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)