abundant life
00:06:22.14 out there in our galaxy.
00:06:25.03 On the other hand, it could be that our experiments show
00:06:29.14 that there are some steps in that pathway that are extremely difficult,
00:06:35.01 there are bottlenecks that might be very hard to overcome,
00:06:38.29 in which case the emergence of life might actually be a very rare phenomenon.
00:06:44.04 And in the extreme, we could be it.
00:06:47.23 This could be the only place in our galaxy or even the universe where life has emerged.
00:06:53.28 So we would like to try to get some insight into these questions
00:06:58.11 by doing simple laboratory experiments.
00:07:00.20 Now, there's a related question, which is shown down here.
00:07:06.03 If there is life out there,
00:07:07.25 is it likely to be pretty similar to what we're familiar with on our planet?
00:07:16.12 Will life that evolved independently elsewhere have the same
00:07:23.25 fundamental kind of biochemistry?
00:07:25.24 Will it be cells that are living in water,
00:07:29.00 using if not RNA and DNA, some nucleic acid to mediate heredity?
00:07:35.20 Will they use protein-like molecules to carry out biochemical functions?
00:07:41.18 Or could there be forms of life that are actually much different, much more diverse,
00:07:49.14 maybe using completely different kinds of molecules to mediate heredity
00:07:52.25 and to mediate function?
00:07:55.04 Or even forms of life that live in very different environments,
00:08:00.06 for example, in solvents other than water.
00:08:03.23 Again, this is the kind of thing that we can address by going into the lab
00:08:09.17 and doing simple experiments, and trying to build structures,
00:08:12.26 and assess the possibility of having living systems in
00:08:18.09 different kinds of environments and with different molecular bases.
00:08:25.16 So, let's try to think, then,
00:08:30.21 about how we can deduce something about early forms of life.
00:08:37.04 After all, if we want to experimentally investigate the beginnings of life,
00:08:41.26 we have to have some idea, some kind of conceptual model,
00:08:45.22 of what very primitive forms of life looked like.
00:08:50.17 And this has been a very difficult thing for people to think about,
00:08:55.03 because we're so biased by our view and our understanding of modern life.
00:09:00.25 So if we look at modern cells, they're incredibly complicated:
00:09:05.16 Just a lot of moving parts, very elaborate structures,
00:09:09.14 such as you can see here in this elaborate structure in a eukaryotic cell,
00:09:15.15 all the machinery involved in cell division.
00:09:19.00 If you go deeper and look at the underlying biochemistry,
00:09:23.09 if anything, it's even more complicated.
00:09:26.17 And this is just a small section of the chart of central metabolism,
00:09:32.09 so there are hundreds or thousands of enzymes that catalyze
00:09:36.22 all of the metabolic reactions that are required for cells to grow and divide.
00:09:43.10 Even the general organizational structure of modern cells is very complicated,
00:09:51.10 in the sense that it's highly self-referential.
00:09:54.24 So every aspect of this process,
00:09:59.03 this central dogma (the transmission of information from DNA to RNA
00:10:03.10 to proteins and then down to building structures with function),
00:10:08.21 every part of that depends on all the other parts.
00:10:12.12 So for example, the replication of DNA requires DNA,
00:10:16.14 but it also requires RNA and proteins, the polymerases.
00:10:20.11 The transcription of RNA requires DNA,
00:10:25.11 which is where the information's stored, but it also requires many proteins,
00:10:29.21 and it also requires many other RNA molecules.
00:10:33.02 And similarly, the formation of proteins occurs on a remarkably complex machine,
00:10:38.24 the ribosome, which is itself composed of RNA and proteins.
00:10:44.02 So, for decades, it was very hard for people to think of any reasonable way
00:10:51.07 in which such an internally self-referential system could emerge
00:10:57.25 spontaneously from a chemical environment.
00:11:01.16 And the answer to that really came from thinking about RNA
00:11:08.27 and the different things that it can do.
00:11:10.21 So this simplification in thinking came from the realization that RNA can not just
00:11:18.04 carry information but can also catalyze chemical reactions.
00:11:23.15 And that realization led immediately to the hypothesis that,
00:11:29.01 in primitive cells, RNA might be able to catalyze its own replication,
00:11:34.05 also carry out biochemical functions for the primitive cell.
00:11:39.10 And so then all you really need to think about is a cell with RNA molecules
00:11:45.14 encapsulated within some kind of primitive cell membrane
00:11:48.25 that itself could be a self-replicating structure.
00:11:52.08 So, the history of this idea actually goes back to the 1960s,
00:11:58.24 and three very smart people, Leslie Orgel, Carl Woese, Francis Crick,
00:12:05.05 hypothesized in part on the basis on the complex folded structure of tRNA,
00:12:13.01 that an early stage of life might've evolved RNA as the
00:12:19.01 sole macromolecular basis of evolved machinery.
00:12:24.25 And so, this lets you think of simple cells emerging with just a single biopolymer,
00:12:31.19 RNA, and that later on, as evolution
00:12:38.03 developed more complex cellular structures, information storage
00:12:42.14 became specialized in DNA,
00:12:44.18 and most functional activities because specialized as the job of proteins.
00:12:51.06 Now, although these ideas were put forth in rather elementary form in the 60s,
00:12:56.14 of course nobody took them seriously at the time,
00:12:59.21 because there was absolutely no experimental evidence for the idea
00:13:04.08 that RNA could catalyze chemical reactions.
00:13:06.23 At the time, people had just started to get very detailed,
00:13:11.19 high-resolution information about how proteins catalyzed reactions,
00:13:16.13 and the idea that a molecule like RNA could do the same thing seemed ludicrous.
00:13:22.26 So it wasn't until almost 20 years later,
00:13:25.05 with the work of Tom Cech and Sid Altman,
00:13:29.10 and the experimental demonstration that RNA molecules could actually
00:13:34.02 very effectively catalyze at least certain types of chemical reactions,
00:13:38.14 that people took this whole idea of an RNA-based early stage of life seriously.
00:13:45.00 And so that hypothesis, the "RNA world hypothesis,"
00:13:48.23 was really summarized by Walter Gilbert in an article in 1986,
00:13:57.18 and this has really become the foundation of a lot of thinking
00:14:02.12 about early stages in the emergence of life.
00:14:08.01 So, apart from the basic facts,
00:14:11.20 that RNA does and can catalyze chemical reactions,
00:14:15.04 is there any other evidence that early life might have been
00:14:19.17 based more exclusively on nucleic acids?
00:14:24.21 And in fact, there are several lines of circumstantial evidence.
00:14:28.24 So one of them is the structure of many cofactors.
00:14:33.07 So here you see acetyl-CoA, just one example.
00:14:39.03 But the working part of the molecule is the thioester out here,
00:14:43.27 and for no obvious reason, there's a nucleotide at the other end.
00:14:48.13 And really the only way to make sense of that is the nucleotide is a "handle,"
00:14:53.11 either a relic of a primitive ribozyme
00:14:56.24 or something that was easy for primitive ribozymes to grab hold of and thereby,
00:15:04.10 using this cofactor, catalyze reactions in a thioester-mediated way.
00:15:11.14 Now there are other examples.
00:15:14.21 Here is vitamin B12, another very important catalyst.
00:15:20.24 Its working part is this complex corrin ring,
00:15:25.07 but down here you see, again, a nucleotide.
00:15:29.22 What's it doing there?
00:15:30.28 It's probably another relic of the RNA world,
00:15:34.02 when all of this complicated biochemistry was being catalyzed by RNA enzymes.
00:15:40.12 Yet another example is the very way that the substrates for
00:15:45.25 DNA synthesis are made, and they're not made de novo,
00:15:50.29 as you might expect if DNA came first.
00:15:53.27 They're actually made from preexisting ribonucleotides,
00:15:58.02 and so the transformation of ribonucleotides to deoxynucleotides
00:16:03.17 is catalyzed by the enzyme ribonucleotide reductase.
00:16:08.13 And this unusual synthetic pathway can be viewed as the relic of the fact that,
00:16:17.04 early in time, metabolism and RNA synthesis used ribonucleotides,
00:16:23.25 and only later when DNA was invented or evolved,
00:16:29.10 was there was requirement to make deoxynucleotides,
00:16:32.15 and so they're from the closest available substrate.
00:16:38.03 Finally, perhaps the most important and dramatic piece of evidence
00:16:44.14 for the early role of RNA in primitive forms of life
00:16:50.06 is the actual structure of the ribosome.
00:16:53.21 And so this is a slide from Tom Steitz showing a
00:16:57.27 view into the active site of the large subunit.
00:17:02.23 So this is the peptidyl transferase center,
00:17:05.06 and this little green structure in here is a transition state analogue
00:17:09.17 that marks it at the place in this giant machine where the chemistry is happening.
00:17:14.28 And what you can see is that it's these gray squiggles,
00:17:17.29 which are the RNA, that completely make up that active site.
00:17:23.23 So all proteins are generated by an RNA machine,
00:17:29.19 the RNA central region of the ribosome itself.
00:17:34.25 So again, this only makes sense in terms of an early stage of biochemistry
00:17:40.00 dominated by RNA functions, which then over time evolved the ability
00:17:45.15 to make proteins, which are now so important in all modern biochemistry.
00:17:52.05 So, if we want to understand the origin of life,
00:17:57.03 what we need to think about is not simply how to make
00:18:02.14 these incredibly complex modern cells, but we need to think about how to go from chemistry
00:18:07.23 to very simple, RNA-based cellular structures.
00:18:13.08 So, what would the process look like?
00:18:16.04 What's the broader picture?
00:18:17.17 When did this all happen on the early Earth?
00:18:20.17 So, what was the timeframe in which these events took place?
00:18:25.07 This is a slide from a review by Gerald Joyce, and it summarizes
00:18:30.02 the broad sweep of events that were important in the origin of life.
00:18:34.20 So we actually need to think of everything from planet formation,
00:18:38.14 the beginning of the Earth itself around 4.5 billion years ago;
00:18:44.07 over time as the Earth cooled, water could condense,
00:18:48.01 we have a stable hydrosphere, we have liquid water on the surface;
00:18:52.06 following that, increasingly complicated organic chemistry going on,
00:18:57.08 probably in many different environments on the early planet;
00:19:01.00 and then somehow that led up to the synthesis of RNA
00:19:06.09 or RNA-like molecules on the Earth,
00:19:09.13 which could start to carry out biochemical functions inside primitive cells;
00:19:14.24 and then eventually lead to the emergence of much more complicated cells
00:19:19.29 that would be biochemically similar to modern life.
00:19:23.14 Now, the first really good evidence we have about
00:19:27.04 the appearance of modern microbial life is roughly 3.5 billion years ago,
00:19:33.17 so there's a billion-year interval between the formation
00:19:38.17 and cooling of the planet and the first good evidence for life.
00:19:43.21 And basically, we have very little hard evidence about
00:19:48.00 where all of these important events that led up to life emerging from chemistry,
00:19:53.01 when they actually happened.
00:19:55.03 And that goes along with the fact that we have very little concrete evidence
00:19:59.25 concerning the environments in which those transitions took place.
00:20:04.27 So, this is one of the difficult aspects of studying this question.
00:20:09.12 We can't actually go back,
00:20:10.28 we can't know for sure what the early environments were really like,
00:20:16.13 we'll never know exactly what really happened.
00:20:19.15 So what's our goal in studying these questions?
00:20:23.00 What we're trying to do is really come up with a plausibly realistic
00:20:29.16 sequence of events so that we understand all of the transitions
00:20:35.00 throughout this whole pathway,
00:20:37.05 and we'd like to understand a complete pathway,
00:20:38.29 from planet formation through early chemistry,
00:20:41.28 more complicated organic chemistry,
00:20:44.05 up to the assembly of those building blocks into the first cells,
00:20:48.29 the emergence of Darwinian evolution,
00:20:51.04 and then the gradual complexification of early life leading up to what we see now.
00:20:58.10 So, let's look a little bit more closely at the chemical steps.
00:21:03.23 So in broad outline, what we think happened is that you
00:21:07.17 start off with very simple molecules such as shown up here.
00:21:14.02 There's still a lot of debate about the nature of the early atmosphere.
00:21:20.00 Scientific opinions have gone back and forth in terms of
00:21:23.09 the structure and how reducing that atmosphere was.
00:21:27.26 But it's also been recognized that there could be very important local variation,
00:21:32.22 so even if the atmosphere was globally fairly neutral
00:21:38.20 or perhaps mildly reducing or mildly oxidizing,
00:21:41.16 there could be local environments that were more reducing.
00:21:46.14 That, together with the input of various forms of energy
00:21:51.21 (for example, from electric discharges, lightning,
00:21:56.06 high-energy ultraviolet radiation, ionizing radiation)
00:22:01.08 these are all forms of very energetic processes that can basically
00:22:06.15 rip these small starting molecules apart into atoms,
00:22:11.26 which can then recombine to generate high-energy intermediates
00:22:15.17 with multiple bonds, molecules like cyanide and acetylene,
00:22:20.19 formaldehyde and so on.
00:22:22.18 And these molecules can then start to interact with each other
00:22:26.20 and gradually build up more complex intermediates,
00:22:29.13 ultimately leading to the things we really care about:
00:22:33.10 the lipids that will make membranes and vesicles,
00:22:37.25 the nucleotides that will assemble into genetic molecules like RNA,
00:22:42.16 amino acids that can assemble into peptides,
00:22:45.29 which may also play roles in primitive cells.
00:22:49.10 And somehow, and this is the question that my lab has really been focused on,
00:22:53.12 somehow all of these molecules come together
00:22:56.17 and assemble into larger structures that look and act like cells
00:23:01.10 that can grow and divide.
00:23:04.04 So how could that possibly happen,
00:23:06.17 and what would such a primitive cell look like?
00:23:09.24 So here is a schematic version of the way that we're thinking
00:23:13.21 about a primitive cell, or "protocell."
00:23:17.11 So what we think are the important components of a primitive cell
00:23:22.14 are basically two things:
00:23:25.01 a cell membrane and inside,
00:23:28.10 some kind of genetic material, maybe RNA, maybe DNA,
00:23:32.05 maybe something simpler, something more stable, we're not really sure.
00:23:37.12 So the first question is how could you assemble such composite structures?
00:23:43.23 So we want to be have a membrane boundary
00:23:46.13 that can keep important molecules encapsulated within
00:23:51.06 and essentially provide a distinction between the cell itself
00:23:54.22 and the rest of the universe.
00:23:57.05 We need to understand how these two components self-assemble,
00:24:02.09 how they come together.
00:24:04.07 And it actually turns out that that part is all fairly straightforward.
00:24:09.26 Self-assembly processes are critical in thinking about all of the steps,
00:24:15.12 and there are multiple different ways in which these components
00:24:19.06 can be made and can come together.
00:24:22.08 A much harder question and more interesting is:
00:24:28.08 Once you have structures like this,
00:24:30.16 how can they grow and then divide without any of the
00:24:34.17 complicated biochemical machinery that's present in all of modern life?
00:24:40.25 So since we're talking about the origin of life,
00:24:43.00 then by definition we didn't have highly evolved biochemical machinery around.
00:24:48.21 So it's sometimes hard to think about these problems
00:24:51.17 because modern cells use so much biochemical machinery
00:24:56.11 to mediate the process of cell growth and cell division.
00:25:00.12 It's almost hard to think of how could that be driven
00:25:06.04 by simple chemical and physical processes.
00:25:10.07 But that's in essence what we need to figure out
00:25:13.00 in order to understand this process.
00:25:15.07 There's no machinery around,
00:25:16.18 so we have to identify the chemical and physical processes
00:25:20.14 that will drive growth and then mediate cell division.
00:25:25.11 So that applies not only to the membrane,
00:25:27.11 but also the genetic material, whether it's RNA or something else.
00:25:31.13 There have to be simple chemical processes
00:25:35.16 that will drive the copying of that information,
00:25:38.12 that will allow the strands to separate
00:25:41.06 so that another round of copying can take place,
00:25:44.05 and that will allow that replicated material to be distributed into daughter cells.
00:25:50.10 So if we can identify chemical and physical processes that do all of that,
00:25:56.25 we would have a situation where essentially the environment
00:26:00.03 is driving a cycle of growth and division
00:26:04.15 that brings us back to this stage,
00:26:07.05 and you can go around and around that cycle again and again,
00:26:12.05 and that would be just very similar to the way in which
00:26:16.22 modern cells grow and divide.
00:26:18.23 The information within would be propagated and transmitted
00:26:22.23 from generation to generation,
00:26:25.08 and the important thing in terms of the emergence of Darwinian evolution is that,
00:26:31.00 during that continuous process of replication,
00:26:35.07 of course mistakes would be made.
00:26:38.18 Over time, more and more of sequence space would be surveyed,
00:26:43.28 and eventually we think, some sequence would emerge that did something
00:26:48.03 useful for the cell as a whole.
00:26:50.14 As soon as that happened, that sequence,
00:26:54.00 by conveying an advantage to its own cell,
00:26:57.24 whether in terms of growth rate or the efficiency of cell division
00:27:03.00 or the efficiency of survival,
00:27:05.22 it would have an advantage and it would gradually over generations
00:27:08.28 take over the population.
00:27:11.09 And so that is really the essence of Darwinian evolution.
00:27:15.05 You have a change in the genetic structure of the population as a result of natural selection.
00:27:21.01 And that is precisely what we would like to see
00:27:24.03 emerge spontaneously in our laboratory experiments.
00:27:27.17 We want to start with a chemical system
00:27:30.18 and watch it transition into the emergence of real Darwinian
00:27:35.18 evolution at a very simple level.
00:27:39.20 So, let's step back again and think about how all of these
00:27:43.25 molecules would be made in the environment of a primitive planet.
00:27:49.02 And of course, the first breakthrough in this research program
00:27:54.03 was the famous Miller-Urey experiment,
00:27:57.23 in which a mixture of reducing gases was subjected to an electric spark discharge,
00:28:02.10 and the products were analyzed.
00:28:04.12 And amazingly, in that mix of products were many of the amino acids,
00:28:11.19 which are major components of the proteins of modern cells.
00:28:16.28 So that was really a revelation.
00:28:19.29 It really took people by surprise that the building blocks of biological structures
00:28:28.05 could be generated in such an easy manner.
00:28:33.01 Now, in fact that result was so powerful
00:28:36.08 that it might have actually been a little bit distracting.
00:28:40.15 Probably the really important thing that's made
00:28:43.19 in this kind of experiment is not amino acids per se,
00:28:48.03 but high-energy intermediates like cyanide and acetylene.
00:28:53.06 Those are the kinds of molecules that can assemble in
00:28:57.25 subsequent steps into nucleotides, the building blocks of genetic materials.
00:29:07.16 Those molecules are thought to have been made in primitive environments,
00:29:14.11 so that was an electric discharge experiment,
00:29:16.08 which is very analogous to the kinds of lightning displays
00:29:21.12 that you get in volcanic scenarios.
00:29:24.12 So this is the lightning that's going on in the ash cloud
00:29:29.14 of a currently erupting volcano in southern Chile.
00:29:34.02 So since the early Earth was thought to be highly volcanically active,
00:29:38.15 this seems like a very reasonable scenario.
00:29:42.13 What about some of the other molecules that we need
00:29:44.22 to build our primitive early cell?
00:29:50.29 We need to have lipid-like molecules, amphiphilic molecules
00:29:55.29 that can self-assemble into membranes and generate compartments spontaneously.
00:30:00.16 So these are molecules that are amphiphilic:
00:30:03.07 They have one part that likes to be in water,
00:30:05.26 and another part that doesn't like to be in water.
00:30:09.11 And the way that those preferences are balanced
00:30:12.29 is by forming membranes in which the nonpolar parts are on the inside
00:30:17.29 and the polar parts of the molecule face out into the water.
00:30:21.16 So it turns out that it's actually, again,
00:30:24.19 very easy to make molecules like that in a variety of different scenarios.
00:30:30.02 In fact, Dave Deamer and his colleagues showed that you can
00:30:34.08 extract molecules from the Murchison meteorite
00:30:37.11 (it's one of these carbonaceous chondrite meteorites that's rich in organic materials),
00:30:41.23 you can extract molecules that will self-assemble into a vesicle,
00:30:46.10 as you can see here.
00:30:48.04 So they spontaneously make membrane sheets that close up into small vesicles.
00:30:53.19 Here's another example.
00:30:56.00 This is an experiment that was done to
00:31:00.11 mimic processes going on in interstellar molecular clouds,
00:31:05.03 where you have various gasses that have condensed
00:31:07.22 on the surface of silica particles.
00:31:10.11 They're subjected to irradiation by ultraviolet light and ionizing radiation.
00:31:16.03 So if you make ices like that in the laboratory,
00:31:19.18 subject them to ultraviolet radiation,
00:31:22.29 you get a lot of complicated chemistry going on,
00:31:25.01 and then in that vast mix of products,
00:31:28.28 you can extract molecules which again will form membranes
00:31:32.01 and self-assemble into these vesicle compartments.
00:31:37.10 Here is yet another scenario.
00:31:39.03 This is a hydrothermal synthesis done by Bob Hazen and Dave Deamer.
00:31:46.05 Again, in hydrothermal processing,
00:31:50.00 you can grow carbon chains with oxygenated groups
00:31:55.05 such as carboxylates at the end,
00:31:57.05 and these self-assemble into membranes and make many compartments,
00:32:02.03 as you can see in this beautiful image.
00:32:05.29 So, what would be an example of an early Earth
00:32:09.29 environment where something like this could take place?
00:32:14.03 There are a series of experiments from the Simoneit Lab that
00:32:19.14 suggest that hydrothermal synthesis could happen deep down
00:32:24.28 in regions with high temperature and high pressure,
00:32:29.25 on the surface of catalytic minerals such as transition metal sulfides or oxides,
00:32:35.27 and those reactions would basically turn hydrogen and carbon monoxide
00:32:40.27 into fatty acids and related compounds.
00:32:45.05 So the next slide here is a movie that was prepared by Janet Iwasa,
00:32:50.29 that illustrates this process.
00:32:52.15 So we're going deep into the Earth,
00:32:54.23 down through the water channels of a geyser,
00:32:59.00 and here we're looking at the surface of these catalytic transition
00:33:03.28 metal minerals, and you can see hydrogen and carbon monoxide molecules
00:33:08.24 bouncing around the surface, and the mineral is catalyzing
00:33:13.11 their assembly into chains, which eventually will be released and float up,
00:33:21.00 and they'll be caught up in the flow of water
00:33:23.02 and thereby brought to the surface, where you can imagine these fatty acids,
00:33:28.15 fatty alcohols, and related molecules being aerosolized
00:33:32.22 and concentrated in droplets and perhaps even
00:33:35.20 building up into large deposits on the land surface.
00:33:41.05 So it doesn't seem like the prebiotic assembly of molecules
00:33:46.07 that could spontaneously form membrane vesicles is all that difficult.
00:33:53.03 It's definitely an understudied area of prebiotic chemistry, it needs more work,
00:33:57.16 but it looks, I think, reasonably plausible.
00:34:01.13 So the most prebiotically likely molecules
00:34:04.15 would be things like capric acid that you see down here.
00:34:08.24 Short chain, saturated fatty acids.
00:34:12.15 So we do experiments in the lab with molecules like this,
00:34:16.28 but we also use longer chain, unsaturated molecules
00:34:22.07 like myristoleic acid and oleic acid,
00:34:25.05 as model systems because they're just generally easier to work with.
00:34:29.16 So what happens if you just take one of these fatty acids
00:34:34.20 and shake it up in water with some salt and buffer?
00:34:38.06 Is it hard to make membranes? No.
00:34:40.20 What you can see if that you just spontaneously make vesicles
00:34:47.27 in a huge variety of complex structures, a huge range of sizes,
00:34:53.25 all the way from 30 microns (this large vesicle)
00:34:57.19 to many, many smaller vesicles ranging down to 30 nanometers.
00:35:02.29 Many of these vesicles are composed of multiple sheets of membrane,
00:35:08.04 so stacks of membranes.
00:35:10.04 You can see some of these vesicles have smaller vesicles inside them.
00:35:14.17 So it's a very heterogeneous, complex mixture.
00:35:19.29 Now, the other thing that's really important about this is that these vesicles,
00:35:25.25 these membranes, have very, very different properties
00:35:29.02 from modern biological membranes.
00:35:31.26 Modern membranes are basically evolved to be good barriers,
00:35:37.09 so that cells can control the flow of all molecules in and out
00:35:42.04 using complicated protein machines.
00:35:47.26 For a primitive cell, you wouldn't want a situation like that...
00:35:51.03 that would be suicidal.
00:35:52.15 These molecules have to let stuff get across,
00:35:55.03 they have to have dynamic properties
00:35:56.26 that can let them grow and equilibrate.
00:36:00.16 So the next slide is actually a movie, again prepared by Janet Iwasa,
00:36:05.13 to illustrate the dynamic properties of these vesicles,
00:36:09.29 which are so different from modern membranes.
00:36:13.06 And so what you can see here is, first of all,
00:36:15.08 the motion on the surface, a lot of oscillations, diffusion.
00:36:20.27 In the membrane itself, these molecules, the individual molecules
00:36:25.05 are rapidly flip-flopping back and forth from inside to outside,
00:36:29.23 they're constantly entering the membrane, leaving the membrane,
00:36:34.16 so there's a lot of exchange reactions that are
00:36:37.29 going on on very rapid timescales, on the order of a second or less.
00:36:42.23 So they're very dynamic structures.
00:36:44.23 And these dynamic motions are also probably
00:36:49.09 very important in terms of permeability.
00:36:51.26 They allow the formation of transient defects in the membrane,
00:36:55.20 which let molecules get across spontaneously
00:36:58.21 without any complicated machinery.
00:37:02.19 There's another property of these vesicles which I find quite fascinating.
00:37:09.02 So as you saw in the illustration, the molecules that make up
00:37:12.28 any given vesicle come and go and therefore exchange between vesicles
00:37:19.07 on the timescale of roughly a second.
00:37:22.09 In this slide what you see are two populations of vesicles
00:37:25.16 that were labeled with phospholipid dyes,
00:37:28.18 so they're not exchanging between vesicles.
00:37:31.18 The picture here was taken after about a day,
00:37:35.18 and so you can see that they haven't all just fused and mixed up,
00:37:39.01 there are still red vesicles and green vesicles.
00:37:42.11 And yet we know from our other experiments that the molecules
00:37:45.27 that make up any one of these vesicles are changing
00:37:50.29 on a very rapid timescale, yet the structures themselves
00:37:54.23 maintain their identity on the timescale of weeks or months.
00:38:02.03 What about the nucleic acids then?
00:38:05.05 We've talked a lot about the building blocks of membranes,
00:38:07.27 the way they self-assemble,
00:38:09.12 and the properties of the membranes that they assemble into...
00:38:12.27 let's go back to the genetic materials and think about
00:38:15.21 what kinds of building blocks we need to assemble molecules like RNA.
00:38:22.06 Now, again, we have a difference between the molecules used in modern life...
00:38:28.12 so these of course are nucleoside triphosphates,
00:38:32.11 they're almost ideal substrates for a highly evolved cell
00:38:37.13 with very, very powerful catalysts.
00:38:41.15 These molecules are kinetically trapped in a high-energy state.
00:38:47.13 They don't spontaneously act very well at all,
00:38:51.29 so it takes a very sophisticated catalyst to
00:38:54.27 use molecules like this as a substrate.
00:38:57.26 They're also of course very polar, the triphosphate group is highly charged,
00:39:02.16 and that prevents these molecules from leaking out of the cell,
00:39:06.23 which would be a bad thing.
00:39:08.14 On the other hand, in a primitive cell,
00:39:11.19 if you imagine that substrates, food molecules,
00:39:15.05 are being made in chemical processes out in environment,
00:39:19.15 it needs to be possible for those molecules to get across the membrane
00:39:23.17 spontaneously and get into the interior of the cell.
00:39:27.18 So then we to think about different kinds of substrates,
00:39:31.19 molecules that are less polar so they can get into the cell,
00:39:36.25 and more chemically reactive, so that they can polymerize without the need
00:39:43.15 for very sophisticated, advanced, highly evolved catalysts.
00:39:47.24 And so molecules like this were first made by Leslie Orgel
00:39:52.20 and his students and colleagues 20-30 years ago,
00:39:57.13 and studied in quite a bit of detail as models for the early replication of RNA.
00:40:06.17 So, this brings us back to the question of
00:40:12.02 what was the first genetic material?
00:40:14.17 Was it RNA, in fact?
00:40:17.14 Or is RNA so complicated,
00:40:19.24 or its building blocks so hard to make,
00:40:23.03 that life more likely began with something simpler,
00:40:27.18 something easier to make,
00:40:29.12 maybe something more stable that could accumulate,
00:40:31.24 like DNA for example?
00:40:34.16 So this is an area of active debate and investigation,
00:40:39.10 we really don't know the answer to this question,
00:40:43.13 but lots of people are doing experiments and trying
00:40:45.22 to work out chemical pathways leading up to RNA,
00:40:49.09 for example, the Sutherland Lab in the UK has made a lot of progress in this area.
00:40:55.16 We're studying how these molecules could be assembled and replicated.
00:41:01.27 So one of the satisfying thinks about thinking about RNA
00:41:04.22 as the first genetic material,
00:41:06.10 is that we actually have two different chemical physical processes
00:41:12.29 that can lead to the polymerization of activated building block
00:41:17.01 into long RNA chains.
00:41:19.16 The first of these was discovered by Jim Ferris, working with Leslie Orgel,
00:41:26.27 and that was the discovery that a common clay mineral
00:41:30.20 known as montmorillonite can catalyze the assembly of
00:41:34.19 nucleotides into RNA chains.
00:41:37.04 So this illustrates the structure of this clay,
00:41:39.25 it's a layered hydroxide mineral.
00:41:43.03 In between the layer, the aluminum silicate layers,
00:41:47.21 there's water, and in these inner layers,
00:41:50.27 organic molecules can accumulate, and when they're brought close together,
00:41:54.23 they can react each other and start to polymerize.
00:41:58.25 So here is some of the experimental data.
00:42:01.29 So over a period of days, you start off with small chains,
00:42:07.08 and then gradually they get longer and longer, up to lengths of roughly 40,
00:42:12.18 and in more recent experiments up to 50 or 60, nucleotides long.
00:42:17.27 So I wanted to illustrate that with this movie,
00:42:20.16 another one of Janet Iwasa's animations,
00:42:24.16 to show roughly how we think this works.
00:42:27.15 So these chemically activated building blocks like to stick to the
00:42:31.13 surface of the clay mineral,
00:42:33.20 and when they stick in such a way that they're lined up with each other,
00:42:38.05 they can react and assemble a chemically linked backbone,
00:42:43.16 as you see here.
00:42:47.28 Now, there is another process that can do that same thing,
00:42:51.05 which is very interesting because it's so counterintuitive.
00:42:54.05 It turns out if you take these same building blocks and just have them
00:42:57.29 in a dilute solution and put that on your bench, nothing happens.
00:43:03.10 But if you take that same solution and put it in the freezer
00:43:07.08 and then come back the next day, you'll find RNA chains.
00:43:11.20 Why is that?
00:43:13.09 It's because when water freezes and forms ice crystals,
00:43:16.26 that during the growth of the ice crystals,
00:43:19.12 other molecules (solutes) are excluded from the growing crystal,
00:43:23.23 and so they end up concentrated as much as a thousand fold
00:43:27.21 in between the grains of ice,
00:43:31.04 and so when they're so concentrated, again they can react and polymerize.
00:43:35.08 So having two different processes that can lead the assembly of
00:43:38.23 RNA chains is actually a very satisfying thing...
00:43:41.29 that's something we look for in this field,
00:43:43.29 if there's more than one way of solving a problem,
00:43:47.04 it makes the whole solution seem more robust.
00:43:51.15 Now, the hardest problem, perhaps,
00:43:54.07 is once you've got RNA chains like this, how can they be replicated?
00:43:59.16 So much of our early thinking was based on RNA catalysis,
00:44:04.19 and in fact the whole basis of the RNA world is the idea
00:44:07.16 that RNA can act as an enzyme that could catalyze its own replication.
00:44:13.18 And Dave Bartel, when he was a student in my lab many years ago,
00:44:19.17 actually evolved an RNA enzyme with a catalytic activity,
00:44:25.11 that can ligate together pieces of RNA.
00:44:29.04 And Dave subsequently evolved this ribozyme into an even more complex structure
00:44:35.07 that is really an RNA polymerase made out of RNA.
00:44:40.09 Now, that's a very impressive proof of principle,
00:44:43.26 but unfortunately, despite many advances over the years,
00:44:47.26 we're still far from having an RNA molecule that can
00:44:51.09 completely catalyze the copying of its own sequence.
00:44:55.28 So, what we've decided to do is to actually again step back
00:45:01.07 and try to look at the underlying chemistry
00:45:04.03 and see if there might be ways of adjusting or playing
00:45:10.05 with the chemistry of RNA polymerization that would simplify this problem.
00:45:17.04 Ideally, perhaps we will be able to find a complete chemical process
00:45:22.00 that could drive RNA replication.
00:45:25.15 Now, that's a very difficult task,
00:45:28.16 Leslie Orgel and his colleagues worked on that for many years,
00:45:32.17 got partway to a solution,
00:45:34.22 but were never able to have complete cycles of replication.
00:45:39.23 But we have decided to go back and look at some model systems
00:45:44.16 and see if we can get some clues as to how to approach that problem,
00:45:48.24 perhaps in some fresh ways.
00:45:51.13 So, just to illustrate what we're really after,
00:45:53.24 I'm going to show another of Janet Iwasa's movies,
00:45:57.16 and so what you see here is an RNA template, a single-stranded molecule,
00:46:02.00 floating in a solution full of activated monomers,
00:46:05.05 which then find their complementary bases,
00:46:07.26 so they use Watson-Crick base pairing to line up on the template,
00:46:11.06 and then they basically click together to build up a complementary strand,
00:46:16.16 generating a duplex product.
00:46:20.25 So we're after some kind of simple,
00:46:25.03 chemical system that would drive that process very efficiently.
00:46:30.18 So, if we could get to that point,
00:46:33.06 then we would be back to being able to assemble this kind of model system,
00:46:39.10 a model protocell, composed of a membrane compartment boundary
00:46:45.07 and replicating genetic material on the inside.
00:46:50.28 Now, when we're thinking of a complex composite system like this,
00:46:56.13 the question often arises as to,
00:46:58.06 well, why really bother with the membrane compartment?
00:47:01.10 Why not just let the RNA molecules replicate in solution?
00:47:05.17 And one way of thinking about that is that,
00:47:09.22 for Darwinian evolution to emerge,
00:47:12.27 molecules that are in some way better than their neighbors
00:47:15.25 have to have an advantage for themselves.
00:47:19.01 So if we think about RNA replicases floating around in solution,
00:47:24.02 so these would be RNA molecules that catalyze the replication
00:47:27.24 of another RNA molecule,
00:47:30.14 it doesn't really help if you have a mutation which is faster or more accurate,
00:47:37.23 if all it's doing is copying random, other RNAs
00:47:41.01 that it bumps into in solution.
00:47:44.01 It has to have an advantage for itself.
00:47:47.18 And the simplest way to imagine that happening
00:47:50.13 is to encapsulate these molecules within a vesicle,
00:47:54.20 so that they're always copying molecules that are related by descent.
00:48:01.03 Now, the self-assembly of these kinds of complex structures
00:48:06.22 is something that's actually quite simple.
00:48:09.28 So, at the lowest level,
00:48:12.26 the formation of a membrane vesicle can just encapsulate
00:48:16.01 whatever is there in the surrounding solution.
00:48:19.17 However, it's intriguing that there are ways of making the process more efficient,
00:48:24.02 and one of the most interesting ways of doing that is
00:48:28.05 to take advantage of that same clay mineral, montmorillonite,
00:48:31.25 that we've already seen can catalyze the assembly of RNA strands.
00:48:36.24 And so what you can see in this picture,
00:48:39.19 which was generated by Shelly Fujikawa and Martin Hanczyc
00:48:44.11 when they were in my lab about eight years ago...
00:48:47.03 what you can see is that we have here a clay particle,
00:48:51.15 which has RNA molecules bound to its surface,
00:48:55.21 so the orange color is a dye-labeled RNA,
00:48:59.02 and it turns out these clay particles can catalyze the
00:49:01.28 assembly of membrane sheets from fatty acids.
00:49:08.15 And what's happened here is that this clay particle has catalyzed
00:49:11.15 the assembly of this large surrounding vesicle
00:49:15.29 as well as the many smaller vesicles encapsulated within.
00:49:20.03 So what we now can see is that a single very common,
00:49:25.11 abundant mineral can catalyze the assembly of a genetic material,
00:49:30.17 it can catalyze the assembly of compartment boundaries (cell membranes),
00:49:34.08 and it can help bring them together.
00:49:36.13 So very intriguing as a way of simplifying the assembly of
00:49:39.14 cell-like structures on the early Earth.
00:49:43.02 Here's another picture: clay particle inside a vesicle.
00:49:48.01 Here the boundary is quite dramatically evident,
00:49:52.07 so this is a stack of many layers of membrane bilayers.
00:49:56.26 Here's yet another example where the large outer vesicle
00:50:00.15 is filled with hundreds of smaller vesicles, all assembled under
00:50:04.08 the catalytic influence of this clay particle in the middle.
00:50:10.11 So, assembling these things looks fairly simple.
00:50:14.02 What about the process of growth and division?
00:50:16.21 After all, that's what we really need to generate
00:50:19.07 cell-like structures that can propagate.
00:50:22.18 And at this point,
00:50:25.01 what I can say is that we've come up with a process that looks fairly robust.
00:50:31.09 We can start with vesicles and food in the form of fatty acid micelles.
00:50:37.16 They grow remarkably into filamentous structure,
00:50:42.13 which can then divide very easily into daughter cells,
00:50:45.19 and this generates a cycle that can go around and around indefinitely.
00:50:51.08 And in the next part of this lecture,
00:50:53.26 I'll go into much more detail about the nature of this process
00:50:57.09 and the mechanism by which this happens.
00:51:01.02 But, putting this cycle together with our
00:51:06.17 thinking about nucleic acid replication,
00:51:09.13 we can actually start to imagine what a
00:51:12.13 primitive cell cycle would have looked like.
00:51:15.12 And so this is shown in this figure from a Scientific American article
00:51:20.04 that I wrote with Alonso Ricardo from my lab,
00:51:23.03 and it summarizes some of our ideas about the ways in which
00:51:28.28 the early Earth environment might help to drive cell growth and division.
00:51:35.22 So the idea is that the general environment should be rather cold,
00:51:40.15 perhaps even an ice-covered pond,
00:51:44.24 something you might find in an arctic or alpine environment.
00:51:48.29 There are many examples on the modern Earth.
00:51:52.16 The reason for wanting a cold environment in general is that the
00:51:56.08 copying chemistry seems to go better at low temperatures.
00:52:01.06 The low temperature helps the building blocks
00:52:03.10 to bind to the template and facilitates the copying process.
00:52:07.10 But then we know that eventually, once copying is complete,
00:52:10.28 you have to get the strands apart so that you can
00:52:13.09 undergo another round of copying.
00:52:15.21 Simplest way for that to happen is to invoke high temperatures.
00:52:19.23 And so what we like to think about are
00:52:21.21 convection cells driven by geothermal energy;
00:52:26.12 so essentially in a hot spring type of environment,
00:52:30.13 you could have a pond that's mostly cold,
00:52:32.20 but every now and then, these particles would get caught up
00:52:35.01 in a plume of hot water rising from a hot spring.
00:52:39.01 They'd be transiently exposed to high temperatures
00:52:41.24 that would result in strand separation.
00:52:45.02 It also allows for a rapid influx of nutrients from the environment
00:52:50.02 to feed growth and replication through the next round.
00:52:54.08 And then that would generate a cycle in which the
00:52:57.25 entire process of growth and replication and division
00:53:01.07 is driven by fluctuations in the environment.
00:53:04.20 This is driving us to talk to geologists and to search
00:53:09.19 for analogues of this kind of environment on the modern Earth.
00:53:13.13 Here is a beautiful image of an Antarctic lake
00:53:18.19 in which you see stromatolites,
00:53:20.15 these mounds here are microbial growths on the surface,
00:53:25.18 and the reason that it's liquid is of course there is heat rising up from
00:53:29.18 below geothermally, so it's not a perfect analogue
00:53:34.15 of the scenario I described.
00:53:36.05 We'd like to find environments like this where there are hot springs
00:53:39.12 generating convection cells that could drive the whole cycle.
00:53:43.05 So that would be very satisfying if we could identify such environments.
00:53:48.29 So, what I've tried to show in this lecture is basically
00:53:54.13 the context of the environment and the chemistry
00:53:57.10 leading up to the assembly of primitive cells,
00:54:00.25 in a way that's plausible on the early Earth.
00:54:03.17 And what we'll head into in the next two parts
00:54:06.18 are a more detailed look at the chemistry of membrane assembly,
00:54:11.11 growth, and division;
00:54:12.16 and the chemistry of nucleic acid replication.
00:54:15.25 And all of this work is of course has been done through many
00:54:20.17 very talented students and postdocs in the lab
00:54:25.03 who you can see here on this slide.
00:54:28.13 Thank you.

00:00:00.00 My name is Jack Szostak,
00:00:02.10 I'm a Professor Genetics at Harvard Medical School,
00:00:05.22 and an Investigator at Massachusetts General Hospital,
00:00:08.22 where my laboratories are.
00:00:10.21 I'm also an Investigator of the Howard Hughes Medical Institute.
00:00:14.07 And in this lecture, I would like to tell you about some of our

00:00:18.21 recent work relevant to the origin of life on Earth,

00:00:22.12 and we're going to focus this time on the membrane component of simple protocells,

00:00:30.03 how those membranes assemble,

00:00:32.03 what their properties are,

00:00:34.03 how those properties are relevant for early forms of life,

00:00:38.08 and how we imagine them gradually changing

00:00:42.27 as a result of the emergence of Darwinian evolutionary processes.

00:00:48.28 So, let's begin by having a look at our conceptual model

00:00:55.26 of what an early protocell looks like.

00:00:59.09 So, again, we think of protocells having two parts:

00:01:03.24 A membrane boundary that closes up into a spherical vesicle

00:01:11.25 that can encapsulate molecules in the internal, aqueous compartment...

00:01:16.21 and the most important of those would be genetic molecules,

00:01:21.25 possibly RNA or DNA or maybe some related kind of nucleic acid.

00:01:27.04 And what we would like to understand is how,

00:01:33.07 in the absence of highly evolved genetic material,

00:01:37.18 this membrane compartment could first grow

00:01:41.22 and subsequently divide into daughter cells.

00:01:47.10 And because we're thinking about this at a very early time,

00:01:53.14 prior to the emergence of Darwinian evolution

00:01:56.16 and advanced, highly evolved cellular machinery,

00:01:59.17 we have to look to simple chemical and physical processes

00:02:04.15 to drive these transformations.

00:02:08.11 So, to think about that,

00:02:10.22 we need to think about the molecules that are going to be used

00:02:14.11 to build these membranes, and our favorite molecules

00:02:18.28 in this regard are the fatty acids.

00:02:22.01 So, these range from short-chain, saturated fatty acids,

00:02:26.20 like capric acid, which are the kinds of molecules

00:02:29.05 that we think could have been easily made in different scenarios

00:02:33.01 on the early Earth.

00:02:35.05 Many times in our laboratory work,

00:02:37.07 we use more modern fatty acids

00:02:40.03 (myristoleic acid or oleic acid) simply for convenience,

00:02:45.01 but in the end, we always come back to more prebiotically reasonable systems.

00:02:52.12 So what are the biophysical properties of these closed-up membranes,

00:03:00.08 in the form of vesicles or liposomes?

00:03:04.12 So, the most important aspect,

00:03:08.13 and something which is quite different from modern membranes

00:03:12.21 that are based on phospholipids, is that fatty acid vesicles...

00:03:19.20 basically, their formation and existence is controlled

00:03:23.08 by a series of phase transitions.

00:03:26.29 And the most important of those depends on the pH of the environment.

00:03:31.21 So, at fairly alkaline pHs,

00:03:35.17 the carboxylates of fatty acids are basically fully ionized,

00:03:40.24 and then charge repulsion wants to keep those groups

00:03:45.00 as far apart from each other as possible,

00:03:48.07 while the hydrophobic forces want to keep the hydrophobic tail

00:03:52.18 of the molecule as close together as possible.

00:03:55.26 And the result is the formation of micelles,

00:03:59.14 which are very small aggregates, a few nanometers in size,

00:04:05.08 and composed of roughly 10 to 100 fatty acid molecules.

00:04:09.20 Now as the pH in the environment gradually

00:04:12.00 drops towards roughly 8.5,

00:04:16.26 which is the point at which half of these carboxylates

00:04:19.21 will be ionized and half-protonated,

00:04:22.06 there's a phase transition that results in the spontaneous

00:04:25.14 assembly of bilayer membranes, as you can see here.

00:04:29.21 And because the curvature is much reduced in these much larger aggregates,

00:04:36.00 these are actually stabilized by hydrogen bonding

00:04:41.19 interactions between adjacent charged and ionized carboxylates.

00:04:47.07 Now, as the pH drops even further into the acidic range,

00:04:51.07 all of the carboxylates will become protonated eventually,

00:04:55.05 and then these structures collapse into oil droplets.

00:04:59.01 So the existence of these bilayer membranes

00:05:02.27 is only possible at an intermediate pH range, close to the pKa.

00:05:08.23 You'll notice this pKa of 8.5 is considerably higher

00:05:12.11 than the pKa of individual fatty acid molecules in solution,

00:05:17.24 which is closer to 4.5.

00:05:19.20 And the reason for that is that these charged head groups

00:05:23.15 are much closer together in this aggregate structure.

00:05:29.16 Now, when you do form these membranes,

00:05:32.14 they have very interesting properties,

00:05:34.18 which again are very different from the

00:05:37.01 properties of phospholipid-based membranes.

00:05:40.27 And one of the things that is very important to us

00:05:44.08 is the dynamic exchange processes that occur in these membranes.

00:05:49.02 So fatty acid molecules in a fatty acid membrane

00:05:52.25 flip-flop back and forth, from the outer leaflet to the inner leaflet,

00:05:57.28 very rapidly, sub-second timescale.

00:06:01.00 They also exchange in and out of the membrane,

00:06:06.03 so into solution, where they can exchange with molecules in micelles,

00:06:10.25 and again this is a very rapid process,

00:06:14.03 so that the molecules that make up any given vesicle

00:06:17.08 are constantly in flux and exchanging with the molecules in other vesicles,

00:06:22.01 again on the second timescale.

00:06:27.13 Another very interesting aspect of these membranes is that

00:06:34.24 they can be quite heterogeneous at a molecule level.

00:06:40.02 And so these membranes can incorporate not just fatty acids

00:06:46.14 but also their glycerol esters, as you see here,

00:06:50.01 and even lyso-phosphatidic acids,

00:06:53.10 so an ester with phosphoglycerate.

00:06:56.12 They can incorporate alkanes and

00:07:00.02 polycyclic aromatic hydrocarbons as well.

00:07:04.00 Now, what happens when you make membranes

00:07:07.00 with these kinds of mixed compositions?

00:07:10.09 It's very interesting that, in many cases,

00:07:14.15 this results in membranes that are much more stable,

00:07:18.15 they're more physically stable, more thermostable, and also,

00:07:22.25 often more permeable to polar nutrients that we would like to

00:07:27.04 bring in from the outside environment.

00:07:30.27 So this slide simply illustrates an experiment

00:07:35.05 to look at the thermostability of some vesicles

00:07:39.18 made from a pure fatty acid, as opposed to mixtures.

00:07:43.01 So in this experiment, what we've done is to encapsulate

00:07:47.18 a short DNA oligonucleotide within vesicles

00:07:52.08 and watch it leak out over the course of about an hour.

00:07:57.07 And now if the membrane is composed purely of myristoleic acid,

00:08:02.23 this 14-carbon, singly unsaturated fatty acid,

00:08:06.11 you can see that up to about 50 degrees,

00:08:09.04 nothing leaks out over an hour.

00:08:11.11 But as the temperature goes up to about 80,

00:08:14.20 they become more leaky, and then at 90 degrees,

00:08:18.09 everything is leaked out over an hour.

00:08:21.00 Now, if you add some of the corresponding alcohol,

00:08:23.26 you can see significant stabilization,

00:08:27.10 but the most dramatic effect is when you add a fraction of the

00:08:31.27 glycerol ester of that fatty acid.

00:08:34.04 And now, you can essentially boil these vesicles for an hour,

00:08:39.00 and none of the contents leak out,

00:08:41.13 at least these oligonucleotide contents are trapped inside.

00:08:46.02 So that's really exactly what we want.

00:08:49.00 We want the genetic material, long oligonucleotides,

00:08:53.18 to stay inside indefinitely,

00:08:56.24 while small molecules (nutrients)

00:08:59.00 can get across the membrane very rapidly.

00:09:03.06 And again, as the temperature goes up,

00:09:05.07 the permeability to small molecules increases dramatically.

00:09:11.02 So, we got into this area about 10 years ago,

00:09:17.16 and started doing some very simple experiments

00:09:21.20 based on the pioneering work of people like Dave Deamer

00:09:26.14 and Pier Luigi Luisi.

00:09:28.14 Now, the Luisi Lab in particular had shown already

00:09:32.05 that you can make vesicles grow simply by

00:09:35.24 adding more fatty acids in the form of alkaline micelles.

00:09:40.05 When the micelles go into a buffered solution,

00:09:42.21 they become a thermodynamically unstable phase,

00:09:45.21 so there's an energetic driving force for those molecules

00:09:49.04 to assemble into new membranes or

00:09:51.21 to integrate into preexisting membranes.

00:09:56.09 And at the time,

00:09:57.27 Martin Hanczyc was a postdoc in the lab,

00:09:59.29 Shelly Fujikawa was a student...

00:10:03.01 they basically repeated those experiments,

00:10:06.01 but using real-time methods of analysis,

00:10:09.23 so that we could watch the process of vesicle growth in real time,

00:10:14.19 after the addition of food molecules.

00:10:17.15 The assay that we used was a fluorescence assay

00:10:21.21 based on energy transfer from donor to acceptor dyes.

00:10:27.02 It's basically a measure of how far apart the dyes are on average.

00:10:31.15 And as the membrane grows by incorporating new molecules,

00:10:34.28 the dyes are diluted, they get further apart,

00:10:37.14 and energy transfer efficiency decreases.

00:10:40.18 So you can use this as a very effective assay for

00:10:43.05 surface area in real time, so we can watch things grow very easily

00:10:48.27 by this simple fluorescence assay.

00:10:52.17 And the essential result from that is that, if you add the food slowly,

00:10:57.02 almost all of it gets incorporated into preexisting membranes;

00:11:00.13 if you add it very quickly, only a fraction, maybe half,

00:11:04.26 will get incorporated, and the rest will form new vesicles.

00:11:08.00 So that takes care of growth at a basic level.

00:11:11.22 What about division?

00:11:13.24 So in the early days, we didn't really have any good idea

00:11:17.27 of how to drive protocell division through environmental fluctuations.

00:11:23.25 So essentially, in desperation,

00:11:26.04 we resorted to a brute force approach of forcing big vesicles

00:11:31.12 through small pores in a filter,

00:11:33.23 and little vesicles come out the other side.

00:11:37.00 So, on the one hand,

00:11:39.00 it's satisfying because it's a proof of principle that

00:11:41.26 cell division could be driven by a physical, environmental force.

00:11:47.00 On the other hand, it's not very satisfying because

00:11:49.22 we didn't think there was any realistic analogue

00:11:53.26 to that process on the early Earth.

00:11:56.07 And furthermore, when you take a large vesicle

00:11:59.16 and physically divide it into smaller spherical vesicles,

00:12:03.18 you necessarily lose some of the contents,

00:12:06.16 which is also not very satisfying.

00:12:09.15 Nonetheless, that process could be turned into a cycle,

00:12:13.19 where we could do growth and division

00:12:15.28 and growth and division, again and again.

00:12:20.04 So then the problem really became:

00:12:22.15 How could we find other pathways that might have

00:12:27.15 been more physically realistic, more robust,

00:12:30.24 and more plausible for the early Earth?

00:12:34.29 So, the first advance in this area came from

00:12:41.21 thinking about an alternative growth pathway.

00:12:46.03 And so this is a pathway of growth that,

00:12:48.08 instead of relying on the environmental influx of new molecules,

00:12:54.23 occurs in a different way,

00:12:56.04 by competition between protocells in a population.

00:13:01.13 So some of are going to grow at the expense of others.

00:13:04.15 And this was work done by Irene Chen when she was a student in my lab,

00:13:09.09 and initiated by discussions with Rich Roberts,

00:13:12.12 when he was a postdoc in the lab.

00:13:14.14 So the basic idea was that,

00:13:17.08 if you think of our basic protocell model,

00:13:21.17 we have genetic molecules trapped inside a semipermeable membrane.

00:13:27.19 Now, large polymers contribute very little to the

00:13:31.06 overall internal osmotic pressure in a system like this.

00:13:36.13 Most of the osmotic pressure actually results from

00:13:40.11 counter-ions that have to be there to neutralize the charge

00:13:44.11 on the polyanionic genetic molecule.

00:13:47.29 And so this is the classical Donnan effect:

00:13:51.25 These ions contribute to an internal osmotic pressure,

00:13:56.03 and we thought that, as a result of this physical phenomenon,

00:14:01.05 this physical effect,

00:14:02.28 vesicles that had more RNA inside of them

00:14:06.01 should have a higher osmotic pressure,

00:14:09.12 and maybe that could actually drive growth competitively.

00:14:14.21 So, the way that we looked at that experimentally

00:14:19.08 was using the same fluorescence assay that we had been using

00:14:24.17 before to monitor membrane growth following

00:14:28.11 the addition of new membrane molecules,

00:14:31.13 but in this case, the idea is that we're going to take

00:14:37.05 osmotically swollen vesicles that have,

00:14:39.28 for example, a lot of RNA inside,

00:14:42.16 and we'll monitor the surface area of those vesicles following

00:14:47.22 mixing with vesicles that are osmotically relaxed,

00:14:51.25 and we'll ask what happens to the surface area.

00:14:56.14 So here's the experiment,

00:14:58.23 where we're monitoring the surface area of the swollen vesicles,

00:15:03.10 these are vesicles that have a lot of RNA on the inside,

00:15:06.28 and by themselves, they're perfectly stable, nothing happens,

00:15:11.02 same thing for the relaxed vesicles, nothing happens.

00:15:13.24 As soon as you mix the swollen vesicles with relaxed vesicles,

00:15:18.02 you can see that their surface area starts to increase

00:15:22.05 over a period of minutes.

00:15:24.09 And in control experiments,

00:15:25.29 where we mix with buffer or other swollen vesicles,

00:15:29.21 there's very little change.

00:15:31.28 So it seems like the swollen vesicles are growing

00:15:35.19 by absorbing molecules from their relaxed neighbors.

00:15:40.11 We can of course do the converse experiment,

00:15:43.11 and monitor the surface area of the relaxed vesicles,

00:15:47.12 and when they are mixed with swollen vesicles,

00:15:49.25 their surface area declines, so molecules are leaving

00:15:53.05 those vesicles and going into the swollen vesicles.

00:15:56.10 Again, control experiments where relaxed vesicles

00:15:58.17 are mixed with more relaxed vesicles, not much happens.

00:16:03.06 So what's going here is that,

00:16:07.09 as soon as we mix these populations of vesicles,

00:16:09.20 the swollen ones are growing, the relaxed ones are shrinking,

00:16:13.28 and the reason that's happening is that this osmotic pressure

00:16:17.10 creates a tension in the membrane,

00:16:20.20 which is only relaxed by the incorporation of more molecules,

00:16:24.21 which allows the surface area to increase.

00:16:28.04 So the exciting implication of that physical process

00:16:32.17 is that it's a potential link between genome replication

00:16:37.09 and faster membrane growth.

00:16:39.08 So, any process that results in faster replication

00:16:43.15 of the genetic material inside a vesicle will automatically,

00:16:48.28 as a result of the Donnan effect,

00:16:51.05 lead to faster growth of the membrane compartment.

00:16:54.07 So it's a coupling between genome replication

00:16:57.01 and cell growth as a whole.

00:16:59.16 Now, that could be particularly interesting if genome replication

00:17:04.22 is an autocatalytic process driven by RNA-catalyzed RNA replication.

00:17:10.11 So now, any mutation that leads to faster,

00:17:14.01 more efficient replication of the genetic material

00:17:17.18 will automatically go along with faster growth

00:17:20.12 of the cell as a whole,

00:17:22.04 and it's a competitive form of growth

00:17:24.10 that results from these cells essentially eating their neighbors.

00:17:30.06 So that was a very exciting conceptual advance,

00:17:34.17 but there was always this nagging problem.

00:17:38.09 That here, we're looking at growth driven by osmotic forces,

00:17:43.15 which means that these are growing as spherical vesicles.

00:17:48.09 And dividing a spherical vesicle,

00:17:50.13 especially one that's osmotically swollen,

00:17:52.25 is something that's actually quite difficult to do.

00:17:55.08 It takes a lot of energy,

00:17:57.10 and as I said in the case of the extrusion experiments,

00:18:00.18 results in loss of some of the contents,

00:18:02.27 which is not very satisfying.

00:18:05.04 So, we were left thinking for a long time

00:18:08.23 about possible alternative membranes

00:18:11.17 and alternative division processes,

00:18:15.07 and that's something we'll come back to later.

00:18:19.28 So, let's think about more natural systems.

00:18:27.24 Everything that we've looked at up till now

00:18:30.26 has been highly constrained, very artificial laboratory system

00:18:38.16 designed so that we can follow what's happening

00:18:42.29 analytically in a very clear and simple way.

00:18:46.12 But of course, the vesicles that form naturally

00:18:51.21 aren't so nicely constrained.

00:18:55.04 They're extremely heterogeneous,

00:18:58.19 and that generates a system which is much harder to study experimentally.

00:19:03.26 So that's illustrated by this image,

00:19:07.24 which shows the range in the diversity of vesicles that are formed

00:19:11.16 when you just shake up oleic acid with some salt and buffer.

00:19:15.18 You get these huge vesicles, tiny vesicles,

00:19:18.29 compound vesicles, it's a terrible mixture,

00:19:23.27 and if you imagine adding some more oleic acid to

00:19:27.08 this in the form of micelles,

00:19:28.25 so that everything grew a little bit,

00:19:31.22 you couldn't tell that anything had happened.

00:19:34.27 So we need new analytical ways of looking at this,

00:19:38.14 or new ways of preparing vesicles so that we can actually

00:19:42.13 watch what's happening.

00:19:44.08 And our major advance in that area came from the

00:19:48.06 work of a new graduate student, Ting Zhu,

00:19:51.28 who had a background in mechanical engineering

00:19:54.14 and proposed a very simple and elegant way of

00:19:57.25 preparing more or less uniform populations of vesicles

00:20:01.23 that were large enough to just look at in the microscope.

00:20:06.04 So for the first time, we were able to do experiments

00:20:09.08 where we take vesicles, add food, and just watch how they grow.

00:20:14.09 And so what you can see in this image is a field of vesicles,

00:20:18.00 they're all roughly four microns in diameter,

00:20:21.24 so big enough to image optically,

00:20:25.29 and what you're actually seeing here is a fluorescent dye

00:20:29.24 encapsulated in the aqueous internal space of each vesicle.

00:20:35.05 So now, what you'll see next is a time-lapse video

00:20:41.01 of how these things actually grow following the addition

00:20:44.17 of more fatty acids.

00:20:47.04 What we expected was simply that these spherical vesicles

00:20:51.26 would swell up a little bit, we'd just watch them get bigger.

00:20:56.19 We thought that, if the surface area grew faster than the volume,

00:21:01.11 they might become somewhat elongated in shape.

00:21:05.24 But we did not expect what actually happened,

00:21:09.05 which is quite dramatic.

00:21:12.16 So, what you see here is that, initially,

00:21:15.24 a faint filament emerging from the parental spherical vesicle,

00:21:20.06 and over time that filament grows and absorbs

00:21:23.21 all of the material in the starting spherical vesicle,

00:21:27.21 which has now grown into a long, thin, branched,

00:21:32.03 filamentous morphology.

00:21:35.13 So, this was a very surprising observation.

00:21:41.05 It immediately raised a host of mechanistic questions,

00:21:45.16 which we're still investigating.

00:21:48.01 But it, very importantly, immediately solved the problem

00:21:52.27 of how cell division might happen in a manner

00:21:56.29 driven by environmental fluctuations.

00:21:59.28 As I said, spherical vesicles are hard to divide,

00:22:02.23 it takes a lot of energy.

00:22:04.15 But these structures are incredibly fragile,

00:22:07.17 and all you have to do it shake them up.

00:22:11.21 Gentle agitation is sufficient to cause them to divide,

00:22:16.09 and you can see that process in action in the next video.

00:22:21.13 So here you see the pearling instability:

00:22:25.07 The filament forms a set of beads,

00:22:28.16 and then they snap apart and generate fragments,

00:22:32.27 which eventually round up into spherical daughter vesicles.

00:22:36.25 And so this process is driven by very gentle perturbations

00:22:43.01 in the fluid, very mild shear forces.

00:22:46.08 You can easily imagine the shear forces generated by

00:22:50.15 wave action on a shallow pond leading to cell division

00:22:54.06 in this kind of process.

00:22:56.27 Now, this process of growth followed by shear-induced division

00:23:03.10 makes a cycle that can be iterated indefinitely.

00:23:07.09 So you can start off with a spherical vesicle,

00:23:09.20 grow it into a filament, divide it into spherical daughter vesicles,

00:23:14.00 which can grow again.

00:23:16.03 If they sit around, the volume increases,

00:23:18.20 they round up, then you can grow again, divide, grow.

00:23:23.22 This cycle of growth and division can be carried on indefinitely.

00:23:29.19 So, let's think for a minute about some of the

00:23:32.29 mechanistic processes that are involved.

00:23:35.10 We don't understand everything that's going on,

00:23:37.13 but at least we have some experiments that tell us

00:23:41.21 what some of the critical factors are.

00:23:45.11 So, what we think is going on,

00:23:47.21 and what the important factors are,

00:23:49.26 is that surface growth is happening much faster than volume growth,

00:23:57.07 and so that makes a shape change inevitable.

00:24:01.15 Volume is conserved on the timescale of growth

00:24:04.16 because most of the solvents are only very slowly permeable

00:24:08.05 across these membranes,

00:24:10.05 and the solvent can be anything from the rather artificial buffer that we use,

00:24:16.13 to a mixture of amino acids,

00:24:18.12 such as you would get from Miller-Urey-type syntheses.

00:24:22.00 The other and somewhat surprising factor that's critical is the

00:24:26.07 multilamellar structure of these vesicles.

00:24:29.03 So these large vesicles tend to have several bilayers closely apposed,

00:24:35.29 as their sort of outer structure.

00:24:38.25 What we think happens is that when we add food molecules,

00:24:41.23 they first integrate into the outermost bilayer,

00:24:46.28 there's very little space between it and the next bilayer,

00:24:51.00 and so that extra surface area is extruded as a

00:24:55.01 thin, initial filament, which then subsequently elongates

00:25:01.27 into this long, tubular, filamentous structure.

00:25:05.21 We really don't understand the physical forces

00:25:08.04 that are involved in those later stages,

00:25:11.25 but we have some ideas about the initiation phases.

00:25:16.04 So I want to show you first an experiment that shows

00:25:19.25 that volume conservation is very important.

00:25:22.23 If you use a buffer that is very, very rapidly permeable,

00:25:27.24 then you don't see this kind of process at all.

00:25:30.22 Instead, what happens is that the outermost bilayer just balloons up.

00:25:36.25 As surface area increases, volume keeps up,

00:25:40.10 the volume expands, and so you end up with

00:25:43.18 most of the bilayers almost unchanged,

00:25:48.12 and trapped within a much larger unilamellar vesicle.

00:25:53.26 Now, the second important aspect is how

00:25:58.18 multilamellar vesicles grow in contrast to how unilamellar vesicles grow.

00:26:04.10 So when you have these multiple bilayers, as I suggested,

00:26:08.17 it looks like, from these confocal images,

00:26:11.00 that it's the outermost bilayer that grows first,

00:26:14.13 and that extra surface area is extruded as a thin tubule.

00:26:18.21 Eventually, you get a long filament,

00:26:21.04 and when you divide that by mild shear forces,

00:26:25.11 you get back small, multilamellar vesicles,

00:26:29.05 and then you can repeat the cycle.

00:26:31.11 Now, something very different happens if you go to the

00:26:33.22 trouble of making a unilamellar initial vesicle.

00:26:38.16 This grows the way we intuitively thought it would grow:

00:26:41.08 It just expands, becomes somewhat elongated.

00:26:45.25 But now these structures are extremely delicate and unstable,

00:26:50.17 and when they're subjected to shear force fluctuations,

00:26:54.19 they tend to rip apart, the contents spill out,

00:26:57.25 and remarkably, they reassemble into multilamellar structures.

00:27:03.19 So, to summarize this,

00:27:05.23 the two important aspects of the overall process

00:27:09.04 are beginning with multilamellar vesicles

00:27:11.28 and having something in the surrounding solution

00:27:16.08 which can only permeate slowly across the membrane,

00:27:20.02 resulting in slow volume increase

00:27:23.26 compared to rapid surface area increase.

00:27:26.19 And then environmental perturbations lead to cell division

00:27:29.25 in a very simple and natural way.

00:27:34.02 So, to summarize this, in a series of stages,

00:27:38.22 we've gone from a very artificial laboratory simulation,

00:27:43.19 to a process that looks simple and robust enough

00:27:47.06 to have occurred on early Earth environments.

00:27:53.04 Now, more recently, we've come back to the idea of competition

00:27:59.17 between protocells as an alternative mechanism of driving growth.

00:28:05.18 And this is particularly interesting in terms

00:28:09.11 of a way that this could emerge from Darwinian processes.

00:28:16.20 We think that one of the first ribozymes to emerge,

00:28:20.10 perhaps THE first, was actually a ribozyme

00:28:23.21 that could catalyze the synthesis of phospholipids.

00:28:28.15 And so why do we think this?

00:28:30.08 This came out of considering the series of evolutionary transitions

00:28:35.24 that must have occurred to get from

00:28:39.20 primitive membranes based on fatty acids,

00:28:42.05 with the appropriate dynamic and exchange processes

00:28:46.13 for a primitive cell,

00:28:48.01 up to modern membranes, which are based on phospholipids,

00:28:52.18 which are good barriers and which rely on

00:28:55.15 highly evolved biochemical machinery to mediate

00:28:59.07 growth and division and the transport of molecules

00:29:03.20 across the membrane.

00:29:04.27 So you couldn't go directly from the primitive state

00:29:08.20 to the modern state.

00:29:10.10 That would be suicidal,

00:29:11.21 because now a cell that depended on the influx

00:29:14.27 of nutrients from the environment

00:29:16.18 would no longer have access to those nutrients

00:29:18.29 because these membranes are so impermeable.

00:29:22.12 So presumably, the transition had to occur in a series of stages,

00:29:27.02 and what would an intermediate stage look like?

00:29:31.19 Probably it would look like a fatty acid membrane

00:29:35.09 with a small amount of phospholipid doped into the structure.

00:29:41.02 So if you think of a protocell containing genetic molecules,

00:29:46.21 if one of those sequences happens to be a simple

00:29:49.26 acyltransfer ribozyme that could make phospholipids

00:29:54.11 (after all, it's a simple, single, one-step acyltransfer reaction),

00:29:59.05 you could imagine making just a little bit of

00:30:02.09 phospholipid as an initial step in this transformation.

00:30:07.14 The thing that puzzled us for a long time is

00:30:11.11 what possible selective advantage could there be to

00:30:16.11 the presence of a small amount of phospholipid

00:30:19.16 in a fatty acid background?

00:30:22.24 It was very hard to imagine any reason why that would be advantageous.

00:30:27.21 It didn't really seem that a little bit of phospholipid

00:30:31.05 would change the physical properties of the membrane

00:30:33.18 in any particularly useful way,

00:30:37.06 and so the answer to this conundrum

00:30:39.29 came from doing experiments,

00:30:42.10 and this is all work done by Itay Budin,

00:30:45.06 who's a graduate student in the lab.

00:30:47.25 So what Itay did was simply to prepare vesicles

00:30:53.13 containing a little bit of phospholipids

00:30:56.28 doped into a fatty acid membrane structure.

00:31:01.11 And then he asked, well,

00:31:03.00 how would that affect the properties of the membrane?

00:31:05.20 And what turned out to be particularly interesting

00:31:08.12 is what happens when these vesicles are mixed with

00:31:11.18 pure fatty acid vesicles.

00:31:14.23 And it turns out, as you can see on the next slide,

00:31:18.14 that even a little bit of phospholipid confers a really dramatic ability

00:31:24.12 on that vesicle to absorb fatty acids from its

00:31:28.02 neighboring fatty acid vesicles.

00:31:31.13 So here's the experiment that Itay did:

00:31:34.11 He prepared vesicles with 10% of a phospholipid

00:31:41.11 in an oleic acid background,

00:31:43.19 and mixed them with a large excess of pure,

00:31:46.02 fatty acid, oleic acid vesicles.

00:31:48.23 So the vesicle starts out as a spherical structure,

00:31:51.13 you can see the encapsulated dye.

00:31:53.26 Within seconds, you see the protrusion of small filaments,

00:31:59.00 and over time, you can see a transformation

00:32:01.17 into a filamentous structure,

00:32:05.04 much as you saw before following the addition

00:32:08.07 of a large excess of fatty acids.

00:32:11.07 But this time the process of growth being driven purely

00:32:16.20 by the initial vesicle absorbing fatty acids

00:32:20.03 from its neighboring molecules.

00:32:21.27 It's essentially growing by eating its neighbors.

00:32:24.26 The mechanism of this phospholipid-driven growth is extremely interesting,

00:32:30.01 and you can see what we think is going on in the next slide.

00:32:34.16 It turns out there are actually two processes:

00:32:37.07 The first is a simple, entropically favored process

00:32:40.17 in which the absorption of fatty acids molecules into the mixed bilayer

00:32:46.04 dilutes the phospholipids,

00:32:50.10 which are extremely insoluble in the surrounding space,

00:32:53.19 so this is an entropically driven dilution.

00:32:58.04 But it turns out that, in addition to that factor,

00:33:01.02 there's actually a quantitatively more important factor,

00:33:05.00 which is the fact that these phospholipid molecules

00:33:09.01 change the properties of the bilayer such that

00:33:13.14 the disassociation of fatty acids from the bilayer is slowed down.

00:33:18.25 As a result, the vesicles grow because the

00:33:24.06 association rate constant is the same,

00:33:27.27 but the dissociation is slowed down,

00:33:30.27 so they hold onto more of their molecules, they grow,

00:33:34.10 and the pure fatty acid vesicles therefore shrink.

00:33:37.21 So how do we demonstrate this experimentally?

00:33:41.06 Itay took advantage of an assay that was developed by Jim Hamilton.

00:33:48.00 It's a rather indirect but very effective assay.

00:33:51.00 So if you have a fatty acid vesicle,

00:33:52.26 you can monitor the dissociation of

00:33:56.04 fatty acid molecules from that membrane, which is a slow step,

00:34:02.11 by looking at how they dissociate into solution,

00:34:07.06 integrate into the membrane of a reporter vesicle,

00:34:10.26 which is a fatty acid vesicle that contains inside it

00:34:15.20 a fluorescent dye that's pH-sensitive.

00:34:18.20 Now when the fatty acid molecules integrate into the outer leaflet,

00:34:23.05 they flip rapidly to the inner leaflet.

00:34:26.10 It's the protonated form that flips because it's neutral,

00:34:29.16 it can flip very rapidly, and then it re-equilibrates on the inside,

00:34:34.10 releasing protons, which acidify the interior.

00:34:38.05 So we have a fluorescence assay for this pH decrease,

00:34:42.16 and that gives us essentially a real-time assay for this

00:34:46.01 initial slow step of dissociation.

00:34:49.17 You can see that this assay works by comparing

00:34:53.25 the dissociation of fatty acids of different chain lengths,

00:34:58.00 and so here you see that oleic acid, which is an 18-carbon chain,

00:35:03.16 dissociates fairly slowly.

00:35:06.13 Palmitoleic acid, two carbons shorter, dissociates more rapidly.

00:35:11.19 And myristoleic acid, 14 carbons, dissociates extremely rapidly.

00:35:17.06 So it gives you a good readout for the dissociation kinetics,

00:35:21.09 and that allows you to monitor the rate of dissociation

00:35:26.21 as a function of phospholipid content of the membrane.

00:35:31.17 And so if you start off with a pure fatty acid membrane,

00:35:35.28 so a pure oleic acid membrane, dissociation is quite rapid.

00:35:40.21 But the more phospholipid that there is in the membrane,

00:35:44.25 the slower the off-rate of the fatty acid is.

00:35:51.17 So, as a result of these two processes,

00:35:56.24 this entropically driven dilution aspect

00:36:00.19 and this off-rate-driven aspect,

00:36:03.11 the presence of even a little bit of phospholipid will drive growth

00:36:08.29 by essentially extracting or absorbing fatty acid molecules

00:36:13.09 from neighboring vesicles that have either no phospholipid

00:36:17.25 or even just less phospholipid.

00:36:20.24 So what would the implications of that process be

00:36:23.15 in an evolutionary scenario?

00:36:27.15 We begin with this idea that phospholipids drive growth.

00:36:31.12 As a result, of course there would be a very strong selection

00:36:35.08 for a ribozyme that has the catalytic ability

00:36:40.08 to make phospholipids, so a simple acyltransferase ribozyme.

00:36:48.07 That kind of primitive cell would rapidly take over

00:36:51.24 the population because it has such a strong selective advantage,

00:36:55.23 but that would lead to an evolutionary arms race.

00:36:59.11 Because now, in order to grow by eating your neighbors,

00:37:03.15 you have to make more phospholipids than they do.

00:37:07.11 So there'd be an evolutionary race to make

00:37:11.29 more and more and more phospholipids.

00:37:14.27 And so now the phospholipid abundance in the membrane

00:37:19.13 would start to increase, you'd have more and more phospholipids,

00:37:23.05 and eventually that would lead to new selective pressures

00:37:26.25 favoring the emergence of metabolism,

00:37:29.09 because now it's actually useful to have internal metabolic reactions

00:37:33.24 because you get to keep the molecules you make

00:37:36.00 instead of having them just leak out,

00:37:38.20 since the phospholipid makes the membrane less permeable.

00:37:42.09 At the same time, there would be selective pressures

00:37:45.03 for the emergence of transport machinery,

00:37:48.23 which would help you absorb molecules that you still want

00:37:52.05 to have access to from the environment,

00:37:54.00 or release molecules that you want to get rid of,

00:37:56.22 so waste products.

00:37:58.15 So we think that this physical process,

00:38:01.26 this unexpected physical process of phospholipid-driven membrane growth,

00:38:06.05 would set in place a whole cascade of events eventually

00:38:11.20 leading to selective forces for much more complicated cells,

00:38:17.25 where we see the emergence of metabolism and

00:38:20.09 membrane transport machinery,

00:38:22.11 and eventually, we think that could drive the emergence of

00:38:26.10 protein synthesis, initially uncoded but eventually coded protein synthesis,

00:38:32.08 and be a major factor in the emergence of modern biochemistry.

00:38:39.25 So from all of these experiments, just to sum up,

00:38:42.23 what can we say that we've learned that's really relevant

00:38:46.03 to the origin of life on Earth?

00:38:47.22 And I think at at general level,

00:38:49.03 there are two things I'd like to point out:

00:38:52.01 One is that, from doing very, very simple laboratory experiments,

00:38:56.14 again and again we've uncovered a surprising new

00:38:59.08 physical phenomenon, things that you would have a hard time

00:39:03.05 thinking up a priori, but which emerge

00:39:07.16 naturally from doing laboratory experiments

00:39:10.08 where you're trying to build systems and see how they work.

00:39:13.08 The second general lesson, I think,

00:39:16.19 is that in our early experiments,

00:39:20.08 we tend to use very highly constrained artificial systems

00:39:24.23 because they're analytically tractable.

00:39:27.20 But they often don't exhibit the properties

00:39:30.28 that we really want to see that are relevant to the prebiotic system.

00:39:35.24 So it's very important to relax those experimental constraints

00:39:41.20 and gradually develop the ability to look at "messier"

00:39:45.00 and more natural systems that give you insight

00:39:48.01 into physical processes that really could explain some of the

00:39:52.21 apparently hard parts in the process by which life emerged

00:39:57.17 from the chemistry of the early planet.

00:40:01.02 So, again, all of this work was done by many very talented

00:40:04.08 students and postdocs.

00:40:06.26 I've mentioned in particular the work of Itay Budin and Ting Zhu,

00:40:10.28 and the early work of Irene Chen, Shelly Fujikawa, Martin Hanczyc.

00:40:17.09 Many people have played a role in developing these systems

00:40:21.09 and gradually giving us the ability to understand what's going on

00:40:26.13 in these model systems and some insight into the origin of life.

00:40:31.02 Thank you.

00:00:00.00 My name is Jack Szostak,
00:00:01.22 I'm a Professor of Genetics at Harvard Medical School,
00:00:05.01 an Investigator at Massachusetts General Hospital,
00:00:07.17 where my lab is,
00:00:09.02 and I am an Investigator of the Howard Hughes Medical Institute.
00:00:12.17 And in this part of my lecture,
00:00:15.00 I'd like to concentrate on the one aspect of the origin of cellular life,
00:00:21.28 which is the chemistry of copying replicating nucleic acid templates.
00:00:29.29 So let's begin by looking at our schematic version
00:00:34.20 of how we're thinking about a simple protocell,
00:00:39.06 so again we have a two-component system:
00:00:43.12 A primitive cell membrane encapsulating some
00:00:47.01 genetic molecules, could be RNA, could be DNA,
00:00:50.12 could be something related.
00:00:54.08 In the previous lecture,
00:00:56.11 we dealt with the growth and division
00:00:58.21 of the membrane compartment,
00:01:00.15 and this time we want to focus on the copying of
00:01:04.24 templates to make a duplex product:
00:01:07.00 The separation of strands,
00:01:08.26 and the copying of those in a subsequent round
00:01:11.22 so that you can distribute that information to daughter cells.
00:01:16.10 And this is an essential aspect of the
00:01:21.23 emergence of Darwinian evolution.
00:01:26.02 There's some kind of informational polymer
00:01:28.11 to code for heritable functions,
00:01:31.24 could be anything that's useful for the cell,
00:01:34.13 something that helps it to grow,
00:01:36.24 something that helps it to divide,
00:01:38.07 something to helps it to survive better in its environment,
00:01:43.01 almost anything.
00:01:44.29 But we need to have a way for that function
00:01:48.23 to be coded and transmitted from generation to generation.
00:01:54.19 Now, there are two general ways
00:01:59.12 of thinking about the process of nucleic acid replication:
00:02:03.27 The first would be some kind of enzymatic or catalytic process,
00:02:08.15 so the classical example of that would be an RNA replicase,
00:02:13.00 an RNA molecule that's an RNA polymerase
00:02:15.15 that's good enough to copy its own sequence.
00:02:19.11 I worked on that for a number of years.
00:02:24.18 Many people have followed up on that work,
00:02:27.22 and there's been a lot of progress made.
00:02:30.12 I do think that eventually we will have molecules
00:02:33.19 that can do that, but at the present time,
00:02:35.19 we're still far from such a solution.
00:02:38.12 And that has driven us to rethink the process
00:02:41.23 and to step back and look at chemical processes
00:02:46.06 that might lead to the replication of nucleic acid templates,
00:02:50.06 whether RNA or some related molecule.
00:02:54.11 So that's what the main focus will be on:
00:02:56.09 Chemical processes leading to efficient copying
00:03:00.07 and replication of templates.
00:03:03.05 Now, there are two really critical factors that apply
00:03:07.03 in any such system, and these are that both the
00:03:11.18 rate of copying and the accuracy, the fidelity,
00:03:14.29 of copying have to exceed critical thresholds.
00:03:18.10 So the rate of replication has to be faster
00:03:22.23 than the rate of degradation, and with a molecule
00:03:26.00 such as RNA, that's a really important factor,
00:03:29.06 because RNA is such a delicate polymer,
00:03:32.00 hydrolyzes relatively rapidly.
00:03:34.17 That imposes a lower limit on an acceptable
00:03:38.03 rate of replication. In addition,
00:03:41.07 the fidelity of that process has to exceed the
00:03:44.27 Eigen error threshold. If we want to propagate
00:03:47.18 useful information from generation to generation,
00:03:50.27 the accuracy of that copying process
00:03:54.08 has to exceed a threshold,
00:03:57.02 which is basically related to the reciprocal of the
00:04:00.10 number of important nucleotides for whatever
00:04:04.13 function we're talking about.
00:04:06.21 In practical terms, that means we probably
00:04:08.24 need to think of accuracies or, shall we say,
00:04:12.07 error rates below a few percent.
00:04:16.16 Typical chemical processes that we study now,
00:04:19.01 the error rates are in the range of 5% to 10% or 15%,
00:04:22.22 so some improvement is required.
00:04:25.08 And we would also like to see improvement
00:04:27.12 in the rate of replication, so that we can do these
00:04:30.16 experiments on a reasonable laboratory timescale.
00:04:35.04 So, the building blocks that we're going to use
00:04:37.11 for these experiments are quite different
00:04:40.04 from the nucleoside triphosphates that are used in all modern cells.
00:04:46.07 So these are modern substrates.
00:04:48.22 They are kinetically trapped in a high-energy state
00:04:53.07 so that they require very sophisticated catalysts,
00:04:56.20 enzymes that can confer the 10^12 rate acceleration
00:05:00.21 that you need in order to make effective use of this kind of substrate.
00:05:06.12 They are also, as I've mentioned before,
00:05:09.00 highly charged because of the triphosphate group,
00:05:13.23 which keeps them from leaking out of cells,
00:05:15.26 which is a good thing for modern cells
00:05:17.13 but a bad thing for primitive cells,
00:05:19.26 which will require their substrates to come in
00:05:23.28 from the environment, get across the membrane spontaneously.
00:05:27.24 And that means we would like to think about less polar substrates.
00:05:33.12 And so that drives us to think of molecules
00:05:36.11 similar to that that you can see down here.
00:05:39.23 These are nucleoside phosphorimidazolides.
00:05:43.05 These kinds of molecules were first synthesized by Leslie Orgel
00:05:47.22 and his students and colleagues,
00:05:50.16 and studied in quite a lot of depth in the 1970s and 80s and 90s.
00:05:58.07 These molecules are much more chemically reactive,
00:06:01.18 we have a much better leaving group,
00:06:03.14 so they're intrinsically more reactive.
00:06:05.19 They can spontaneously polymerize and copy templates
00:06:10.14 without enzymes, and they're also less polar,
00:06:16.04 which means they can get across membranes
00:06:18.17 much more rapidly, without any transport machinery
00:06:21.28 being invoked. So, the early work by Orgel
00:06:29.11 and his colleagues got us partway to copying systems,
00:06:35.24 but there were a series of problems that they ran into,
00:06:38.18 which we'll come to and try to consider individually.
00:06:42.18 But first, I want to step back a bit and
00:06:45.04 think about how these kinds of molecules
00:06:47.08 would've been generated on the early Earth.
00:06:49.23 And that's really a pretty major problem.
00:06:52.04 It's still a major research area,
00:06:54.25 but I think very exciting progress has been made recently.
00:07:00.15 So in the early days,
00:07:04.22 there were self-assembly processes that were
00:07:07.01 discovered that seemed to suggest that
00:07:11.29 the solution might be really easy.
00:07:14.14 In fact, the classical formose reaction, going back to Butlerov,
00:07:19.27 showed that you could make sugars by polymerizing formaldehyde.
00:07:23.25 And so for example, ribose can be viewed as
00:07:26.25 an oligomer of formaldehyde.
00:07:29.12 Five formaldehyde molecules can self-assemble
00:07:31.27 in a series of steps to give you ribose.
00:07:35.19 The problem is making just ribose.
00:07:38.21 In fact, in this kind of chemistry,
00:07:41.04 typically you'll end up with dozens or
00:07:44.00 even hundreds of products.
00:07:46.09 So, part of the problem that's absorbed people
00:07:51.01 has been how to make just the right sugar.
00:07:54.21 A similar problem comes from thinking about the nucleobases.
00:07:59.19 So early on, Juan Oro did very dramatic experiments
00:08:04.00 showing that he could actually made adenine from cyanide.
00:08:09.05 Simply boiling a solution of cyanide gave you some adenine,
00:08:12.24 along with a lot of other products.
00:08:15.18 But it's very striking that adenine can be viewed
00:08:18.20 as a pentamer of hydrogen cyanide.
00:08:25.06 So again we have the same problem of how do we get just the
00:08:28.08 building blocks we want (the A, G, C, and U),
00:08:31.21 as opposed to all of the other related heterocycles
00:08:35.11 that come out of this kind of chemistry.
00:08:38.19 Now, when people like Orgel and many of his colleagues
00:08:44.26 started to look at the synthesis of pyrimidines,
00:08:47.11 again it looks, superficially, very easy.
00:08:52.03 For example, a cytosine can be viewed as the product
00:08:55.18 of reaction of cyanoacetylene and urea.
00:09:00.21 And in fact there are variations on this chemistry
00:09:02.19 that are extremely efficient.
00:09:05.19 So it was starting to look like you could make sugars,
00:09:11.04 you could make nucleobases,
00:09:13.22 maybe it would actually turn out to be easy
00:09:16.24 to make nucleosides and nucleotides
00:09:18.28 and get us all the way to RNA.
00:09:21.05 But it turned out that there was a major problem,
00:09:26.07 even apart from the problem of making just the molecules we want.
00:09:30.12 If you could have ribose and, say, cytosine,
00:09:35.03 you would need to join them together by making
00:09:37.18 the glycosidic bond that links them,
00:09:40.28 and that chemistry basically just doesn't work,
00:09:43.27 no matter how hard people tried,
00:09:45.20 this was a roadblock for a long time.
00:09:49.16 So one of the most exciting advances in prebiotic chemistry
00:09:52.29 in recent years has come from the laboratory of John Sutherland
00:09:56.01 in the UK, who basically followed up
00:10:00.08 on much earlier work of several other labs,
00:10:03.17 and showed that there's an alternative pathway
00:10:06.13 that can get you to the final product
00:10:08.24 without ever having to make this particular
00:10:12.05 glycosidic linkage by joining together a base and a sugar.
00:10:17.03 And the solution basically comes by making this intermediate,
00:10:22.05 2-aminooxazole, from cyanamide and glycolaldehyde.
00:10:28.10 In a series of very simple and actually remarkably efficient steps,
00:10:32.16 this intermediate can be elaborated into cytosine,
00:10:36.08 and then deamination can give you U.
00:10:40.06 So it looks like there might be a reasonable pathway
00:10:43.00 to at least getting to the pyrimidine nucleosides.
00:10:46.11 The synthesis of the purine nucleosides
00:10:48.20 by an analogous pathway
00:10:51.11 is a topic of active research.
00:10:54.03 And if it turns out that there is a similarly efficient pathway,
00:10:59.29 that will certainly be very satisfying in the sense
00:11:02.07 of providing very efficient and regiospecific chemistry
00:11:08.08 that gives us a restricted set of building blocks
00:11:11.12 leading up to RNA.
00:11:13.29 Now, there's still many gaps in our understanding
00:11:17.14 of how we would make pure, concentrated starting materials.
00:11:22.06 There are some steps leading up to activated nucleotides
00:11:24.23 that are far from clear.
00:11:26.08 So there's a lot of work to be done,
00:11:28.17 but I think this new chemistry has really advanced the field a lot.
00:11:33.09 So, let's skip those missing link steps for the time being,
00:11:39.01 and assume that we can make activated nucleotides.
00:11:42.27 The next problem we have to think about:
00:11:45.01 Is there polymerization into RNA chains,
00:11:48.08 and how could that happen?
00:11:49.21 Well, here, we're in a good situation,
00:11:52.10 because we actually have two solutions to the problem.
00:11:55.19 The first is the finding of Jim Ferris and colleagues,
00:12:02.23 including Leslie Orgel,
00:12:05.02 and this is a finding that a common clay mineral,
00:12:08.05 montmorillonite, illustrated here,
00:12:10.17 is a really effective catalyst for that
00:12:13.03 kind of polymerization.
00:12:15.05 So this clay mineral is a layered hydroxide,
00:12:19.01 aluminosilicate, and in between the layers
00:12:21.27 there are water molecules.
00:12:24.14 And organic molecules also tend to accumulate
00:12:28.03 in the inner layers of the clay mineral,
00:12:31.04 and as they accumulate there and become concentrated
00:12:33.15 and oriented relative to each other,
00:12:35.29 their polymerization is catalyzed.
00:12:40.13 And the nice thing is that this is not the only way of doing it.
00:12:46.09 You can get essentially the same result
00:12:49.29 simply by taking a solution of these activated nucleotides,
00:12:53.25 these phosphorimidazolides.
00:12:56.03 And as a dilute solution at room temperature,
00:12:58.17 almost nothing happens, they don't polymerize.
00:13:01.19 But if you put that solution in the freezer
00:13:04.28 and allow the water to freeze and generate ice crystals,
00:13:09.08 what you see is that the solutes, including these nucleotides,
00:13:13.00 get concentrated in thin layers in between the ice crystals,
00:13:18.02 and when molecules are concentrated that much,
00:13:21.07 even at low temperature, they can start to react with each other,
00:13:24.22 and you will see the spontaneous formation of long RNA
00:13:28.27 chains as a result of freezing.
00:13:31.25 So this is very nice,
00:13:33.21 because now we have two plausible, natural scenarios
00:13:38.25 where either a common mineral or just the process of freezing
00:13:41.25 could generate RNA chains.
00:13:45.21 So the next problem we have to deal with,
00:13:48.00 assuming we can make sets of essentially
00:13:50.26 random RNA polymers, how could they be copied?
00:13:56.05 And so here is where we run into a fresh set of difficulties.
00:14:00.29 So the partial copying of RNA templates
00:14:03.29 has been known for decades, as I said,
00:14:06.10 from the work of Leslie Orgel and his colleagues.
00:14:09.11 This is an example of that kind of chemistry
00:14:11.20 done by David Horning, when he was an undergrad in my lab.
00:14:15.29 We start off with a substrate here, a guanosine nucleoside,
00:14:21.19 activated as a phosphorimidazolide,
00:14:25.08 and we supply that to a primer-template complex
00:14:28.11 in which the template contains a region of Cs
00:14:32.28 where the G nucleoside can bind and result in primer extension.
00:14:38.24 So here would be the starting material,
00:14:40.27 and then over time we want to see
00:14:43.02 the incorporation of Gs to elongate the primer.
00:14:47.04 And that process is shown down here in timecourse,
00:14:51.12 where we start off with just the primer,
00:14:54.17 and then over the first, say, six hours,
00:14:57.21 we observe the incorporation of the first nucleotide,
00:15:02.11 and then over the next day or two,
00:15:04.06 we see the second nucleotide come in,
00:15:07.06 but even after two days, there's very little of the third nucleotide.
00:15:12.04 So that illustrates the first problem:
00:15:15.06 This process is intrinsically rather slow.
00:15:18.07 And because this chemistry requires
00:15:21.26 a very high concentration of magnesium
00:15:25.03 to catalyze the reaction,
00:15:27.15 this rate of synthesis is actually on the same timescale
00:15:32.05 as the rate of degradation of the RNA template.
00:15:36.11 So that's a problem.
00:15:38.24 There are other problems, you can see,
00:15:41.20 in the structure of the ribonucleotide,
00:15:44.16 that there are two hydroxyls on the sugar,
00:15:47.27 and either of them can react to generate either the
00:15:51.16 correct 3'-5' linkage or the incorrect 2'-5' linkage.
00:15:57.17 And so that means you inevitably get a mixture of linkages,
00:16:02.28 some of which are the natural, correct linkage found in RNA,
00:16:06.13 and others which are not.
00:16:09.24 The next slide really summarizes the numerous problems
00:16:14.29 or challenges that must be solved if we're ever
00:16:18.12 to think of a complete chemical process for the replication of RNA.
00:16:24.07 So, we begin with these problems of rate and fidelity.
00:16:29.21 The fidelity is actually closely related to the problem of rate.
00:16:36.27 It turns out that, when you make a mistake
00:16:40.27 in incorporating a nucleotide, so as a chain is growing,
00:16:45.10 you put in the wrong base, make a mismatch,
00:16:47.19 the addition of the next base can be dramatically slowed.
00:16:51.25 We call this the stalling effect,
00:16:53.21 and therefore it slows down the overall rate of synthesis.
00:16:58.00 If we could make the chemistry more accurate,
00:17:01.06 the rate of synthesis would be much better.
00:17:04.11 There's the regiospecificity problem that I mentioned,
00:17:06.26 2' versus 3' linkages.
00:17:10.09 There is a problem that we need
00:17:13.14 very high concentrations of these monomers.
00:17:16.28 They apparently need to be very pure,
00:17:19.18 so if you have other kinds of nucleotides in there, say,
00:17:23.14 with different sugars or different stereochemistry,
00:17:26.03 they will also get incorporated,
00:17:27.27 and that will mess up the product that's made.
00:17:30.23 Also, these monomers, when they're activated as imidazolides,
00:17:36.06 are rather unstable, they're quite susceptible
00:17:40.09 to hydrolysis or cyclization, so those are undesired side reactions.
00:17:47.11 They could be solved if we had the right kind of chemistry
00:17:51.01 to reintroduce the activated state,
00:17:54.14 but that's something that's missing so far.
00:17:57.29 The requirement for a very high concentration of magnesium
00:18:02.03 is extremely problematic both because it's
00:18:04.17 geochemically unrealistic and because it leads to RNA hydrolysis.
00:18:10.20 There's another problem with RNA, which is that,
00:18:14.26 even if you could replicate a strand of any significant length,
00:18:19.14 the melting temperature of that duplex is so high
00:18:23.12 that it's almost impossible to pull the strands apart.
00:18:26.16 Thermally, it becomes impossible to melt them.
00:18:29.27 Even if you could melt them,
00:18:31.21 the strands will come back together again extremely rapidly.
00:18:36.15 This rapid reannealing rate is something that will
00:18:39.15 compete with the much slower template copying chemistry,
00:18:43.11 so this is another problem that has to be solved.
00:18:45.28 And finally, in our experiments,
00:18:48.26 we use primers and watch them grow,
00:18:51.12 simply because that's analytically very easy to do,
00:18:56.14 but of course there weren't primers around on the early Earth,
00:18:59.16 and so we need to think of a primer-independent process
00:19:02.06 for copying templates.
00:19:04.01 So all of these problems together have made it very difficult
00:19:09.12 to think of a plausible pathway
00:19:13.01 for the overall replication of RNA templates.
00:19:18.00 So, what we decided to do was essentially to step back
00:19:22.29 from this and think about other polymers,
00:19:27.08 maybe it would be easier to replicate something else.
00:19:31.04 And in the process of figuring that out,
00:19:33.06 maybe we would get clues that would let us come back
00:19:38.02 to RNA and think about how to solve some of these problems.
00:19:41.25 Now in fact, Leslie Orgel concluded a couple of decades ago
00:19:49.26 that, even though RNA replication looked really hard,
00:19:54.09 he thought that the replication of some kind of
00:19:57.04 informational polymer would be achieved fairly readily,
00:20:01.18 and that in the process of doing that,
00:20:04.27 we would learn something about either RNA replication
00:20:09.21 or how to replicate something that might be
00:20:12.08 relevant to the origin of life.
00:20:14.17 Now, unfortunately, despite that challenge
00:20:17.18 to the chemistry community, few people have addressed
00:20:20.16 the problem, and there is still no example
00:20:24.06 of the chemical replication of any informational polymer.
00:20:27.17 So I think this is a major challenge.
00:20:29.08 It's a really interesting and fun thing to investigate.
00:20:32.09 And this is really the focus of a lot of our attention
00:20:36.02 at the moment. So what can we look at?
00:20:39.19 What would other interesting nucleic acids be?
00:20:44.22 So, what I'm going to do is just show you a set of the
00:20:48.09 kinds of molecules that we've been studying in my lab
00:20:50.26 over the last couple of years.
00:20:53.05 And we're concentrating on
00:20:55.20 phosphoramidate-linked genetic polymers,
00:20:58.22 so these have nitrogen-phosphorus bonds
00:21:01.25 in place of the oxygen-phosphorus bond you see
00:21:05.01 in normal phosphodiesters.
00:21:07.04 The reason for that is that the building blocks, the monomers,
00:21:11.23 for making these polymers are aminosugars,
00:21:14.23 so we now have a much better nucleophile than a hydroxyl group,
00:21:20.02 so that speeds up the chemistry again,
00:21:22.03 giving us another boost in rate.
00:21:24.26 So, here are three phosphoramidate backbones.
00:21:28.07 Here's an acyclic, open-chain backbone with
00:21:32.15 essentially a glycerol nucleic acid backbone.
00:21:37.09 Here is a 2'-5'-linked polymer,
00:21:43.18 so the phosphoramidate version of DNA,
00:21:46.14 with 2' linkages.
00:21:48.16 And here's the molecule closest to DNA.
00:21:51.08 All that's been done is to change to normal oxygen
00:21:53.25 atom here to an NH group,
00:21:57.06 so this is phosphoramidate DNA.
00:22:01.12 And then there are two other molecules that have captured
00:22:03.25 our attention recently, and these are somewhat
00:22:07.10 more conformationally constrained molecules.
00:22:10.19 So this is the phosphoramidate version of TNA,
00:22:13.24 threose nucleic acid;
00:22:15.23 here, the sugar is a four-carbon sugar, threose.
00:22:20.17 And here we have a 2' linkage.
00:22:25.13 These molecules were first made in the Eschenmoser Group
00:22:29.18 and studied. They're perfectly good base-pairing systems.
00:22:34.13 And finally, here you see a morpholino backbone,
00:22:39.08 another conformationally constrained backbone.
00:22:42.19 So in the case of the threose,
00:22:45.01 the conformational constraint comes from the fact
00:22:47.23 that there are only five atoms in the backbone repeat unit,
00:22:51.15 so there's one less rotatable bond,
00:22:53.26 so it's entropically constrained.
00:22:56.09 Here, the constraint is different.
00:22:58.17 It comes from the fact that we have the
00:23:00.09 six-membered morpholino ring,
00:23:03.02 which likes to sit in this chair conformation,
00:23:05.13 so it's conformationally constrained
00:23:07.09 in a very different manner.
00:23:09.11 So, what we've been trying to do is
00:23:11.22 systematically study all of these different kinds of templates
00:23:18.01 and see if we can learn anything from this process that might
00:23:22.07 eventually feed back and teach us about RNA replication.
00:23:26.21 So, at this stage, we're really still heavily involved
00:23:30.17 in studying the copying of these templates.
00:23:33.25 In many cases,
00:23:34.28 these are actually quite challenging to prepare synthetically,
00:23:38.26 so that takes a lot of time and effort.
00:23:41.08 But I'll take you through what we've done so far.
00:23:45.00 So we began by studying the simplest template
00:23:51.26 from a structural point of view,
00:23:53.20 so this is the glycerol nucleic acid backbone.
00:23:56.29 So no cyclic sugar, just an open-chain backbone.
00:24:02.22 And here's the corresponding monomer.
00:24:05.27 So we have the amino nucleophile,
00:24:07.27 we have the activated phosphate,
00:24:10.16 these look very simple from a structural standpoint,
00:24:14.29 but in fact, there's a major problem:
00:24:18.17 That lack of constraint from the cyclic sugar
00:24:24.04 means that the amine nucleophile
00:24:27.25 can directly reach the phosphorus electrophile,
00:24:32.00 and as a result, the activated monomer cyclizes
00:24:36.09 to give this useless product here faster than we can measure.
00:24:42.07 So that tells you right away that this system is not
00:24:45.11 chemically a good system to look at
00:24:47.20 in terms of replication.
00:24:49.15 But, the speed of this reaction actually told us
00:24:53.16 something kind of interesting, which is that
00:24:56.20 the intrinsic chemistry, it can be very fast.
00:25:00.19 So, if you can position the nucleophile
00:25:04.17 in just the right position and orientation
00:25:09.07 relative to the electrophile,
00:25:12.26 polymerization could in principle go very rapidly,
00:25:16.14 even without an external catalyst.
00:25:21.16 The system that we've actually spent the most time
00:25:24.20 studying so far and learned the most about,
00:25:28.14 is the 2'-5'-linked phosphoramidate version of DNA,
00:25:34.21 so here's the polymer.
00:25:36.15 A series a of 2'-5' phosphoramidate linkages,
00:25:40.09 and here's the corresponding monomer,
00:25:42.19 shown as the G nucleoside version, but amine nucleophile.
00:25:48.20 Phosphorimidazolide, so good leaving group, good nucleophile...
00:25:53.22 we should get very fast polymerization chemistry.
00:25:58.09 And in fact, that's exactly what we observed.
00:26:01.05 In our first experiments, after we learned how to make
00:26:03.14 these molecules, we set up the following system,
00:26:07.16 where we have a primer-template complex.
00:26:11.05 The template contains this region of Cs,
00:26:14.01 where the G monomers can bind,
00:26:17.29 and we can then observe the primer being
00:26:20.29 elongated by the sequential incorporation of
00:26:23.23 multiple G residues.
00:26:26.14 The result experimentally is shown here.
00:26:30.14 We start off with the primer, and over the course of hours,
00:26:34.04 we can see the complete copying of the template
00:26:38.12 and the accumulation of the full-length, extended primer.
00:26:44.13 So this reaction is so efficient that, I think,
00:26:47.17 if you didn't know this was just chemistry,
00:26:50.00 you would think this an enzymatically catalyzed reaction,
00:26:53.17 but there's no enzyme, there's no polymerase,
00:26:56.26 this is just the intrinsic chemistry of activated nucleotides
00:27:01.13 binding to a template and extending a primer.
00:27:05.04 So, if we could do this in a more general way,
00:27:09.22 so that we could copy templates of arbitrary sequence,
00:27:14.09 we would basically have the kind of system that we want.
00:27:18.06 We would be able to copy sequences
00:27:20.10 that could carry out functional tasks.
00:27:25.00 One of the nice things about this overall system is that,
00:27:30.11 because of the 2'-5' linkages,
00:27:33.08 the duplex that's formed has a relatively low melting temperature,
00:27:38.28 it's relatively easy to thermally separate the strands,
00:27:42.09 and we could imagine a cycle of complete steps
00:27:47.14 of copying and full replication.
00:27:50.07 So, unfortunately, things are not so simple.
00:27:53.25 This copying chemistry works very well
00:27:56.23 with C templates driving G incorporation,
00:28:00.19 works very well with G templates driving C incorporation,
00:28:04.22 but when we went to try to copy templates
00:28:07.29 that contain As and Us, it basically didn't work at all.
00:28:13.16 So we assumed that the problem was that the AU base pair
00:28:17.16 is much weaker than the GC base pair, which is true.
00:28:22.01 And so the solution was simply to go back
00:28:24.29 to the chemical drawing board and
00:28:27.25 look at different nucleobases that make an AU-like base pair,
00:28:35.14 but that is just as strong as a GC base pair.
00:28:39.14 So it turns out, in fact,
00:28:40.23 this has already been done in other contexts.
00:28:44.04 So, people like Chris Switzer, for example,
00:28:48.10 have looked at the base pair made between D,
00:28:51.26 which is short for diaminopurine, and propynyl-U,
00:28:56.00 which you see over here.
00:28:57.08 So we have a propynyl group at the 5' position of U,
00:29:00.02 this contributes extra stacking energy.
00:29:03.09 The extra amino group in diaminopurine
00:29:07.02 gives us back the third hydrogen bond,
00:29:09.17 and this base pair in the context of DNA
00:29:12.19 is essentially just as strong as a GC base pair.
00:29:16.24 So, we made the corresponding activated monomers,
00:29:22.11 and sure enough, it solves the rate problem.
00:29:25.18 We can now, using this activated propynyl-U nucleotide,
00:29:31.02 we can copy a template consisting of four D residues,
00:29:34.06 and in fact, it's very fast.
00:29:36.02 The reaction's finished within the first ten minutes.
00:29:40.01 We can copy using the activated D monomer,
00:29:45.06 a template consisting of propynyl-Us,
00:29:48.02 it's a little bit slower, but still mostly done within an hour.
00:29:51.12 Not too bad!
00:29:52.27 So we thought, okay, maybe we've really solved the problem.
00:29:56.12 Let's get a little bit more ambitious
00:29:58.18 and try to copy progressively longer template sequences
00:30:03.04 that include progressively more, different nucleotides
00:30:07.21 in the sequence.
00:30:09.16 So, the first step looked pretty good.
00:30:13.02 Here we have a template that consists of three Ds and three Cs,
00:30:17.18 so we're incorporating three propynyl-Us followed by three Gs,
00:30:21.26 and the reaction goes pretty well, within a few hours.
00:30:25.26 If you leave out the G, you stall where you should,
00:30:30.05 if you leave out the U, you basically stall almost at the beginning.
00:30:35.09 So that looks encouraging.
00:30:37.26 You do see a few shorter products here,
00:30:40.16 which made us worry a little bit about the
00:30:42.13 accuracy of the overall process, but it's not too bad.
00:30:46.15 As we go to longer templates, so here a mix of Gs and Cs,
00:30:51.13 we can still copy the whole thing, but it does take longer,
00:30:55.24 and you do see more of these intermediate sequence
00:31:00.10 accumulating. And that actually gets much worse,
00:31:05.02 when we go to an even longer sequence of 15 or 16 nucleotides
00:31:09.21 incorporating all four bases in the template.
00:31:13.23 And now we still get some full-length product,
00:31:17.17 but it takes a long time to accumulate,
00:31:19.27 and we see a lot of these stalled intermediate products
00:31:25.13 accumulating over the course of the reaction.
00:31:28.11 So we don't know for sure,
00:31:29.22 but we suspect that these stalled intermediates
00:31:32.22 are the result of mistakes in the template-copying process,
00:31:39.27 such that a chain is growing, a mismatch is formed
00:31:45.11 by a mistaken incorporation,
00:31:47.19 and that drastically slows down the subsequent polymerization.
00:31:52.03 So in fact, we think that,
00:31:56.06 in order to get more efficient copying,
00:31:59.29 in order to speed up the overall reaction,
00:32:02.24 we need to solve the fidelity problem,
00:32:06.09 and that that might help solve the rate problem.
00:32:08.21 So how could we do that?
00:32:10.10 Well, we could look at different nucleobases,
00:32:13.24 maybe our choice of D and propynyl-U was not so great.
00:32:17.19 In fact, there are chemical reasons to think that that's true.
00:32:21.17 In particular, the propynyl group on U changes the pKa of its N1,
00:32:26.26 which can lead to the formation of other tautomers
00:32:29.18 and mismatched base pairs.
00:32:32.05 We could look at other backbones, which we are doing.
00:32:36.14 So there's the possibility that, if the backbone is
00:32:39.02 conformationally constrained in just the right way,
00:32:43.02 it will favor the incorporation of the right bases
00:32:45.20 and disfavor the incorporation of mistaken bases.
00:32:49.26 We could also consider looking at oligonucleotide substrates,
00:32:53.06 which actually turns out to be a really good idea.
00:32:55.14 There are probably a lot of reasons why this would be helpful.
00:32:58.28 And we could also consider looking at catalysis,
00:33:02.00 either ribozyme-mediated catalysis
00:33:04.26 or perhaps catalysis by small molecules or short peptides
00:33:09.13 that might've been lying around,
00:33:11.23 and that's another approach that we're starting to take.
00:33:16.23 So, let's go back to this idea of looking at different nucleobases.
00:33:22.29 So, it turns out there's actually a very simple substitution
00:33:28.15 that looks extremely promising at this stage.
00:33:31.10 So here's the D-propynyl-U base pair
00:33:34.12 which we think is causing problems with fidelity.
00:33:39.06 An alternative is to just replace U with 2-thio-U,
00:33:44.08 so U with a sulfur in place of the oxygen
00:33:47.01 normally at the 2' position.
00:33:50.03 This is an analogue of U that's actually found in nature,
00:33:55.08 in modern biology, it's a common substituent in tRNAs,
00:33:59.04 where it plays the role of stabilizing an AU base pair
00:34:02.21 and increasing the fidelity of that interaction.
00:34:06.07 And the reason that works is because the much
00:34:08.22 larger and polarizable sulfur
00:34:11.05 contributes to stacking interactions.
00:34:15.18 The larger size is accommodated in a base pair with A,
00:34:19.14 but is not accommodated in a wobble base pair with U.
00:34:24.08 So it seems to both stabilize the AU base pair
00:34:27.14 and disfavor the incorrect wobble base pairing.
00:34:35.03 So, we have preliminary experiments that suggest that
00:34:40.18 this is a promising approach, and we're continuing to look at that.
00:34:44.02 Meanwhile, we're also looking at a number of these other backbones,
00:34:48.21 and so here we come back to the
00:34:52.01 3'-linked phosphoramidate version of DNA.
00:34:56.08 Here is the corresponding monomer.
00:35:00.12 We like this system for different reasons than we like the 2' system.
00:35:06.27 Here we're making the natural 3'-5' linkage,
00:35:12.08 so we're a little bit closer to making an RNA-like product.
00:35:16.28 In fact, duplexes of this 3' phosphoramidate version
00:35:21.26 of DNA have a very RNA-like geometry,
00:35:25.06 so that's kind of nice.
00:35:27.06 These monomers can cyclize,
00:35:29.03 so this amino group can reach the phosphorus,
00:35:31.29 but that reaction is fairly slow, so it's not a fatal problem.
00:35:36.12 What may, in the long run, be more of an issue,
00:35:40.29 is that those duplexes have a very high melting temperature.
00:35:45.00 Nonetheless, it's an interesting system to study
00:35:49.12 because this chemistry seems to go very effectively, and in fact,
00:35:54.14 we see efficient incorporation of all four natural nucleotides
00:36:00.22 using this backbone. So it shows that
00:36:02.19 there's a very important coupling between backbone
00:36:05.18 chemistry and the bases that are involved
00:36:09.12 in forming the Watson-Crick paired structure of the duplex.
00:36:13.28 We don't really fully understand the nature of that coupling.
00:36:20.15 I mentioned before two conformationally constrained
00:36:24.05 phosphoramidate-linked nucleic acids,
00:36:26.21 the threose nucleic acid (TNA) and the morpholino backbone,
00:36:32.25 which we abbreviate as MoNA.
00:36:35.03 These are very interesting systems,
00:36:38.02 and the hope here is that conformational constraint
00:36:44.03 might be a way of increasing both the accuracy
00:36:46.28 and the rate of chemical copying.
00:36:49.26 So why do we really think that?
00:36:52.00 Here's an experiment done by Jason Schrum
00:36:55.02 when he was a graduate student in the lab,
00:36:58.03 and we're here looking at the incorporation of 2' amino
00:37:03.06 nucleosides, extending primers where the template
00:37:09.03 is composed of a series of different polymers.
00:37:11.29 So DNA over here, RNA template,
00:37:15.16 an LNA template (LNA stands for "locked nucleic acid,"
00:37:19.14 this is a relative of RNA where the sugar conformation is locked
00:37:24.06 into the RNA-like conformation by a cross-link
00:37:27.21 underneath the sugar),
00:37:29.14 and over here is a 2'-5'-linked DNA template.
00:37:33.01 So what you see in the timecourse of this primer extension reaction,
00:37:37.16 is that the reaction goes fairly slowly on a DNA template,
00:37:41.17 a lot more rapidly on an RNA template,
00:37:44.19 but even more rapidly on the conformationally
00:37:47.25 constrained LNA template.
00:37:52.10 So this was our first real experimental hint
00:37:55.20 that conformational constraint of a template could
00:37:58.22 really have a useful and significant effect
00:38:04.22 on the rate of polymerization.
00:38:08.20 And so that's encouraged us to go ahead and
00:38:12.15 make these conformationally constrained templates,
00:38:16.02 even though the synthetic chemistry is rather challenging.
00:38:21.18 So here is the threose nucleic acid backbone,
00:38:26.12 so again a four-carbon sugar.
00:38:28.11 Here's the corresponding monomer.
00:38:31.09 There has been some structural work done from the Egli Lab.
00:38:36.04 You can see here that a TNA duplex looks, at a gross level,
00:38:40.25 very similar in overall geometry to an RNA duplex.
00:38:45.01 So this, I think, is encouraging for the possibility that this
00:38:49.17 constrained backbone might help to position
00:38:53.19 the incoming nucleoside correctly, so as to speed up
00:38:58.10 polymerization and potentially make it more accurate.
00:39:02.05 So we hope to do those experiments
00:39:04.17 over the next few years.
00:39:07.14 Here is the morpholino phosphoramidate backbone,
00:39:10.16 again conformationally constrained
00:39:12.05 in a very different way
00:39:13.22 because of the six-membered morpholino ring.
00:39:17.25 These molecules are actually much easier
00:39:20.14 to make than the TNA molecules.
00:39:23.29 And so we've been able to start looking at the copying
00:39:27.10 of morpholino templates by morpholino monomers,
00:39:30.07 so we think this is another very promising system
00:39:33.20 in which to investigate experimentally the effects
00:39:36.24 of conformational constraint on the rate
00:39:39.11 and fidelity of copying.
00:39:43.07 So, even though we're still far from having
00:39:48.13 a complete chemical system that could drive the replication
00:39:52.08 of any nucleic acid or any genetic material,
00:39:56.16 we can use what we've found so far to learn about
00:40:01.03 the compatibility of the chemistry of genetic replication
00:40:05.26 with our replicating vesicle systems.
00:40:09.09 So we can do experiments where we encapsulate
00:40:12.00 nucleic acids inside vesicles and look at the copying chemistry.
00:40:17.06 So an example of that is shown here.
00:40:19.14 This was work done by Sheref Mansy and Jason Schrum
00:40:23.04 and other people in my lab, a few years ago.
00:40:26.16 The basic experiment is to take the same kind
00:40:28.23 of primer-template complex that you've seen before,
00:40:32.13 and monitor the extension of the primer by a
00:40:36.04 template-directed synthesis.
00:40:38.08 But this time, the primer-template is inside
00:40:42.06 one of these vesicles,
00:40:43.25 so we're going to add the activated monomer to the outside.
00:40:47.25 It has to get across the membrane spontaneously,
00:40:50.19 without any transport machinery, to get to the inside,
00:40:53.22 where it can do this template-copying chemistry.
00:40:58.01 So, here's the experimental result:
00:41:00.22 On this side, you see the control reaction done in solution,
00:41:04.19 you see the accumulation of full-length material over 12 to 24 hours.
00:41:08.23 Here is the same experiment with an encapsulated
00:41:12.00 primer-template, and you can see that the reaction is slowed
00:41:15.02 down a little bit, but still by 24 hours
00:41:18.10 you can see the accumulation of mostly full-length product.
00:41:22.26 So that was actually extremely encouraging for us.
00:41:26.00 This was a real major advance, because it said that,
00:41:29.08 yes, the chemistry is compatible;
00:41:32.05 these building blocks can get across the membrane;
00:41:34.26 when they get to the inside of the vesicle they can copy templates;
00:41:38.17 and once we developed a more general
00:41:42.08 template-copying chemistry, we should be able to
00:41:45.05 combine it with the replicating vesicles
00:41:48.03 and have the composite system that has been our goal all along.
00:41:54.04 Now, in this experiment, the membrane was made
00:42:00.06 from a convenient laboratory system,
00:42:03.27 these unsaturated C14 fatty acids.
00:42:07.16 We can do the same experiments with a much more
00:42:10.17 prebiotically realistic mixture of fatty acids, fatty alcohols,
00:42:15.24 their glycerol esters, saturated ten-carbon chains,
00:42:20.18 and we see the same thing.
00:42:21.27 Essentially over a period of 12 to 24 hours,
00:42:25.25 the nucleotides can get across these membranes
00:42:29.03 and copy templates. In contrast,
00:42:32.23 when the vesicle is made from more modern molecules,
00:42:36.25 from phospholipids, those are a complete barrier
00:42:40.21 to the penetration of these nucleotides,
00:42:43.12 so nothing happens. You see no primer extension.
00:42:47.21 So what this is telling us is that,
00:42:49.16 for this to work, for this protocell model to work,
00:42:52.27 you need the membrane to be made of the
00:42:55.23 right kinds of primitive molecules,
00:42:57.23 so simple fatty acids and related molecules,
00:43:01.01 and the nucleotides have to be the right kind of
00:43:04.17 primitive molecules, not triphosphates,
00:43:07.21 but something like phosphorimidazolides,
00:43:09.27 something less polar.
00:43:14.10 All right, so with all of this work,
00:43:16.03 are we actually any closer to coming back to RNA
00:43:20.04 with new ideas for complete replication?
00:43:24.05 So, I think that we are,
00:43:26.21 and I'll tell you about one of the way of the conceptual advances
00:43:30.14 that we've made recently.
00:43:32.04 This actually comes from a selection experiment
00:43:35.27 that was done by Simon Trevino when he was a
00:43:38.02 graduate student in the lab,
00:43:40.02 and it addressed this question of monomer homogeneity:
00:43:43.13 How important is it really in a prebiotic setting
00:43:47.01 that the monomers be really pure and concentrated,
00:43:51.20 so that we don't make backbones that have different kinds
00:43:56.01 of linkages, different kinds of sugars, etc.?
00:44:00.17 So the experiment that we could do in the lab
00:44:03.29 is a little bit more limited,
00:44:06.06 but what Simon worked out was a way of taking a
00:44:09.09 library of DNA sequences and transcribing that
00:44:15.01 into molecules that are not just RNA,
00:44:21.08 but a mixture of RNA and DNA.
00:44:23.28 In fact, in every position in these transcripts,
00:44:28.00 there's roughly a 50-50 chance of that linkage
00:44:32.03 being a ribonucleotide or a deoxyribonucleotide.
00:44:36.26 So we have extreme backbone heterogeneity,
00:44:39.16 ribo- and deoxyribonucleotide linkages,
00:44:42.25 and that variation is not heritable.
00:44:47.29 The experiment was to take this library
00:44:50.17 and then select for functional molecules,
00:44:53.01 we select for aptamers just by binding to a target molecule.
00:44:58.01 The targets were used were ATP and GTP,
00:45:00.08 because we'd done this many times years ago.
00:45:03.11 It's easy to evolve RNA molecules or DNA molecules
00:45:07.24 that specifically recognize these nucleotides,
00:45:11.15 but in this experiment, the pool isn't pure RNA or DNA,
00:45:15.23 it's this mixed backbone polymer.
00:45:19.25 So what Simon found is that he could go around
00:45:23.04 cycles of selection and amplification.
00:45:27.00 Every time we do the amplification,
00:45:28.24 we reintroduce this backbone heterogeneity,
00:45:31.25 but of course the molecules get shuffled.
00:45:34.10 The exact order of ribo- and deoxyribo- linkages is randomized
00:45:38.29 at every round of the selection process.
00:45:41.29 Nonetheless, after a few rounds of selection,
00:45:45.04 Simon was able to obtain aptamers
00:45:47.21 that bound to their target with great specificity.
00:45:53.02 They weren't quite as good in terms of affinity
00:45:56.09 as the aptamers we get from pure RNA or pure DNA,
00:46:00.25 but they still work.
00:46:02.21 So this told us that maybe monomer heterogeneity
00:46:06.26 wasn't as important as we'd been thinking.
00:46:09.29 Maybe you could actually evolve functional molecules,
00:46:13.23 ribozymes, in the face of nonheritable
00:46:17.12 backbone heterogeneity.
00:46:19.17 So why is that important in the context of RNA?
00:46:24.23 Well, one of the big problems with RNA is this regiospecificity,
00:46:30.17 the fact that, in a chemical system,
00:46:33.19 it seems almost unavoidable that some fraction of
00:46:37.08 2'-5' linkages will be formed.
00:46:41.00 I think Simon's experiment hints that this
00:46:44.05 may still allow for the evolution of ribozymes,
00:46:48.01 so this is something that needs to be experimentally investigated,
00:46:51.13 something we're doing now.
00:46:53.10 Now, if that turns out to be true,
00:46:55.26 and you can still get functional molecules in the face
00:46:58.11 of this backbone heterogeneity,
00:47:00.18 then the important implication comes from the fact that
00:47:03.23 we already know the 2'-5' linkages in the backbone
00:47:08.00 drastically lower the melting temperature of an RNA duplex.
00:47:12.20 And so, as a result, it would now become possible
00:47:16.21 to thermally separate the strands after the copying
00:47:20.02 of an RNA template.
00:47:21.28 So, it's possible that this 2' versus 3' heterogeneity
00:47:27.07 that we used to think was such a huge problem with RNA,
00:47:31.09 is actually what allowed RNA to work
00:47:34.16 as the primordial genetic material,
00:47:37.16 because it allows for thermal strand separation,
00:47:40.15 and therefore, the repeated cycles of
00:47:44.13 template copying and strand separation that
00:47:46.20 give you overall replication.
00:47:49.17 So this is the kind of thing that we're actively studying.
00:47:53.13 A few more points about more
00:47:56.25 primitive scenarios for template copying...
00:48:03.28 All of the work that we've done,
00:48:06.12 and many other people have done over the decades,
00:48:09.03 has tended to focus on primer extension reaction
00:48:12.13 with monomers, because this is a very simple
00:48:15.28 and analytically tractable approach to the problem
00:48:19.00 of template copying. You get a lot of information,
00:48:21.20 it's easy to analyze the products by simple methods
00:48:25.24 such as gel electrophoresis,
00:48:28.29 but it's probably completely unrealistic
00:48:32.19 as a prebiotic scenario.
00:48:34.22 So what we're being driven to think about is template
00:48:37.16 copying by mixtures of short, random-sequence oligomers,
00:48:42.10 along with monomers, dimers, etc.
00:48:46.02 And so it's a much messier situation,
00:48:48.15 you have a large number of different types of substrates,
00:48:52.29 the number of partial products of template copying
00:48:56.10 becomes enormous, and so the analytical problem
00:48:59.03 gets much worse. But nowadays,
00:49:01.26 we have much more advanced analytical techniques,
00:49:04.23 and with advanced methods of mass spectrometry,
00:49:07.14 we can actually hope to analyze these
00:49:09.23 kinds of reactions and perhaps, we'll see that
00:49:14.08 this kind of system gives unexpected benefits.
00:49:19.23 We have the possibility of nucleating the copying chemistry
00:49:23.24 of multiple sites, the incorporation of oligomers
00:49:27.04 means that fewer catalytic or chemical steps are required,
00:49:32.08 so we're very excited about following up on this kind of
00:49:36.01 more natural, "messier" but more natural scenario,
00:49:40.03 in the hope that this will actually lead us closer
00:49:43.20 to a realistic scenario for full replication.
00:49:47.23 So I just want to end by pointing out that,
00:49:50.07 in this much messier scenario,
00:49:53.15 there are completely new ways in which we can think
00:49:56.29 of ribozymes, catalytic RNAs, contributing
00:50:01.10 to the overall process of replication.
00:50:04.02 Up till now, we have exclusively thought about RNA-catalyzed
00:50:09.03 RNA replication occurring through RNA catalysts
00:50:14.26 that are RNA polymerases.
00:50:18.03 But in these scenarios,
00:50:19.26 I think that it's actually possible
00:50:21.26 that the primordial replicase might've been a nuclease.
00:50:26.13 For example, if an oligomer binds and then gets extended,
00:50:30.25 but a mistake is made, you make a mismatch.
00:50:34.04 As we've discussed, that slows down
00:50:36.17 subsequent primer extension,
00:50:40.05 then that can be a drastic effect.
00:50:42.25 So, if there was a ribozyme nuclease
00:50:46.04 that could chew off that mistake,
00:50:49.18 it would allow chemical copying to go back to normal.
00:50:54.00 So that would be one way of speeding the process up.
00:50:57.20 Another scenario comes from thinking about
00:51:01.16 the use of oligonucleotide substrates.
00:51:04.15 It could be that overlapping oligonucleotides
00:51:07.17 bind to a template, and so this
00:51:09.16 would be a kind of a dead-end situation,
00:51:12.02 unless you have a nuclease that can come along,
00:51:15.10 trim away the overlap, and allow chemical ligation
00:51:18.20 to complete the process of template copying.
00:51:21.22 So, I think these kinds of changes in the way
00:51:25.02 we're thinking about the process have really
00:51:27.15 opened up a lot of new experiments
00:51:30.02 and have made me very optimistic about the possibility
00:51:33.22 of attaining a complete replication system,
00:51:36.14 either purely chemically, or by a combination of
00:51:39.21 chemical and RNA-catalyzed reactions.
00:51:46.07 So, just to sum up then,
00:51:50.00 I think that these considerations tell us that
00:51:52.14 monomer purity may not be as important.
00:51:56.01 It's possible that some backbone heterogeneity
00:51:58.28 may not be fatal.
00:52:02.05 The incomplete regiospecificity may be fine;
00:52:08.15 2' linkages may solve the melting issue for RNA.
00:52:13.18 And we're very excited about studying 2-thio-U
00:52:18.00 as a simple nucleotide substitution prebiotically plausible,
00:52:25.05 something that might enhance both rate and fidelity.
00:52:28.25 So by putting all these things together,
00:52:30.16 we're hopeful that over the coming years,
00:52:33.03 we'll eventually converge on a complete chemical system
00:52:38.02 for the replication of either RNA or maybe some related polymer.
00:52:42.11 And if we can get to that point,
00:52:45.00 combining it with the replicating vesicle system should
00:52:47.21 allow us to observe the spontaneous emergence
00:52:51.21 of Darwinian processes from a purely chemical system.
00:52:55.16 And that's really the major goal of this whole thing,
00:52:57.29 and the part of the project that's most relevant
00:53:01.04 to the emergence of biology from the
00:53:03.21 chemistry of the early Earth.
00:53:05.26 So, again, I've tried to mention people as I've gone along.
00:53:10.21 In terms of the chemistry,
00:53:13.09 many people have contributed to this over the years:
00:53:16.10 Jason Schrum, Alonso Ricardo, Matt Powner, Na Zhang,
00:53:20.22 Ben Heuberger, Craig Blain, Shenglong Zhang.
00:53:24.10 Many people have contributed to this work,
00:53:27.29 and so they've played a very important role in developing
00:53:30.28 all of these new ideas that are leading us, hopefully,
00:53:34.13 towards a solution to this major problem
00:53:37.00 in thinking about the origin of life.
00:53:39.06 Thank you.