Szostak begins his lecture with examples of the extreme environments in which life exists on Earth. He postulates that given the large number of earth-like planets orbiting sun-like stars, and the ability of microbial life to exist in a wide range of environments, it is probable that an environment that could support life exists somewhere in our galaxy. However, whether or not life does exist elsewhere, depends on the answer to the question of how difficult it is for life to arise from the chemistry of the early planets. Szostak proceeds to demonstrate that by starting with simple molecules and conditions found on the early earth, it may in fact be possible to generate a primitive, self-replicating protocell.
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.
- What insights from the modern world/modern ecosystems lead us to believe that life could emerge even in harsh, extreme environments?
- What experimental evidence or results might indicate that life is “easy” to emerge in harsh environments? What evidence or results might indicate that life is “hard” to emerge in harsh environments?
- Explain the protocell. How could these cells undergo Darwinian evolution?
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.
- What are some characteristics of a protocell membrane? How do these differ from a modern cell membrane?
- What is the proposed link between genome replication and membrane growth? Describe the experiments that support this model.
- What proposed evolutionary pressure drove the incorporation of phospholipids into protocell membranes? Describe experiments that support this model.
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.
- What is structurally and catalytically different about the substrates required for chemistry-driven RNA/DNA replication? How could they have been generated on early Earth?
- Describe some of the challenges for chemical replication of protocell genetic material. What evidence strongly suggests chemical replication is possible?
- Give examples of current research efforts to solve the fidelity problem with RNA replication.
- What is the major takeaway about monomer (or nucleotide base) homogeneity?
Paper for this Session’s Discussion
Discussion Questions for the Paper
- What was the “roadblock” in non-enzymatic RNA replication chemistry that the authors addressed?
- How are the experiments shown in figures 2A and 2B similar? What is different about them
- Talk through Figure 2. What did they test, and how? What could be some biases in their primer- extension assay? Describe the main conclusion(s). What properties of chelators are beneficial/harmful for a primitive protocell?
- Comment on the time scale for RNA replication vs. RNA degradation in a fatty acid vesicle containing Mg2+ and citrate. Does this seem plausible for early life?
- Recall the model for protocell growth/division (iBioSeminar). To what extent does this paper piece together critical elements of the model? What aspects of the model remain to be tested?
Early in his research career, Dr. Szostak made important contributions to the field of genetics. These included construction of the first yeast artificial chromosome and furthering our understanding of the function of telomeres, work for which he shared the Nobel Prize in Physiology or Medicine in 2009. By the 1990s, however, Szostak had redirected his… Continue Reading