oo
o
o
O
CHH
H C NHO
C C
O
HHH
Paleogenetics
A path to the simplest first life
C C C N
Eucarya
interstellar organics
Life as a universal
HO
CNH2H
backwardsin time tosimplerlife
forwards from chemistry
Archaea Bacteria
infer ancestral life forms; resurrect for laboratory study
PrebioticChemistry
Synthetic biologyBaross, Benner et al. (2007) Natl. Res. Councl.Limits toLife
Ricardo et al. (2004) Science 303, 196
Benner et al. (2007) Adv. Enzymol. Mol. Biol. Protein Evol. 75, 1-132
Benner (2004) Acc. Chem. Res. 37, 784-797
N
N
OR
RN
N N
NNN
O
RH
H
H
H
H
N N
N
RN
H H
N N
N NO
O
R
H
N
NR
RO
N N
N NO
N
R
H H
H
H
H
HH
N
OR
RN
N N
N NN
O
R
H
H
H
H
H
Construct life in the lab
Search cosmos
discover alien life
independent genesis?
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 InstituteLooking for life on Titan, a moon of Saturn
ESA-NASA Cassini-Huygens mission• Rich in organics.• Subsurface liquid water-ammonia.
Inspiration: Perhaps Titan’s 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.
oo
o
o
O
CHH
H C NHO
C C
O
HHH
Paleogenetics
A path to the simplest first life
C C C N
Eucarya
interstellar organics
Life as a universal
HO
CNH2H
backwardsin time tosimplerlife
forwards from chemistry
Archaea Bacteria
infer ancestral life forms; resurrect for laboratory study
PrebioticChemistry
Synthetic biologyBaross, Benner et al. (2007) Natl. Res. Councl.Limits toLife
Ricardo et al. (2004) Science 303, 196
Benner et al. (2007) Adv. Enzymol. Mol. Biol. Protein Evol. 75, 1-132
Benner (2004) Acc. Chem. Res. 37, 784-797
N
N
OR
RN
N N
NNN
O
RH
H
H
H
H
N N
N
RN
H H
N N
N NO
O
R
H
N
NR
RO
N N
N NO
N
R
H H
H
H
H
HH
N
OR
RN
N N
N NN
O
R
H
H
H
H
H
Construct life in the lab
Search cosmos
discover alien life
independent genesis?
Qu’est-ce que c’est “la vie universelle”? Quatres rues
xx x x x xxx x x x xx
>3000 B.C.
Proto-Germanic
MiddleEnglish
Old English
Old HighGerman
Gothicsnaiws
snow
snaw
sneoChurchSlavonic
snegu
Old Irish
Proto-Indoeuropean
Old Norse
Greekφιν
.Old Fr
Latin
OldPrussian
snechte
œsn rnoif
, nix nivus
α
*snigw -h
xx
Slavic Germanic Romance Celtic
Reconstruction says something about the Proto-IndoeuropeansThey 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)
The historical past captured in sequences
Paleogenetics:Use recombinant DNA technology to bring ancient proteins back to life for studyLinus Pauling, Emile Zuckerkandl
Resurrect ancestral protein sequences
10 20 . | . |ox KETAAAKFERQHMDSSTSAA || ||||||||||||||| |sheep KESAAAKFERQHMDSSTSSAcamel SETAAEKFERQHMDSYSSSSAncestor KERAAAKFERQHMDSSTSSA
closer homologs
more distant homologs
present
past
both reconstructions require one mutation
SerThr
Ser?
position 3
Thr?
Thr Ser
Thr
Thr Ser Thr
add outgroup; best reconstruction clear
orThrposition 1
Lys
Lys Lys
1 reconstruction no mutation
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.
“Old school?” synthetic biologists “explore basic questions” !?
2004. I discovered that I had helped found the field of synthetic biology 20 years earlier.
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
Mars climate orbiter
But synthesis provides more…Human instinct. If an observation contradicts a theory, discard the observation.Mars climate orbiterGuidance 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 mustsolve 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.
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.
OO
OO
P
O O
H
P
O
P
O O
O
NN
NN
N ON
NN OOO N
O
N
OOP
O
N
O
N
O
O
N
OPO
N
XXX
N
N
O OO N
ON
O
O
O
N
OOOP
N
O O
O
NN
N ON
N
HH
HH
H
H
P
O
HH
H
H
H H
H
O
NN
N N
O
N
OO
X
OO
P
O O
H
H
O
O
R
R
-
base pairs
charged phosphate backbone (!?)
T/U
G
T/U
G
sugar as scaffold (!?)
C
AC
- - -
----
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???
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 challengeSynthesize a charge-neutral analog of DNA
O
O
N
N
N
N
NH
O
NH2
N
NH2
H
N
N
O
NH2
ONO
P
OP
OO
O
ONO
P
O
O
O
O
O
O
O
N
N
N
N
NH
O
NH2
N
NH2
H
N
N
O
NH2
ON
S
S
O
ON
S
O
O
O
O
Oligosulfone
-
-
-
NaturalDNA
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.
0 10 20 30 40 50 60 70 80 90 100
100
200
temperature (0°C)
Lots of uncharged linker = oligo folds
ASO2USO2GSO2GSO2USO2CSO2ASO2U
An oligo that folds does not bind to its complement
Clemens RichertUniv. Stuttgart
Melting curve shows presence of folded form
Why genetic systems must be polyelectrolytes in water
neutral polymer
Radius = length
(1/2)
polyanionic polymer
Radius = length
>> (1/2)
-
-
-
-
-
-
- -
-
-
-
-
-
-
-
-
-
-
-
-
-
-
--
-
-
-
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
O
O
N
N
N
N
NH
O
NH2
N
NH2
H
N
N
O
NH2
ONO
P
OP
OO
O
ONO
P
O
O
O
O
O
O
O
N
N
N
N
NH
O
NH2
N
NH2
H
N
N
O
NH2
ON
S
S
O
ON
S
O
O
O
O
-
-
-
strand-strand interactions canbe anywhere
Such a universal helps search for life in the cosmos
Not sufficient that a molecular system direct its reproductionThe reproduction must be allowed to include errorsThose errors must themselves be replicable Not like crystallization, with defects in the crystal structure “cured”
plasmonresonancedetection
+ + + + + + + + + +
regularly spacedcharges on surface
biopolymer with complementary charges
- - - - - - - - -
SO4=
Br-
Cl-
HPO4=
Repeating charge “easy” to detect in situ.
A polyelectrolyte is a universal feature of genetic polymers in waterCharges 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
oo
o
o
O
CHH
H C NHO
C C
O
HHH
Paleogenetics
A path to the simplest first life
C C C N
Eucarya
interstellar organics
Life as a universal
HO
CNH2H
backwardsin time tosimplerlife
forwards from chemistry
Archaea Bacteria
infer ancestral life forms; resurrect for laboratory study
PrebioticChemistry
Synthetic biologyBaross, Benner et al. (2007) Natl. Res. Councl.Limits toLife
Ricardo et al. (2004) Science 303, 196
Benner et al. (2007) Adv. Enzymol. Mol. Biol. Protein Evol. 75, 1-132
Benner (2004) Acc. Chem. Res. 37, 784-797
N
N
OR
RN
N N
NNN
O
RH
H
H
H
H
N N
N
RN
H H
N N
N NO
O
R
H
N
NR
RO
N N
N NO
N
R
H H
H
H
H
HH
N
OR
RN
N N
N NN
O
R
H
H
H
H
H
Construct life in the lab
Search cosmos
discover alien life
independent genesis?
Synthese: “la vie universelle” a dessein?
Nucleobases as interchangeable parts
CC
HC
N C
N
O
O
C C
C
NC
N
N
N
H
H
H
N
CHN
R
R
H
Acceptor
Donor
Acceptor
Donor
Acceptor
Donor
H
H3C
CHC
HC
N C
N
N
O
C C
C
NC
N
O
N
H
H
H
N
CHN
R
R
H
H
Donor
Acceptor
Acceptor
Acceptor
Donor
Donor
cytosine guanine
thymine aminoadenine
Chemical reasons for Watson-Crick complementarity
Size:Small pairs with large
Hydrogen bonding:Donors pair to acceptors
Shuffling donor/acceptors gives orthogonal pairs
... including the two that terrean DNA already has
CHC
HCN
C
N:
CC
NC
N
CN
HCN
:O RH
N
R
H C
O:H
NH
C
HCN
C
H
N
CC
:NC
N
CN
HCN
N RH
O:
R
H
H
HN
HO:
CH3
aminoadenine
thymine
guanine
cytosine
pyADA
puDADpuADD
pyDAA
Acceptor
Donor
Donor
Donor
Acceptor
Donor
Acceptor
Donor
Acceptor
Donor
Acceptor
Acceptor
.. ..
Do these support synthetic genetics?
CC
HCN
C
N:
CC
NC
N
CN
CHN
N RH
O:
R
H
C
H:O
H
N
HCC
C H
N:
NC
NC
N
CN
CHHC
:O RH
N
R
H
H
H
:O
N
H
CC
HCC
C
N
NC
:NC
N
CN
CHHC
:O RH
N
R
H
C
O:H
NH
C
HCC
C
H
N
NC
:NC
N
CN
CHHC
HR
N
O:
R
H
H:O
H
H
N
N
R
R
R
pyAAD
puDDA
Acceptor
Acceptor
Donor
Donor
Donor
Acceptor
pyDDA
puAAD
pyADD
puDAA
Acceptor
Acceptor
Donor
Donor
Acceptor
Acceptor
Acceptor
Donor
Acceptor
Acceptor
Donor
Donor
Donor
Donor
Acceptor
Donor
Acceptor
DonorpyDAD
puADA
Yes. 3 hydrogen bonds are better than 2 hydrogen bonds, which are better than 1 hydrogen bond
Thomas Battersby,C. RonaldGeyer (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
Synthetic genes encode synthetic proteins with 21 amino acids
ONH3
+
O
AI
OH
GG
GG G G
G
G GG
G
G
dC
G
GGG
.
.
.
.
.
.
.
GG
GG.
.
.
.G
GG
.
U
U
ACA C
A
UU
AA C
U ACU U C
ACU U C
C
UUCUCC
AA
U
U
C
A
U
C
AA
NU
U
C
UC
C
A
C
UC
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
O
O
O
O
P
O
O
H
OO
O
P
O
OOO
P
O
O
N
N
N
O
N
N
OO
NO
N
O
O
O
P
O
N
O
O
O
O
OO
P
O
O
O
OH
OH
OH
N
N
O
O
O
N
P
HO
HO
HO
N
N
N
NN
N N
H
H
H
H
H
N
H
H
H
O
N
N
N
N
HH
H
H
65th anticodon
-
-
iso-C
G
iso-G
65th codon
-
-
-
-
A
U
C
Yes, we can. It is just as simple as Watson-Crick said.
iodotyrosine
C-U-isoG
isoC-A-G
minor groove
major groove
N
N
O
O
N
N
N
N
H
H
H
N
N
R
R
H
unshared pair of electrons
H
N
N
N
O
N
N
O
N
H
H
H
N
N
R
R
H
H
unshared pair of electrons
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 l’evolution Darwinienne?
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.
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?
N
N
O
N
N
N
O
N
H
H
H
N
R
R
H
unshared pair of electrons
HpyDDA
puAADO2N
standard template
standard template
synthetic template
standard synthetic syntheticsynthetic standardstandardprimer
no kids
no kids
no kids
standardkids
standardkids
synthetic kids
Yes it can. GACTZAP directs the formation of CTGTPAZ children using synthetic polymerases
Zunyi Yang
CC
HCN
C
N
CC
:NC
N
CN
CHN
N RH
O:
R
H
H
H
O
CH3
pyADA
puDAD
Donor
Acceptor
Donor
Acceptor
Donor
Acceptor
CHC
HCN
C
N:
CC
NC
N
CN
CHN
:O RH
N
R
H
O:H
NH
H
puADD
pyDAA
Acceptor
Donor
Donor
Donor
Acceptor
Acceptor
..
HCC
HCC
C
N
NC
:NC
N
CN
CHHC
:O RH
N
R
O:H
NH
H
O2N
pyDDA
puAAD
Acceptor
Acceptor
Donor
Donor
Donor
Acceptor
GACTZAP
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.
Gtransversion
transversion
A T
C
Errors, of course, occur when copying natural DNA
This is also true with GACTZAP DNA
Gtransversion
transversion
A T
C
transversionZP
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.
5-CTAGGACGACGGACTGC5-CTAGGACGACGGACTGCCCATGGGAGACCGCGGTGGGCCCGGCCGGGTACCATCGATACGCGTTGCGATCGCTCCTTCCTG-3 CGCTAGCGAGGAAGGAC-5
GTGGGCCCGGCACCCGGGCC
Apa 1 restriction siteApa site lost as G or C is replaced by P or C after many PCR cycles.PCR cleavage product no longer seen.
GTGPGCZCGGCACZCGPGCC
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
p H d e p e n d e n c e w i t h T a q
0
1 0
2 0
3 0
4 0
5 0
6 0
p H
Retention of Z:P pair
p H 7 . 5
p H 7 . 8
p H 8 . 0
p H 8 . 5
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.
NN
N
R O
HH
NN
N
N
O
N
RH
HH
N
NO2N
R O
NN
NN
O
N
R
HH
HH
H
deprotonated dZ pairs with dG protonated dC pairs with dP
CC
HCN
C
N
CC
:NC
N
CN
CHN
N RH
O:
R
H
H
H
O
CH3
pyADA
puDAD
Donor
Acceptor
Donor
Acceptor
Donor
Acceptor
CHC
HCN
C
N:
CC
NC
N
CN
CHN
:O RH
N
R
H
O:H
NH
H
puADD
pyDAA
Acceptor
Donor
Donor
Donor
Acceptor
Acceptor
..
HCC
HCC
C
N
NC
:NC
N
CN
CHHC
:O RH
N
R
O:H
NH
H
O2N
pyDDA
puAAD
Acceptor
Acceptor
Donor
Donor
Donor
Acceptor
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
Raising the barMany theories of life are in common use
Gene theory
Can we get our synthetic genetic system into a cell?
Evolution theory Cell theory
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.
Synthetic cells (water cells in hydrocarbonsolvent 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 ShawRoberto Laos
Amplification (with replicable mutation) in artificial cells of GACTZAP system
13’th generation GACTZAP children
Now is this a synthetic biology?
Life: A self-sustaining chemical system capable of Darwinian evolution
This is not self-sustaining
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.
Many discoveriesRibosome synthesis of proteins with21 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, hepatitisB and C, respiratory viruses.
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 AEGIS
analyte DNAor RNA
capture strand
branched DNA
signal molecules
artificial nucleobases here improve signal-to-noise
CC
HCN
C
N:
CC
NC
N
CN
CHN
N RH
O:
R
H
H:O
H
H
N
R
pyAAD
puDDA
Acceptor
Acceptor
Donor
Donor
Donor
Acceptor
oo
o
o
O
CHH
H C NHO
C C
O
HHH
Paleogenetics
A path to the simplest first life
C C C N
Eucarya
interstellar organics
Life as a universal
HO
CNH2H
backwardsin time tosimplerlife
forwards from chemistry
Archaea Bacteria
infer ancestral life forms; resurrect for laboratory study
PrebioticChemistry
Synthetic biologyBaross, Benner et al. (2007) Natl. Res. Councl.Limits toLife
Ricardo et al. (2004) Science 303, 196
Benner et al. (2007) Adv. Enzymol. Mol. Biol. Protein Evol. 75, 1-132
Benner (2004) Acc. Chem. Res. 37, 784-797
N
N
OR
RN
N N
NNN
O
RH
H
H
H
H
N N
N
RN
H H
N N
N NO
O
R
H
N
NR
RO
N N
N NO
N
R
H H
H
H
H
HH
N
OR
RN
N N
N NN
O
R
H
H
H
H
H
Construct life in the lab
Search cosmos
discover alien life
independent genesis?
A general theory of life as a universal? No, but we are constraining the black box.
standard terran biochemistry(parasitism)
self sustaining
capable of evolving
not self sustaining(needs to be fed)
not standard terran biochemistry
not capable of evolving
RISK
Venter-Smith artificial cell
most engineeringsynthetic biol
SB Risk DiagramCopyright 2007Benner (2007) Synthetic BiologyFoundation Press
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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