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Quest-ce que cest la vie universelle? Quatres rues pour linvestigation de quelque chose que nous ne pouvons pas voir directement. Slide 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. Inspiration: Perhaps Titans cells are water drops emulsion in hydrocarbon solvents, but it will be some time before we can detect them. If life is a natural produt of organic reactivity, then Titan is a candidate spot for life. Baross, Benner et al. Slide 3 Quest-ce que cest la vie universelle? Quatres rues Slide 4 Reconstruction says something about the Proto-Indoeuropeans They lived where it snowed. No gold. But dogs (*kwn-), horses (*kwo-), sheep (* H3 wi-), ox (*g w w-), pigs (*su H -), grain (*yewo), vehicles (*wogho-) with wheels (*k w ek w lo-); Count to 100 (*kmtm) The historical past captured in sequences Slide 5 Paleogenetics: Use recombinant DNA technology to bring ancient proteins back to life for study Linus Pauling, Emile Zuckerkandl Resurrect ancestral protein sequences 10 20. |. | ox KETAAAKFERQHMDSSTSAA || ||||||||||||||| | sheep KESAAAKFERQHMDSSTSSA camel SETAAEKFERQHMDSYSSSS Ancestor KERAAAKFERQHMDSSTSSA Slide 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. Slide 7 Old school? synthetic biologists explore basic questions !? 2004. I discovered that I had helped found the field of synthetic biology 20 years earlier. Slide 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. If we reproduce a biological behavior with a different molecular structure, we demonstrate our understanding of the chemistry behind the behavior. 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 Slide 9 Mars climate orbiter 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 Synthesis drives paradigm changes in ways analysis cannot. 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. Slide 10 What is a really big challenge for synthetic biology? 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. Building artificial life. 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. Slide 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??? Slide 12 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 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. Slide 13 Lots of uncharged linker = oligo folds A SO 2 U SO 2 G SO 2 G SO 2 U SO 2 C SO 2 A SO 2 U An oligo that folds does not bind to its complement Clemens Richert Univ. Stuttgart Melting curve shows presence of folded form Slide 14 Why genetic systems must be polyelectrolytes in water 1.Keeps DNA soluble in water. 2.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) 4.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 Slide 15 Such a universal helps search for life in the cosmos 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 Repeating charge easy to detect in situ. 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. Synthesis drove a paradigm change that is now helping build instruments to detect la vie universelle. Self-sustaining chemical system capable of Darwinian evolution Slide 16 Synthese: la vie universelle a dessein? Slide 17 Nucleobases as interchangeable parts Chemical reasons for Watson-Crick complementarity Size: Small pairs with large Hydrogen bonding: Donors pair to acceptors Slide 18 Shuffling donor/acceptors gives orthogonal pairs... including the two that terrean DNA already has Do these support synthetic genetics? Slide 19 Yes. 3 hydrogen bonds are better than 2 hydrogen bonds, which are better than 1 hydrogen bond Thomas Battersby, C. Ronald Geyer (2003) Structure 11, 1485-1498. Size complementary > large rungs or short rungs Battersby- Geyer plot; 2 hydrogen bonds OK; one is not. No base at all Slide 20 Synthetic genes encode synthetic proteins with 21 amino acids Bain, et al. (1992) Ribosome-mediated incorporation of non-standard amino acids into a peptide through expansion of the genetic code. Nature 356, 537-539 Yes, we can. It is just as simple as Watson-Crick said. iodotyrosine C-U-isoG isoC-A-G Slide 21 minor groove major groove Get natural polymerases to accept synthetic genetics Standard nucleobases present unshared electron pairs to the minor groove. These are the only common pharmacophore. Many nucleobases in our genetic system lack these pairs. Peut notre biologie synthetique faire levolution Darwinienne? Slide 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. Slide 23 Time is short, so let us consider the Darwinian potential of just one synthetic nucleobase pair minor groove major groove Z P Can G, C, T, Z, A, P support Darwinian evolution like G, C, T, A in the laboratory? Slide 24 standard template synthetic template standardsynthetic standard primer no kids standard kids standard kids synthetic kids Yes it can. GACTZAP directs the formation of CTGTPAZ children using synthetic polymerases Zunyi Yang GACTZAP Slide 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 Slide 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. Slide 27 5-CTAGGACGACGGACTGC 5-CTAGGACGACGGACTGCCCATGGGAGACCGCGGTGGGCCCGGCCGGGTACCATCGATACGCGTTGCGATCGCTCCTTCCTG-3 CGCTAGCGAGGAAGGAC-5 GTGGGCCCGG CACCCGGGCC Apa 1 restriction site Apa site lost as G or C is replaced by P or C after many PCR cycles. PCR cleavage product no longer seen. GTGPGCZCGG CACZCGPGCC G:C can mutate to P:Z via transitions Can P:Z convert back to G:C? Yes it can; multiple PCR cycles used to detect infrequent mutation Slide 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. Slide 29 A synthetic chemical system capable of Darwinian evolution with 6-letter "RNA" Is this synthetic life? A self-sustaining chemical system capable of Darwinian evolution? ET Slide 30 Raising the bar Many theories of life are in common use Gene theory Can we get our synthetic genetic system into a cell? Evolution theory Cell theory Slide 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. Slide 32 Synthetic cells (water cells in hydrocarbon solvent holding) synthetic genetic system (GACTZAP) capable of Darwinian evolution. Ca. 2 microns Artificial genetic system placed in water droplet cells to amplify (by 6- letter GACTZAP-PCR) a synthetic polymerase that accepts synthetic genetic systems. Ryan Shaw Roberto Laos Slide 33 Amplification (with replicable mutation) in artificial cells of GACTZAP system 13th generation GACTZAP children Now is this a synthetic biology? Slide 34 Life: A self-sustaining chemical system capable of Darwinian evolution This is not self-sustaining Slide 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. Slide 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. Nature 356, 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. Slide 37 Orthogonality allows analyte recognition distinct from movement of the complex, signaling, etc. 8 molecules/mL The branched DNA architecture measures viral load 400,000 patients last year; $100 million product Personalizing healthcare with A EGIS Slide 38 A general theory of life as a universal? No, but we are constraining the black box. Slide 39 Slide 40 Potential hazards? Risk elements. Standard biochemistry (parasitism) Self-sustenance (otherwise tied to lab food) Ability to evolve (otherwise hazard is stationary)