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)