How far can biology be redesigned? Orthogonality as a biosafety tool Dr. Vitor B. Pinheiro Lecturer in Synthetic Biology Institute of Structural Molecular.

How far can biology be redesigned?

Orthogonality as a biosafety tool

Dr. Vitor B. PinheiroLecturer in Synthetic Biology

Institute of Structural Molecular Biology

BVL 2015 Challenges Symposium – 05.11.14UK Parliamentary copyright:

Reproduced with the permission of Parliament

Synthetic Biology – more than genetic engineering

• Traditional scientific approach:– Real systems are complex– Minimise complexity to make system tractable – top-down approach

• Synthetic Biology– Biology as engineering – bottom-up approach– Can complex systems be assembled from parts?– Can we redesign biological systems?

“What I cannot create, I cannot understand.”

Richard Feynman

Synthetic Biology – more than genetic engineering N

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adapted from de Lorenzo (2010) Bioessays10.1002/bies.201000099

Genetic engineering

Synthetic Biology

Design

Nat

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Synt

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Limitations of our existing tools

• Biological systems are the result of an evolutionary process– Probabilistic nature– Extensively but not thoroughly explored– Not conscious, not targeted, not designed– N = 1 problem

• So, why the systems we can observe have settled to their current arrangement?

• Is the natural setup an intrinsic limitation or a frozen accident?

• Can we try something different?

Nat

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Synt

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Xenobiology – unnatural biological systemsN

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adapted from de Lorenzo (2010) Bioessays10.1002/bies.201000099

Nat

ural

Synt

hetic

Genetic engineering

Synthetic Biology

Xenobiology

Design

Orthogonality

Information transfer in biology

• Information storage and propagation are essential for life

• Information only accessible from DNA and RNA in biological systems– The Central Dogma

• There is a change in information media between RNA and proteins– The Genetic code

DNA

RNA

Proteins

Information transfer in biology – The central dogma

• Information storage and propagation are essential for life

• Information only accessible from DNA and RNA in biological systems– The Central Dogma

• Propagation is viable because of the efficient and unambiguous base pairing

DNA

RNA

Proteins

Nucleic acid structure and function

Ribofuranose sugar

Nucleobase

Phosphate

-

O

OO

Base

DNA

O O

O

P

Nucleic acid structure and function

• All three chemical moieties contribute to nucleic acid chemical properties, structure and function

• Modification in any of the moieties generates a synthetic nucleic acid (XNA)– Range of compatibility with natural

systems– Range of chemical and biological

stability

Ribofuranose sugar

Nucleobase

Phosphate

-

O

OO

Base

DNA

O O

O

P

Xenobiotic nucleic acidsNucleobase substitutions Alternative base pairings

Alternative internucleotide linkagesAlternative sugar backbonePinheiro and Holliger (2014) Trends in Biotechnology

10.1016/j.tibtech.2014.03.010

Serum

h : 0 2 12 24 48

h : 0 24 48

DNA

HNA

DNA

HNA

DNase I

RNase I

BAL-31

No treatm

ent

Hexitol nucleic acids

• Can base pair with natural nucleic acids

• Not naturally synthesised• Increased chemical and biological

resistance– Still susceptible to oxidative and UV

damage

• Low toxicity• Poorly incorporated by natural

polymerases

-

O

OOBase

HNA

O O

O

P-

O

OO Base

DNA

O O

O

P

Expanding the central dogma

• Establish an XNA as a genetic material– Code (DNA XNA) and decode (XNA

DNA) information from a synthetic backbone using biocompatible routes

• Engineering DNA polymerases for XNA synthesis and reverse transcription

DNA

RNA

Proteins

XNA

From XNA to Xenobiology

• XNA genetic material is the first step towards an XNA episome– XNA genetic element stored

independently and maintained stably within an organism

• Make information in XNA inaccessible to general biology

• XNA maintenance in vivo depends on XNA information being required for cell survival– Link to metabolism

• Many viable topologies integrating XNA information to the cellular function

DNA

RNA

Proteins

XNA

From XNA to Xenobiology

XNA as a biosafety tool

• XNA as a dead man’s switch– Synthetic precursors– XNA traits are isolated from biology– Cell’s dependence on XNA limits its

ecological impact

• Containment failure depends on shortest evolutionary distance– Archaeal XNA RT was a single mutation

• Xenobiology systems are additive and can be systematically integrated to increase containment likelihood

DNA

RNA

Proteins

Metabolism

XNA

Information transfer in biology – The genetic code

• Information storage and propagation are essential for life

• Information only accessible from DNA and RNA in biological systems– The Central Dogma

• There is a change in information media between RNA and proteins– The Genetic code

DNA

RNA

Proteins

The genetic code

• Information in RNA is stored in nucleotides while information in proteins is stored in amino acids

The genetic code

• Genetic code emerged early in evolution

• Universal bar a handful of exceptions

• Can it be modified?

tRNA synthetase

Protein biosynthesis

RNA

Ribosome

tRNA

Nascentprotein

Synthetase orthogonality

• For the genetic code to be specific, aaRS must be orthogonal– Charge only its cognate amino

acid– Charge only its cognate tRNAs

• Multiple interactions ensure aaRS specificity– Amino acid and enzyme– tRNA and enzyme– Downstream interactions also

reported

X

X

Expanding the genetic code• Make use of unused rare codons

to introduce unnatural amino acids– E. coli only uses TAG as a stop

codon in 8% of its genes– It can be modified without

greatly affecting the bacteria– It can be removed by systematic

genomic editing Jackson et al. (2006) JACS10.1021/ja061099y

Rewriting the genetic code

STCC

STCC

SATG

• In vivo and in vitro approaches currently being developed– Multiple reassignments need to be carried out

to regenerate a viable code

Rewriting the genetic code

S

L

M

ETCC

CTG

ATG

GAG

ATT

… ATG TCC ATT CTG GAG TAG …

… MSILE

… ATG TCC ATT CTG GAG TAG …

… SMILE

I… SMILE

… TCC ATG ATT CTG GAG TAG …

Proteins

Metabolism

Rewriting the genetic code as a biosafety tool

• A genetically recoded organism (GRO) would not be able to exchange information with natural organisms– GRO Nature will not generate

viable proteins– Nature GRO will not generate

viable proteins

• A GRO auxotroph would be contained– Semantic firewall

DNA

RNA

Proteins

Metabolism

Notrepis

Xenobiology as a biosafety tool• Biosafety can be introduced in

multiple layers– Genetic firewall– Semantic firewall– Metabolic firewall

• These can be introduced in parallel

Proteins

Metabolism

DNA

RNA

XNA

Proteins

Metabolism

Notrepis

Human risk of Xenobiology

• ‘Xeno’-organisms are still biological systems– As a class, broadly similar risks and hazards as posed by GMOs

• Additional considerations required depending on modification, its implementation and purpose:– Input compounds – e.g. XNA precursors – chemical toxicity of

precursors, contaminants from precursor synthesis, abiotic precursor breakdown

– Intermediates and side reactions – e.g. unnatural amino acids – biological modification or misuse of input compounds, pathway intermediates, truncation products, biologically accessible bypass alternatives

– Output compounds – e.g. XNAs – biological activity or toxicity of intended products or molecules, and of their breakdown products by natural metabolic or environmental routes, co-option by cellular mechanisms

Acknowledgements

“The farther [from biology], the safer.”

Philippe Marliere

• Leticia Torres• Antje Krüger• Eszter Csibra• Pinheiro Group

• Phil Holliger• Piet Herdewijn• Philippe Marliere• Chris Cozens

• John Ward• Helen Hailes• Gary Lye• Jack Stilgoe

Environmental risk of GMOs

• GMOs pose two potential risks:– Ecological – the direct risk of a GMO interfering with other organisms

and altering ecological niches– Informational – the risk that at least part of the genetic information of

a GMO can spread to natural organisms, conferring an ecological advantage

• Reports of controlled release of GMOs for bioremediation suggest GMOs pose negligible risk to the environment– GMOs could not establish themselves in the tested niches

Environmental risk of GMOs

• Risk is only one element in looking at potential hazards

Sterling (2010) Nature10.1038/4681029a

Routes to safe bioprocessing

Bioprocessingfocus

Componentfocus

Chassisfocus

Naturalorganisms

Genome ablation

Cell-freechassis

Semantic firewallGenetic firewall

Metaboliccontainment

Auxotrophy

Inducible expressionsystems

1st containment(equipment design)

2nd containment(facility design)