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

Transcript of Part 3: Non-Enzymatic Copying of Nucleic Acid Templates

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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

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