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Qu’est-ce que c’est “la vie universelle”?

Qu’est-ce que c’est “la vie universelle”? Quatres rues pour l’investigation de quelque chose que nous ne pouvons pas voir directement. Exploration: NASA Astrobiology Institute. Looking for life on Titan, a moon of Saturn. ESA-NASA Cassini-Huygens mission • Rich in organics.

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Qu’est-ce que c’est “la vie universelle”?

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  1. Qu’est-ce que c’est “la vie universelle”? Quatres rues pour l’investigation de quelque chose que nous ne pouvons pas voir directement.

  2. Exploration: NASA Astrobiology Institute Looking for life on Titan, a moon of Saturn ESA-NASA Cassini-Huygens mission • Rich in organics. • Subsurface liquid water-ammonia. If life is a natural produt of organic reactivity, then Titan is a candidate spot for life. Inspiration: Perhaps Titan’s cells are water drops emulsion in hydrocarbon solvents, but it will be some time before we can detect them. Baross, Benner et al.

  3. Qu’est-ce que c’est “la vie universelle”? Quatres rues

  4. The historical past captured in sequences Reconstruction says something about the Proto-Indoeuropeans They lived where it snowed. No gold. But dogs (*kwón-), horses (*ékwo-), sheep (*H3éwi-), ox (*gwów-), pigs (*suH-), grain (*yewo), vehicles (*wogho-) with wheels (*kwekwlo-); Count to 100 (*kmtóm)

  5. Resurrect ancestral protein sequences 10 20 . | . | ox KETAAAKFERQHMDSSTSAA || ||||||||||||||| | sheep KESAAAKFERQHMDSSTSSA camel SETAAEKFERQHMDSYSSSS Ancestor KERAAAKFERQHMDSSTSSA Paleogenetics: Use recombinant DNA technology to bring ancient proteins back to life for study Linus Pauling, Emile Zuckerkandl

  6. One learns much about chance and necessity, but only in the life on Earth that we know To support paleogenetics, we did something new: a total synthesis of a gene for a protein.

  7. 2004. I discovered that I had helped found the field of synthetic biology 20 years earlier. “Old school?” synthetic biologists “explore basic questions” !?

  8. In fact, “synthetic biology” is older Waclaw Szybalski (1974) Test hypotheses by constructing living systems with new arrangments of natural genes and proteins. Direct line to Jay Kiesling, Craig Venter, Ham Smith. Meaning 2: Using natural biomolecules to do unnatural things (digital math, oscillators) Adleman (1994); now with validated parts (Endy). -----> Toy projects (make E. coli smell like a banana). Meaning 3. Using unnatural molecules to do natural things that hitherto only life could do. Biomimetic chemistry. Lehn (1987) Binds cation like a protein but not as a protein If we reproduce a biological behavior with a different molecular structure, we demonstrate our understanding of the chemistry behind the behavior.

  9. But synthesis provides more… Human instinct. If an observation contradicts a theory, discard the observation. Mars climate orbiter Guidance hardware: English system (feet) Guidance software: Metric system (meters) In transit: Observations were rationalized away By targeting a "grand challenge" synthesis forces scientists across uncharted ground where they must solve unscripted problems in a way that does not allow self-deception. If the theory is wrong, the rocket crashes. If our designed E. coli does not smell like a banana, something is wrong with the design theory, and we cannot avoid this fact. Mars climate orbiter Synthesis drives paradigm changes in ways analysis cannot.

  10. What is a really big challenge for synthetic biology? Building artificial life. Definition-theory of life: A self-sustaining chemical system capable of Darwinian evolution. The theory that allows us to synthesize a chemical system capable of Darwinian evolution will be the theory that explains life and provides the language of understanding. And if we cannot get from our synthetic Darwinian system all of the behaviors that we expect from life, then something must be wrong with our theory of life.

  11. Parts of Darwinian evolution appear very simple Big pairs with small. Hydrogen bond donors pair with hydrogen bond acceptors. Scaffolding unimportant. Is it this simple???

  12. Failed grand challenge Synthesize a charge-neutral analog of DNA The repeating negative charge is a problem in biotechnology. Site for enzymatic DNA degradation. Prevents crossing membrane. If the scaffold is unimportant, we should be able to change it. Rule-based molecular evolution fails n > 6. Richert, C., Roughton, A. L., Benner, S. A. (1996) Nonionic analogs of RNA with dimethylene sulfone bridges. J. Am. Chem. Soc.118, 4518

  13. Lots of uncharged linker = oligo folds Clemens Richert Univ. Stuttgart Melting curve shows presence of folded form ASO2USO2GSO2GSO2USO2CSO2ASO2U An oligo that folds does not bind to its complement

  14. Why genetic systems must be polyelectrolytes in water Keeps DNA soluble in water. Backbone-backbone coulombic interactions force strand-strand contacts to Watson-Crick edges of the nucleobases (= rules). 3. Repeating charges discourages folding; the “excluded volume” effect (Paul Flory, Stanford) Repeating monopole dominates the physical properties, allowing mutation to occur without changing the bulk properties of the molecule (very unusual) A backbone charge is essential for Darwinian evolution

  15. Such a universal helps search for life in the cosmos Self-sustaining chemical system capable of Darwinian evolution Not sufficient that a molecular system direct its reproduction The reproduction must be allowed to include errors Those errors must themselves be replicable Not like crystallization, with defects in the crystal structure “cured” A polyelectrolyte is a universal feature of genetic polymers in water Charges dominate the physical property of a molecule. Backbone mutation does not change physical properties given those charges. Therefore DNA physical properties can be stable even as its information content changes. Repeating charge “easy” to detect in situ. Synthesis drove a paradigm change that is now helping build instruments to detect “la vie universelle”.

  16. Synthese: “la vie universelle” a dessein?

  17. Nucleobases as interchangeable parts Chemical reasons for Watson-Crick complementarity Size: Small pairs with large Hydrogen bonding: Donors pair to acceptors

  18. Shuffling donor/acceptors gives orthogonal pairs ... including the two that terrean DNA already has Do these support synthetic genetics?

  19. Yes.3hydrogen bonds are better than2hydrogen bonds, which are better than1hydrogen bond Size complementary > large rungs or short rungs Thomas Battersby, C. Ronald Geyer(2003) Structure 11, 1485-1498. No base at all Battersby-Geyer plot; 2 hydrogen bonds OK; one is not.

  20. Synthetic genes encode synthetic proteins with 21 amino acids iodotyrosine C-U-isoG isoC-A-G Bain, et al. (1992) Ribosome-mediated incorporation of non-standard amino acids into a peptide through expansion of the genetic code. Nature356, 537-539 Yes, we can. It is just as simple as Watson-Crick said.

  21. Peut notre biologie synthetique faire l’evolution Darwinienne? Get natural polymerases to accept synthetic genetics major groove minor groove Standard nucleobases presentunshared electron pairsto the minor groove. These are the only common “pharmacophore”. Many nucleobases in our genetic system lack these pairs.

  22. Polymerases have evolved to look for the unshared electron pairs in minor groove No problem. We know where the contact are. Make synthetic polymerases that accept synthetic genetic systems.

  23. Time is short, so let us consider the Darwinian potential of just one synthetic nucleobase pair major groove P minor groove Z Can “G, C, T, Z, A, P” support Darwinian evolution like “G, C, T, A” in the laboratory?

  24. Yes it can. GACTZAP directs the formation of CTGTPAZ children using synthetic polymerases standard template standard template synthetic template GACTZAP primer standard synthetic standard synthetic standard synthetic no kids no kids synthetic kids no kids standard kids standard kids Zunyi Yang

  25. Remember, reproduction alone is not sufficient for a chemical system to be Darwinian. The system must support reproduction with errors. The errors must themselves be reproducible. Errors, of course, occur when copying natural DNA

  26. This is also true with GACTZAP DNA Can study the mutation in the GACTZAP system just as we do with natural genetic material. Remember, this is a chemical system capable of Darwinian evolution; we can study it just like we study living systems.

  27. G:C can mutate to P:Z via transitions Yes it can; multiple PCR cycles used to detect infrequent mutation 5-CTAGGACGACGGACTGC 5-CTAGGACGACGGACTGCCCATGGGAGACCGCGGTGGGCCCGGCCGGGTACCATCGATACGCGTTGCGATCGCTCCTTCCTG-3 CGCTAGCGAGGAAGGAC-5 Apa site lost as G or C is replaced by P or C after many PCR cycles. PCR cleavage product no longer seen. Apa 1 restriction site GTGGGCCCGG CACCCGGGCC GTGPGCZCGG CACZCGPGCC Can P:Z convert back to G:C?

  28. Yes it can. We know the mechanism of mutation Mutation of P:Z back to G:C is pH dependent. Mutations facilitated at high and low pH, just like natural DNA.

  29. A synthetic chemical system capable of Darwinian evolution with 6-letter "RNA" ET Is this synthetic life? A self-sustaining chemical system capable of Darwinian evolution?

  30. Raising the bar Many theories of life are in common use Cell theory Gene theory Evolution theory Can we get our synthetic genetic system into a cell?

  31. Remember the inspiration from Titan? Cells as water drops emulsified in hydrocarbon ESA-NASA Cassini-Huygens mission Put our synthetic genetic system into synthetic cells like those that might be found subsurface on Titan.

  32. Synthetic cells (water cells in hydrocarbon solvent holding) synthetic genetic system (GACTZAP) capable of Darwinian evolution. Ryan Shaw Roberto Laos Artificial genetic system placed in water droplet cells to amplify (by 6-letter GACTZAP-PCR) a synthetic polymerase that accepts synthetic genetic systems. Ca. 2 microns

  33. Amplification (with replicable mutation) in artificial cells of GACTZAP system 13’th generation GACTZAP children Now is this a synthetic biology?

  34. Life: A self-sustaining chemical system capable of Darwinian evolution This is not self-sustaining

  35. Life: A self-sustaining chemical system capable of Darwinian evolution The differences between this and a 6-letter RNA system capable of assisted Darwinian evolution in an artificial cell in a test tube are more than obvious.

  36. Many discoveries Ribosome synthesis of proteins with 21 amino acids using synthetic genetics systems determined the role of release factors in natural biology. Bain, et al. (1992) Ribosome-mediated incorporation of non-standard amino acids into a peptide through expansion of the genetic code. Nature356, 537-539 Attempts to get prebiotic ribose generated a production-scale synthesis of sugars from one carbon feedstocks. Benner, S. A. (2007) Borate Moderated Carbohydrate Synthesis, US Prov. Pat. Appl. 60/997135 Synthetic genetic systems allow re-sequencing of personal genomes. Synthetic genetic systems annually improve the care of some 400,000 patients infected with HIV, hepatitis B and C, respiratory viruses.

  37. Personalizing healthcare with AEGIS The branched DNA architecture measures viral load 400,000 patients last year; $100 million product Orthogonality allows analyte recognition distinct from movement of the complex, signaling, etc. 8 molecules/mL

  38. A general theory of life as a universal? No, but we are constraining the black box.

  39. Potential hazards? Risk elements. Standard biochemistry (parasitism) Self-sustenance (otherwise tied to lab food) Ability to evolve (otherwise hazard is stationary)

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