Qu’est-ce que c’est “la vie universelle”?
Quatres rues pour l’investigation de quelque chose que
nous ne pouvons pas voir directement.
Prebiotic
Chemistry
HO
O H C C C N
C C
HH
H
Ricardo et al. (2004)
Science 303, 196
O H C N O
interstellar
organics
C
C
H
NH2
H
H
forwards
from
chemistry
Benner (2004) Acc.
Chem. Res. 37, 784-797
A path to the
simplest first life
discover alien life
independent genesis?
Search
cosmos
Life as a
universal
backwards
in time to
simpler
life
O
R
Construct
life in the lab
R
H
HN
NH
NR
N
N
N
H H
N
O
N
H
N
NR
N
N
N
O
O
NH
NH
R H
R H
H N
NR
H H O N NR
HN
N
O
N N
R
HN N
NH
N
N
O
O H H
N NH
R
R H
Synthetic biology
Baross, Benner et
al. (2007) Natl.
Res. Councl.
infer ancestral
Limits to
life forms; resurrect
Life
for laboratory study
Eucarya Archaea Bacteria Paleogenetics
Benner et al. (2007) Adv. Enzymol. Mol. Biol. Protein Evol. 75, 1-132
o
o
o
o
Exploration: NASA Astrobiology Institute
Looking for life on Titan, a moon of Saturn
ESA-NASA CassiniHuygens 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.
Qu’est-ce que c’est “la vie universelle”?
Quatres rues
Prebiotic
Chemistry
HO
O H C C C N
C C
HH
H
Ricardo et al. (2004)
Science 303, 196
O H C N O
interstellar
organics
C
C
H
NH2
H
H
forwards
from
chemistry
Benner (2004) Acc.
Chem. Res. 37, 784-797
A path to the
simplest first life
discover alien life
independent genesis?
Search
cosmos
Life as a
universal
backwards
in time to
simpler
life
O
R
Construct
life in the lab
R
H
HN
NH
NR
N
N
N
H H
N
O
N
H
N
NR
N
N
N
O
O
NH
NH
R H
R H
H N
NR
H H O N NR
HN
N
O
N N
R
HN N
NH
N
N
O
O H H
N NH
R
R H
Synthetic biology
Baross, Benner et
al. (2007) Natl.
Res. Councl.
infer ancestral
Limits to
life forms; resurrect
Life
for laboratory study
Eucarya Archaea Bacteria Paleogenetics
Benner et al. (2007) Adv. Enzymol. Mol. Biol. Protein Evol. 75, 1-132
o
o
o
o
The historical past captured in sequences
Proto-Indoeuropean >3000 B.C.
*snig wh-
nix, nivus
  
snaiws snaw
Old
Prussian
Church
Slavonic
x
x
Old High
German
x
x
x
x
snow
x
noif
snœr
Middle
English
Old Fr.
Old Norse
x
x
x
x
x
x
x
tu
ch sh
ea Iri
sn e h
ig enc
ne Fr
e ish
ev an
ni Sp
n
ve lia
ne Ita n
an
a
ne Rum
ia
Germanic
x
an
gi
ø we
sn Nor sh
i
ö ed
sn S w
e ish
sn Dan
sh
ow gl i
sn En
n
ee rma
hn e
sc sG
ga th.
ie Li
sn
h
gs tti s
ie Le
sn
an
eg ssi
sn Ru
g an
ije rbi
sn Se an
i
ih em
sn Boh
Slavic
Greek
Gothic Old English
sneo
Old Irish
Latin
Proto-Germanic
snegu
snechte
Romance
Celtic
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)
Resurrect ancestral protein sequences
past
evolutionary time
Lys Lys
position 3
Thr? or Ser?
Thr Ser
Thr
Thr
el
m
ca
p
ee
sh
both reconstructions
require one mutation
ox
p
ee
sh
present
1 reconstruction
no mutation
Thr Ser Thr
Thr Ser
ox
p
ee
sh
ox
p
ee
sh
ox
man
camel
sheep
ox
clos er homologs
position 1
Lys
add outgroup; best
reconstruction clear
more distant homologs
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
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.
2004. I discovered that I had
helped found the field of synthetic
biology 20 years earlier.
“Old school?” synthetic
biologists “explore basic
questions” !?
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.
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
Mars climate orbiter
uncharted ground where they must
solve unscripted problems in a way that does not allow selfdeception. 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.
Synthesis drives paradigm changes in ways analysis cannot.
What is a really big challenge for
synthetic biology?
Building artificial life.
Definition-theory of life: A selfsustaining 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.
Parts of Darwinian evolution appear very simple
sugar as scaffold (!?)
O O
O
P
O
O
X
O
O
O
A
N
H
N
O
H N
N
T/U
O
X
O
T/U
N
R
O
O
N
O O
-
N
G
O O
P
O
G
O
N
N
H
N
A
N
N
N
H
H
H
O
N
N
O
P
X
H
O
O
O
N
N
P
O
H
O
O O
H
N
R
O
-
H
H
N
N
O
H
H
O
N
N
O
H
O
C
O
P
N
N
N
O
O
O
N
base pairs
O
P
X
N
-
charged phosphate backbone (!?)
-
O
O
-
P
O O
N
N
O
H
N
H
C
O
O
-
O
P
O O
Big pairs with small. Hydrogen bond donors pair with hydrogen
bond acceptors. Scaffolding unimportant. Is it this simple???
Failed grand challenge
Synthesize a charge-neutral analog of DNA
O
O
-O
O
O
-
N
P
O
O
NH
N
NH2
N
NH2
P O
N
O
N
O
-O
Natural
DNA
P
O
O
N
N
O
N O
O
N
S
O
H
O
NH
N
NH2
N
NH2
O
N
NH2
O
O
O
N
S
N
O
N
H
NH2
O
O
N
N
S
O
N O
Oligosulfone
O
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
Lots of uncharged linker = oligo folds
200
Clemens Richert
Univ. Stuttgart
p
er
ce
n
t
h
y
p
er
ch
ro
m
ic
it
y
100
0
10
20
Melting curve shows
presence of folded form
30
40
50
60
70
80
90
100
temperature (0°C)
ASO2USO2GSO2GSO2USO2CSO2ASO2U
An oligo that folds does not bind to its complement
Why genetic systems must be polyelectrolytes
in water
O
O
1. Keeps DNA soluble in water.
O
O
N
N
NH
NH
S
-O P
O
2. Backbone-backbone coulombic
O
N N NH
N N NH
O
O
interactions force strand-strand
NH
NH
O
O
N
N
N
N
- P O
S
contacts to Watson-Crick edges
O
O
N N H
N N H
O
O
O
of the nucleobases (= rules).
NH
NH
3. Repeating charges discourages
O
O
O
-O P
S
N
N
O
folding; the “excluded volume”
O
N O
N O
O
O
strand-strand
effect (Paul Flory, Stanford)
interactions can
O
be anywhere
4. Repeating monopole dominates the
physical properties, allowing
neutral polymer
mutation to occur without changing
Radius = length (1/2)
the bulk properties of the molecule
(very unusual)
2
2
2
2
2
A backbone charge is essential
for Darwinian evolution
-
-
-
-
-
-
-
2
- polyanionic polymer
Radius = length >>(1/2)
-
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”.
biopolymer with
complementary charges
SO 4=
- - - - - - - - -
ClBr-
HPO4=
+ + + + + + + + + +
regularly spaced
charges on surface
plasmon
resonance
detection
Synthese: “la vie universelle” a dessein?
Prebiotic
Chemistry
HO
O H C C C N
C C
HH
H
Ricardo et al. (2004)
Science 303, 196
O H C N O
interstellar
organics
C
C
H
NH2
H
H
forwards
from
chemistry
Benner (2004) Acc.
Chem. Res. 37, 784-797
A path to the
simplest first life
discover alien life
independent genesis?
Search
cosmos
Life as a
universal
backwards
in time to
simpler
life
O
R
Construct
life in the lab
R
H
HN
NH
NR
N
N
N
H H
N
O
N
H
N
NR
N
N
N
O
O
NH
NH
R H
R H
H N
NR
H H O N NR
HN
N
O
N N
R
HN N
NH
N
N
O
O H H
N NH
R
R H
Synthetic biology
Baross, Benner et
al. (2007) Natl.
Res. Councl.
infer ancestral
Limits to
life forms; resurrect
Life
for laboratory study
Eucarya Archaea Bacteria Paleogenetics
Benner et al. (2007) Adv. Enzymol. Mol. Biol. Protein Evol. 75, 1-132
o
o
o
o
Nucleobases as interchangeable parts
Chemical reasons for Watson-Crick complementarity
H
N
Donor
HC
Acceptor
Acceptor
H
N
C
C
H
O
C
N
C
R
N
C
C
HC
N
O
H
CH
N
R
N
N
Donor
Donor
H
cytosine
Acceptor
guanine
H
Acceptor H3C
C
Donor
thymine
R
N
N
C
C
HC
N
N
Acceptor
H
O
N
H
C
C
C
O
C
H
N
N
CH
N
R
Donor
Acceptor
Donor
H
aminoadenine
Size:
Small pairs
with large
Hydrogen
bonding:
Donors pair to
acceptors
Shuffling donor/acceptors gives orthogonal pairs
N CH
N R
C
N
C
H
C
HC CH
N R
N
N
C
H
C
H
O:
Acceptor
Acceptor
R
C
C
HC
Donor
N
pyAAD
Donor
Acceptor
Donor
H
N
N:
C
R
C
Donor
Acceptor
puDDA
H
Acceptor
O:
Donor
C
R
R
Donor
C
HC
pyADD
H
HC CH
N R
N
:O
C
C
H
H
N
N
N
H
C
C
N:
N
:O
C
HC
H
puADA
C
N
pyDAD
Donor
N
:O
N
H
Acceptor
Donor
Donor
Donor
Acceptor
Acceptor
C
C
R
N
:N
H
C
Acceptor
N
puDAA Acceptor
:O
H
HC CH
N R
N
:O
C
C
H
H
N
N
:N
C
C
H
R
N
C
puAAD
N
H
H
C
HC
C
O:
pyDDA
H
N
H
Donor
Acceptor
Acceptor
Donor
R
... including the two that terrean DNA already has
Donor
Acceptor
Acceptor
pyDAA
H
N
H
:O
H
C
C
N
N:
HC
HC
C
H
C
N
R
O:
..
N
C
N
C
N
guani ne
R Acceptor
Donor
thymine
H
Acceptor
H
N
N
C
Donor
H
puADD
Donor CH3
Acceptor
pyADA
C
C
HC
N
H
:N
N
O:
..
R
Do these support synthetic genetics?
C
N
aminoadeni ne
N
C
H
C
N
H
C
C
O:
..
cytosine
H
C
R Donor
Acceptor
..
N
Donor
H
puDAD
Yes. 3 hydrogen bonds are better than 2 hydrogen
bonds, which are better than 1 hydrogen bond
Size complementary > large rungs or short rungs
Thomas
Battersby,
C. Ronald
Geyer (2003)
Structure 11,
1485-1498.
No base at all
BattersbyGeyer plot;
2 hydrogen
bonds OK;
one is not.
Synthetic genes encode synthetic proteins with 21
amino acids
iso-G
H
iodotyrosine
O
NH3
A O
dC
I
C
U
G C
OH
G C
G C
A U
G C
A U
A U
UGA
U
U
G
C
U
C
G
U A
G
C U C GA
U
C
G
A
G
C
G
UU
G
G A GC
C
A
A
G
GU
C G G GG
G C
A U
C U
C G
A
U
U
A
CUN
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
N
H
+
N
O P
- O
N
O
-
O
O
O
H
N
H
H
OH
O
O
N
N
O
N
H
-
65th codon
OH
O
O
O
N
P
N
O
O
O
HO
H
O
N
O
C
P
N
N
N
N
O
N
O
isoC-A-G
U
O
H
A
O
O
O
O
C-U-isoG
N
N
N
-
P
HO
P
O
65th anticodon
O
N
H
H
iso-C
O
O
N
O
HO
N
N
H
O
N
O
O
O
H
O
H
O
P
G
O
OH
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.
Peut notre biologie synthetique faire
l’evolution Darwinienne?
Get natural polymerases to accept synthetic genetics
major groove
H
H
O
H
N
N
H
N
N
N
N
R
N
O
H
R
unshared pair
of electrons
N
N
H
O
N
H
N
N
N
N
R
H
minor groove
N
O
H
R
unshared pair
of electrons
N
H
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.
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
major groove
pyDDA
O2N
H
N
H
O
puAAD
N
N
H
N
N
R
N
R
O
H
unshared pair
of electrons
N
H
Z
minor groove
P
Can “G, C, T, Z, A, P” support Darwinian evolution
like “G, C, T, A” in the laboratory?
Yes it can. GACTZAP directs the formation of
CTGTPAZ children using synthetic polymerases
standard
template
primer
standard
synthetic
standard
template
standard
synthetic
synthetic
template
standard
GACTZAP
synthetic
N CH
N
C
N
C
H
C
H
standard
kids
no
kids
standard
kids
no
kids
no
kids
synthetic
kids
Acceptor
Donor CH3
Acceptor
O:
C
C
HC
pyADA
Acceptor
Acceptor
C
R
H
Donor
N
N
HC
HC
pyDAA
N
C
N
H
H
:N
puDAD
O
N CH
N
C
:O
C
C
N
N:
R
Acceptor
Donor
H
H
C
C
N ..
R Donor
H
O:
C
N
N..
R Acceptor
Donor
Donor
H
puADD
..
HC CH
N R
N
:O
Acceptor
C
C
H
H
Donor
N
N
:N
Acceptor
Donor O2N
C
C
H
N
C
puAAD Donor
N
Acceptor HC
H
H
C
C
O:
pyDDA
R
Zunyi
Yang
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.
transversion
tr
an
si
ti
o
n
T
tr
an
si
ti
o
n
A
G
C
transversion
Errors, of course, occur when copying natural DNA
This is also true with GACTZAP DNA
transversion
tr
an
si
ti
o
n
T
tr
an
si
ti
o
n
A
G
C
tr
an
si
ti
o
n
tr
an
si
ti
o
n
transversion
P
Z
transversion
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.
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.
Can P:Z convert back to G:C?
Apa 1 restriction site
GTGGGCCCGG
CACCCGGGCC
GTGPGCZCGG
CACZCGPGCC
Yes it can. We know the mechanism of mutation
Mutation of P:Z back to G:C is pH dependent.
pH de pendence with Taq
Retention of Z:P pair
60
Mutations facilitated at high
and low pH, just like
natural DNA.
50
40
pH 7.5
pH 7.8
30
pH 8.0
20
pH 8.5
10
0
pH
O2N
H
N H
N
O
H
N H
N
N
R
O
N
N
H N
H N
H
deprotonated dZ pairs with dG
O
N H
R
N
N
R
N
N
O
H N
H
protonated dC pairs with dP
R
A synthetic chemical system capable
of Darwinian evolution with 6-letter "RNA"
N CH
N
C
N
C
H
C
H
Acceptor
O:
Donor CH3
Acceptor
C
C
N
HC
C
pyADA
Donor
Acceptor
Acceptor
N
R
H
HC
HC
pyDAA
N
C
N
H
H
:N
Acceptor
Donor
H
puDAD
O
N CH
N
C
:O
C
C
H
N
N:
C
C
N ..
R Donor
H
O:
C
N
N..
R Acceptor
Donor
Donor
H
puADD
..
R
HC CH
N R
N
:O
Acceptor
C
C
H
H
Donor
N
N
:N
Acceptor
Donor O2N
C
C
H
N
C
puAAD Donor
N
Acceptor HC
H
H
C
C
O:
pyDDA
ET
R
Is this synthetic life?
A self-sustaining chemical
system capable of
Darwinian evolution?
Raising the bar
Many theories of life are in common use
Gene theory
Evolution theory
Cell theory
Can we get our synthetic genetic system into a cell?
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 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 6letter GACTZAP-PCR)
a synthetic polymerase
that accepts synthetic
genetic systems.
Ca. 2 microns
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 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 resequencing of personal genomes.
Synthetic genetic systems annually
improve the care of some 400,000
patients infected with HIV, hepatitis
B and C, respiratory viruses.
Personalizing healthcare with AEGIS
The branched DNA architecture measures viral load
400,000 patients last year; $100 million product
signal
molecules
O:
Acceptor
Acceptor
branched
DNA
N CH
N R
C
N
C
H
C
H
Donor
R
C
HC
pyAAD
C
N
R
H
N:
C
N
C
:O
N
H
Donor
Donor
N
Acceptor
puDDA
H
su
pp
o
rt
so
li
d
capture strand
artificial nucleobases
here improve signal-to-noise
analyte DNA
or RNA
Orthogonality allows analyte recognition distinct from movement
of the complex, signaling, etc. 8 molecules/mL
A general theory of life as a universal?
No, but we are constraining the black box.
Prebiotic
Chemistry
HO
O H C C C N
C C
HH
H
Ricardo et al. (2004)
Science 303, 196
O H C N O
interstellar
organics
C
C
H
NH2
H
H
forwards
from
chemistry
Benner (2004) Acc.
Chem. Res. 37, 784-797
A path to the
simplest first life
discover alien life
independent genesis?
Search
cosmos
Life as a
universal
backwards
in time to
simpler
life
H
HN
O
R
NH
Construct
life in the lab
O
R
N
N
R
N
NH
H
H
HN
R
N
H H
N
O
N
H
N
O
N
NR
N
H
N
R
N
O
H
NH
O
O
NR
N
NH
H
R
HN
NH
H
NR
N
N
O
NR
N
N
H
N
N
H
R
Synthetic biology
Baross, Benner et
al. (2007) Natl.
Res. Councl.
infer ancestral
Limits to
life forms; resurrect
Life
for laboratory study
Eucarya Archaea Bacteria Paleogenetics
Benner et al. (2007) Adv. Enzymol. Mol. Biol. Protein Evol. 75, 1-132
o
o
o
o
Potential hazards? Risk elements.
Standard biochemistry (parasitism)
Self-sustenance (otherwise tied to lab food)
Ability to evolve (otherwise hazard is stationary)
SB Risk Diagram
Copyright 2007
Benner (2007) Synthetic Biology
Foundation Press
not self sustaining
(needs to be fed)
most engineering
synthetic biol
capable of
evolving
standard terran
biochemistry
(parasitism)
RISK
Benner-Sism our
synthetic biology
Venter-Smith
artificial cell
not capable
of evolving
self sustaining
not standard
terran biochemistry