Seminar: Artificial cell design and the origin of life (by Dr. Jack Szostak, 2009 Nobel Laureate of Physiology or Medicine)


Dr. Jack Szostak

Nobel Prize Laureate in Physiology or Medicine 2009
Investigator, Howard Hughes Medical Institute
Professor of Genetics, Harvard Medical School
Alex Rich Distinguished Investigator, Massachusetts General Hospital
Dept. of Molecular Biology, and Center for Computational and Integrative Biology 7215
Simches Research Center
Massachusetts General Hospital

The Szostak lab is currently focused on the laboratory synthesis of self-replicating systems, with the goal of understanding how life emerged from the chemistry of the early earth. This approach is based on the view that the two key components of a primitive cell are a self-replicating nucleic acid genome, and a self-replicating cell membrane. The lab has recently discovered a simple and robust pathway for the coupled growth and division of a model primitive cell membrane, and has made considerable experimental progress towards the synthesis of self-replicating nucleic acids. It is hoped that future progress will lead to the spontaneous emergence of Darwinian evolutionary behavior from replicating chemical systems.
Dr. Szostak is an Investigator of the Howard Hughes Medical Institute, Professor of Genetics at Harvard Medical School, and the Alex Rich Distinguished Investigator in the Dept. of Molecular Biology and the Center for Computational and Integrative Biology at the Massachusetts General Hospital. Dr. Szostak is a member of the National Academy of Sciences, and a Fellow of the American Academy of Arts and Sciences and the American Association for the Advancement of Science. Dr. Szostak’s early research was on telomere structure and function, and the role of telomere maintenance in preventing cellular senescence. For this work Dr. Szostak shared, with Drs. Elizabeth Blackburn and Carol Greider, the 2006 Albert Lasker Basic Medical Research Award and the 2009 Nobel Prize in Physiology or Medicine.

LECTURE 1
OCT. 13, 2010 – 6:00 PM TO 8:00 PM
Public Lecture: The Roles for Diverse Physical Phenomena during the Origin of Life.
Department of Physics

Presentation Title: Surprising physical phenomena and the origin of life.
Jack W. Szostack, HHMI, MGH, HMS

This focus of this lecture is on examples of very simple physical phenomena that have possibly set the stage for early life.

Life: is it all the same or is life diverse? In our case it’s based on water, FA, NA’s and proteins doing the cellular work. Or could it be based on a completely different biochemical foundation.
If we go to the lab and try to build structures we can lean a lot about the evolution of life.

Timeline
4.5BYA Formation of earth, 4.2BYA stable hydrosphere, 4.2-4 BYA prebiotic chemistry, 4BYA pre RNA world, 3.8 BYA RNA world, 3.6 BYA first DNA/protein life, ~3.6 diversification of life.
4.5 BYA to 3.6 BYA is a foggy region (Anything could be anywhere).

Evolution of life began with simple molecules/ basic building blocks
Chemical Evolution: Small molecules (CO,H2, H2O, NH3 etc) to lipids, sugars, AA’s, nucelotides etc..
These complex molecules become protocells.

In order to get extremely complex molecules to form from building blocks you need:
1. High concentration. How did they compartmentalize instead of diffuse.
2. Pure substances: How did specific molecules assemble if everything was a mixture.
3. Chirality: How did a Racemic Mixture that’s optically inactive transform in to a pure-chiral and active mixture.

1. Concentration.
a. Concentration by freezing:
Freezing stops reactions. When you freeze an aqueous solution, ice crystals form and molecules concentrate multifold. Example: The very simple experiment of polymerization of nucleotides by freezing.

b. Concentration by temperature gradients:
As in hydrothermal vents. Small molecule tend to concentrate near lower part of the temperature gradient. (Article: Finite element stimulation by Baaske, 2007)

2. Purification of defect free NA’s by liquid crystal phase transition.
Liquid crystal domains of dsDNA in an isotropic ssDNA background.
Simple way to separate what we want from all the other garbage.

3. Chirality
Chiral molecules: Racemic mixtures, asymmetric processes.
NA’s in meteorites have a small bias for one of the chiral molecules. (L-AA’s )

a. Chiral enrichment by crystallization
`Racemic mixture  Solution of one chiral molecule + solid crystals with 1:1 ratio of L and D chirality.

b. Convergence to homochirality by crystal grinding.
Crystals of chiral molecules form mirror image crystal forms.
Cycle of grinding and growth.
Crystal grinding force can be used to draw all molecules to pure chirality.
Pure chirality is the basis of complex molecule build up which is the precursor of life.
Self-assembly of multinuclear complexes with enantiomerically pure chiral binaphthoxy imine ligands, (Maedo, 2006)

Schematic Model of a Protocel
Simple cell might be based on a replicating vesicle for compartmentalization, and a replicating genome to encode heritable information. Complex environment provides NA, lipid, and energy (Mechanical, Chemical, etc…).

Properties of fatty acid vesicles
Its reasonable to assume they have self-assembled in early life give the structure of the membrane bilayer.
Model protocell membranes: fatty acid vesicles self-assemble. These vesicle’s maintain their identity despite rapid molecular exchange.

Protocell assembly
Protocells can self-assemble.
Ex. Montromoriollonite can bring RNA into vesicles. Self-assembly is auto catalytic.
Ex. Thermal diffusion columns can be used to concentrate FA and DNA near the bottom of the scale.

So now we have vesicles filled with genetic material, but how do they divide.

Vesicle growth.
Happens through a cycles of growth and division.
Adding micelles to vesicles  growth (filamentous growth)  agitation  division  repeated cycle.

How does division of micelles happen?
Pearling instability induces vesicle division.

Different mechanism of vesicle growth and division
1. Faster genomic replication brings about faster membrane growth.
Consider a tense membrane (full of encapsulated RNA, DNA) and another empty vesicle.
The osmotic pressure creates membrane tension such that the contents of one squeeze out from one to the other. This relaxes the osmotic pressure allowing the first vesicle to grow and the other to shrink
2. Division driven by phase separation.

Transition from primitive to modern membranes
Membranes with some amount of phospholipid drive vesicle growth. The strong selective pressure for phospholipid synthesis is driven by genetically coded, heritable acetyltransferase. These leads on an evolutionary arms race. Leading to increasing selection for membrane transporters and internal metabolic activity

Summary
Unexpected physical phenomena play an important role.
-RNA polymerization in ice.
-Concentration in thermal gradients.
-Selective sugar permeability favoring ribose. (not included in talk)
-Osmotic pressure can drive vesicle growth
-Vesicle growth into fragile filaments
-Thermostability of fatty acids.
-Permeability of membrane.

LECTURE 2
OCT. 14, 2010 – 4:15 PM
McIntyre Medical Building, Room 504 (Martin), 3655 Promenade Sir William Osler
“Learning About the Origin of Life from Efforts to Design an Artificial Cell”
Department of Biochemistry

Jack W. Szostack
HHMI, MGH, HMS
Title: Learning about the origin of life from efforts to design an artificial cell

Research focused on a narrow slice of building blocks that have the potential to evolve into life.
We have a list of 400-500 planets that are looking more and more earthlike. (Ozone, rotating around star).
“earthlike planets will be discovered soon”.
Is it easy or hard to emerge from chemistry?
Is all life based on the biochemistry of earth (water, DNA, proteins etc) or is it based on a totally different biochemical foundation?
These questions can be answered by attempting to build life in the lab.

Schematic Model of a Protocel
Simple cell might be based on a replicating vesicle for compartmentalization, and a replicating genome to encode heritable information. Complex environment provides NA, lipid, and various sources of energy (Mechanical for division, Chemical for nucleotide activation, phase transfer and osmotic gradient (for growth)). (Mansy, nature 2008)

Two aspects must be considered: 1. Assembly. 2. Growth

Replicating vesicles
Model protocell membranes are made of FA vesicles (oleic, capric etc…) Spontaneous assembly into bilayer membranes by shaking in water. These vesicles exhibit rapid exchange and dynamic motion.

Vesicle growth.
Spherical vesicles transform into tubular structures upon addition of Fa’s. Filaments are very fragile thus solving the problem of division.
Cycles of growth and division.
Adding micelles to vesicles  growth (filamentous growth)  fragile filaments divide upon agitation  divided vesicles  repeated cycle.
Forces of these transitions are not well understood. However this can be used as a system for self replication.

Chemical Replication of Nucleic Acids
Modern substrates are very polar and have low chemical reactivity (nucleoside TP’s)
Prebiotic model substrates are less polar and are more permeable and have high chemical reactivity. (Nucleoside phosphorimidazolides).
RNA: spontaneous primer extension. Replicating RNA alone isint possible according to Lesley (father of artificial cells field). Maybe Ribosyme catalyzed RNA replication.

Phosphoramidate-linked NA’s: self assemble into Watson crick double helix model. (2’NP-DNA)

2’NP-DNA: is a typical monomer for spontaneous synthesis. Why?
1. Monomers can cyclize
2. 2-5 linkage favored in RNA system (defaulat system)
3. Duplex melts at lower temps (less thermally stable

2’NP-DNA: experiments show that these molecules can assemble into a DNA backbone.
Template geometry plays a big role in positioning the incoming monomer. If u can constrain the geometry in the right way, you can make the reaction go better (flopping around conformations won’t work). DNA, RNA, LNA(locked NA, very hard to produce).

Second generation monomers that constrain template geometry: NP-TNA, MoNA.
Replication with these monomers is faster and more accurate.

Replicating NA’s inside Replicating vesicles
Template and primer inside a vesicle. Primer: template 2:1 at 4C, monomyrsiol vesicles.
Only way this works is with a primitive membrane and a primitive template.
Encapsulated templates can separate and divide by thermocyclin.

Emergence of Darwinian evolution
How did the transition from a primitive (dynamic/permeable lipid membrane) to modern (phospholipid bilayer) membranes?
Membranes with some amount of phospholipid drive vesicle growth. The strong selective pressure for phospholipid synthesis is driven by genetically coded, heritable acetyltransferase. This leads on an evolutionary arms race. Leading to increasing selection for membrane transporters and internal metabolic activity. Simple phospholipid effect can drive a whole series of evolutionary processes.

Summary
Unexpected physical phenomena play an important role.
-RNA polymerization in ice.
-Concentration in thermal gradients.
-Selective sugar permeability favoring ribose. (not included in talk)
-Osmotic pressure can drive vesicle growth
-Vesicle growth into fragile filaments
-Thermostability of fatty acids.
-Permeability of simple membrane to nucleotides.
– Membrane Growth can lead to pH gradients.

Compiled by Ahmad Kanaan

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