The Origin of Life

• http://genetics.mgh.harvard.edu/szostakweb/research/protein/research-pro.html

Other possible candidate include peptide nucleic acids (PNA).   These can also form double stranded structures with DNA, RNA, or PNA single strands.  They were initially designed to bind to dsDNA in the major grove forming a triple-stranded structure.  Binding could alter DNA activity, possibly by inhibiting transcription, for example.   The structure of a single-stranded PNA is shown.  Note that the backbone, a polymer of N-(2-aminoethyl)glycine (AEG) which can be made in prebiotic soups, is not charged, making it easier to bind to dsDNA.   AEG polymerizes at 100^oC to form the backbone.  

In addition to changing the backbone, additional bases other than A, C, T, G, and U can be accommodated into dsDNA and ssRNA molecules (Brenner, 2004)

Von Kiedrowski, in an experiment similar to the self-replication of peptides described above, has shown that a single stranded 14 mer DNA strand, when immobilized on a surface, can serve as a template for the binding of complementary 7 mers and their conversion to 14 mers.  When released by base, this process can occur with exponential growth of the complementary 14 mers. (von Kiedrowski Nature, 396, Nov 1998).  Ferris has shown that if the clay montmorillonite is added to an aqueous solution of diadensosine pyrophosphate, polymerization occurs to produce 10 mers which are 85% linked in a 5' to 3' direction.

Why a polyanion as the carrier of genetic information?

There are other reasons why polyanions are useful genetic molecules, other than their resistance to nucleophilic attack.  The biological form of DNA is a large double stranded polyanionic polymer, in contrast to RNA which is a single-stranded polyanion polymer and protein which are polymers with varying combination of anionic, cationic, and hydrophobic properties.  Even with counterions, it would be difficult to fold DNA into complicated and compact 3D structures as occurs for proteins, given the large electrostatic repulsions among the charged phosphates.  Rather it forms a elongated double stranded rod, not unlike the rod-like structure of proteins denatured with sodium dodecyl sulfate (used in SDS PAGE gels).  The elonged rod-shaped structure of ds-DNA is critical for the molecule which is the main carrier of our genetic information since mutations in the bases (leading to a switch in base pairs) causes no change in the overall structure of dsDNA.  This enables evolutionary changes in the genetic material to produce new functionalities.   A single change an amino acid of a protein, however, can cause a large change in the structure of a whole protein, a feature unacceptable for a carrier of genetic information.  RNA structure effectively lies between that of DNA and proteins.  Since it has less charge density than dsDNA, it can actually form dsRNA helices, so it can carry genetic information, as well as form complex 3D shapes necessary for its activity as an ribozyme.  Perhaps more importantly, steric interference prevents ribose in RNA from adopting the 2'endo conformation, and allows only the 3'endo form, precluding the occurrences of extended ds-B-RNA helices.

The Lipid World

Let's assume that abiological precursors would react to form polymer-like molecules that might be complex enough to fold to structures that would allow binding, catalysis, and rudimentary replication.   All this would be worthless unless they could be sequestered in a small volume which would limit diffusion and increase their local concentration.  What is required is a membrane structure. Amphiphilic molecules, like lipids, with which we started this book, would be prime candidates since they spontaneously assembly to form micelles and bilayers, as shown in the review diagram below.

As mentioned in Chapter 1, alternative lipid phases are possible. Bilayers can also be formed from single chain amphiphiles, such as certain fatty acids, as illustrated in the equilibrium shown below.  This occurs more readily at pH values close to the the pKa of the fatty acid, at which the fatty acids are not all deprotonated with full maximal negative charges.  Single chain amphiphiles like fatty acids, which were more likely to formed in abiotic conditions, have been found in meteorites. 

Clay surfaces, which have been shown to facilitate the formation of nucleic acids polymers, can also promote the conversion of fatty acid micelles to bilayers (Szostak).  One such clay surface, montmorillonite, whose structure is shown below, promotes bilayer formation. 

Chime Molecule Modeling: montmorillonite 

The effect of montmorillonite on vesicle formation can be shown by simple measurements of turbidity with time.  Microscopy of fluorophore-encapsulated vesicles also shows encapsulated montmorillonite.  The fatty acids presumably absorb to the cation layer of the clay particles.  Time studies using light scattering also indicate that the vesicles grow in the presence of fatty acid micelles.  To differentiate between the formation of new vesicles and the increase in size of pre-existing vesicles (which couldn't be done by simple light scattering without separation of the vesicles), investigators used two different fluorescent molecules to label fatty acid vesicles.  The two probes were selected such that if the two probes came in close contact, energy transfer from the excited state of one fluorophore to the other fluorophore could occur, an example of fluorescence resonance energy transfer (FRET).  FRET is observed when emission of the second dye occurs after excitation of the first dye, at a wavelength outside of the excitation wavelength of the second dye.  If unlabeled vesicles where added to either labeled vesicles, no changes in FRET were observed, suggesting that the dyes did not move between vesicles.  If fatty acid micelles were added, a decrease in FRET was observed, suggesting that new fatty acids were transferred to the doubly-labeled vesicles, effectively diluting the dye concentrations in the bilayer and their relative proximity, both which would decrease FRET.    Most of the new fatty acid was incorporated into pre-existing vesicles which grew.  The vesicles could also divide if extruded through a small pore.  Later we will see that the energy to grow the vesicles can derive in part from a transmembrane proton concentration collapse.  Division of vesicles might be promoted by bilayer assymetries associated with addition of substances to the outer leaflet, causing membrane distortion.

• Szostak's Lab

Protocells:  Membrane encapsulated nucleic acids

At some point, early genetic material must have been encapsulated in a membranous vesicles.  Would new properties emerge from this mixture that might have a competitive (evolutionary) advantage over either component alone, and thus be a step on the way to the formation of a "living" cell?  The answer appears to be yes. Chen et al. have incorporated RNA into fatty acid vesicles with interest effects.  They asked the question as whether those vesicles could grow at the expense of vesicles without encapsulated RNA.  RNA, with a high charge density and its associated counter ions would create osmotic stress on the vesicles membranes.  To relieve that stress they could acquire fatty acids from other fatty acid vesicles (or fatty acid micelles), increasing their surface area, and concomitantly reducing tension in the membrane.   

Oleic acids vesicles were first placed under stress by encapsulating 1 M sucrose in the vesicle and then diluting it in hypotonic media.  Water would enter and swell the vesicle (but without bursting and resealing, as evident from control experiments).   Then they prepared stressed and unstressed oleic acid vesicles in the presence of two nonpolar flurophores, NBD-PE (excitation at 430 nm, emission at 530 nm) and Rh-DHPE (emission at 586 nm).  These fluorophores were chosen for fluorescence resonance energy transfer measurements.  If the membrane vesicles changed size, the FRET signal would change, based on the relative concentration and proximity of the dual fluorophores.  If the separation between probe molecules increased, the FRET signal would decrease.  Conversely, if the vesicle shrunk, the FRET signal would increase.  

Now what about vesicles swollen with encapsulated RNA?  RNA, with its associated charge and charged counter ions also placed an osmotic stress on the vesicles.  FRET labels (the two fluorophores) were place in vesicles without RNA.  Fatty acids were removed from isotonic labeled vesicles in the presence of unlabeled tRNA swollen vesicles (left panel below).  Labeled vesicles swollen with glycerol took fatty acids from unswollen vesicles (without tRNA), but not from tRNA swollen vesicles, as both were swollen so no net drive to reduce swelling by lipid exchange was present.  

These results show the vesicles with encapsulated RNA have a competitive (evolutionary) advantage over normal  vesicles.   This data suggests that having a polyanion as the source of genetic material is actually advantageous  to the protocell.  In addition the move in modern membranes to phospholipids with esterified fatty acids (instead of free ones) may actually have stabilized membranes, given the movement of free fatty acids to different membranes.

Energy Transduction in protocells

In addition to a genetic macromolecule and a semipermeable membrane, a source of energy to drive intracellular processes must be present.  A common source of free energy used in many cells to drive unfavorable processes is a proton gradient, whose formation in modern cells can be coupled to energy input from oxidation, ATP cleavage, light, or the collapse of another gradient.  Could a proton gradient be formed in protocells?  It can, quite easily, when coupled to the growth of fatty acid vesicles.  If a fatty acid vesicle is to grow, more fatty acid must be added to the outer leaflet.  The protonated, uncharged form of the fatty acid would preferentially be added, since it would lead to less electrostatic repulsion between adjacent head groups.  The protonated, uncharged form of the fatty acid would also be most likely to flip to the inner leaflet to minimize stress asymmetries in the leaflets.  Once in the inner leaflet, it could deprotonated to form H⁺(aq) in the inside of the membrane, creating a transmembrane proton gradient and transmembrane potential.  The energy released on growth of the membrane is partly captured in the formation of a proton gradient, as shown in the figure below.

The proton gradient would soon inhibit its own formation since further movement of protons into the cell would be attenuated by the positive transmembrane potential unless metal ions inside moved outside.  In addition, the gradient would collapse after growth stopped.  The investigators made fatty acids vesicles in the presence of pH 8.5 buffers whose pH was adjusted with an alkali metal hydroxide.  The external pH was reduced to 8.0, resulting in a 0.5 pH unit proton concentration gradient.  (Changes in intravesicular pH were measured with pH-sensitive fluorophore, HPTS.   Inward movement of protons down a concentration gradient, as shown in the figure below, would occur with time, collapsing the imposed concentration gradient.

With fatty acid vesicles, this artificial pH gradient collapsed quickly, suggesting the vesicle permeability to protons was high.  The rate was too high for simple flip-flop diffusion.  Inward movement of protons appeared to be facilitated by outward movement of the M⁺  ions.  The rate of decay of the proton gradient was exponential, and the resulting first order rate constant was easily determined.  A graph of the rate constant for pH gradient collapse vs unsolvated ionic radius of M⁺ decreased with increasing radius (i.e. k_Na > k_K > K_Rb > K_Cs, suggesting that the pH gradient would be more stable if large, impermeable or otherwise trapped cations were encapsulated.  When vesicles were made with encapsulated Arg⁺, the imposed pH gradient did not collapse for hours.  If oleic acid micelles were added to oleic acid vesicles with encapsulated Arg⁺, with no artificial pH gradient induced across the membrane, the vesicle grew with concomitant movement of protons into the vesicle, producing a pH gradient of 0.3 within seconds. 

These experiments show that membrane growth and energy storage could be coupled, and the right composition of encapsulated material could lead to a stable transmembrane pH gradient, a source of energy to drive biological processes.  It even suggests that a charge polyanion would be beneficial as a genetic carrier.