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FOCUS ON SYNTHETIC commentary

A synthetic approach to James Attwater & Philipp Holliger

Synthetic biology seeks to probe fundamental aspects of biological form and by construction (resynthesis) rather than deconstruction (analysis). Here we discuss how such an approach could be applied to assemble synthetic quasibiological systems able to replicate and evolve, illuminating universal properties of and the search for its origins.

Four billion years of on Earth new metabolites3 or carry out computa- activity within model membranes13–16 but has yielded a packed with tion4. Advances in DNA solid-phase syn- cannot yet regenerate the or trans- exquisitely optimized molecular compo- thesis and assembly have culminated in the lation factors needed to support replication. nents that enable life to evolve and thrive. first synthesis of a of a unicellular To achieve this, a minimal would need Traditionally, molecular seek to organism5. Such can explore to encapsulate a sufficient set of cellular analyze their function and interconnect- the arrangement and context of in replication, and edness in order to better understand the on an unprecedented scale, open- machinery components together with their of living systems. Yet ultimately these ing up biotechnological opportunities encoding genome (Fig. 1b). This could components and systems are all representa- through our growing mastery over the cen- define the core set of natural components tives of a single interrelated biology deriving tral dogma (Fig. 1a). required for propagation and expression of from the last universal common This naturally leads to questions as to how genetic information, establishing (LUCA), a breakthrough (or set far we can stray beyond life’s present molecu- and providing a stable chassis for bioengi- of genes1) already reminiscent of modern lar paradigms. New chemical functionalities neering17. However, although clearly much . In the absence of the discov- such as unnatural amino acids and carbohy- simpler than any extant cellular organism, ery of other biologies (on Earth or beyond2) drates can be added to both unicellular and such a semisynthetic cell is still estimated 6,7

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature not related to LUCA, it is challenging to multicellular , and in one case, to require between 100 and 150 genes establish universal principles and laws of one of the bases of the genome has been expressed from a >100-kilobase genome. biology. To paraphrase Carl Sagan: our biol- entirely replaced by an unnatural analog8. Is such complexity a prerequisite of life, or ogy, although amazingly diverse, is ‘provin- engineering9 or global recod- is it merely a consequence of using compo- npg cial’—in contrast, for example, to the laws of ing10 allows reassignment of the genetic nents firmly rooted in extant biology? In physics, whose generality can be observed code, and expansion of the genetic alphabet other words, is life more complicated than throughout the cosmos. However, by going itself might be possible11. The challenge of all it needs to be? beyond the simple analysis and deconstruc- these approaches in supporting stable aug- An appreciation of the minimal require- tion of extant life and building ‘new biolo- mentation is to fit the new functionalities ments of life as a self-perpetuating phenom- gies’ through modification, reconstruction into preexisting biological networks through enon may emerge from even more radical and de novo construction, either replacement or the establishment of strategies. Constructing systems exhibiting promises a fresh perspective and ultimately orthogonal pathways or chemistries12. Such key traits of life, using synthetic compo- a better understanding of the unifying prin- efforts promise to reveal the limits (if any) to nents not found in biology, will challenge ciples of living systems. life’s tolerance of expansion into new chemi- our preconceptions of what constitutes life. Our ability to modify existing biol- cal and informational space. Abandoning the superbly refined molecular ogy has grown rapidly since the advent of These approaches, however, remain machinery of extant biology may seem an . Biological tools allow defined by and embedded in preexisting inauspicious step to take but may prove both the , and modification of biology and thus are likely to reflect its instructive and liberating. Much of the cen- genes at will, enabling the rewiring of path- constraints. The underlying principles of tral complexity of biology arises from the ways to generate new , produce our biology may be more clearly exposed need to support and integrate three sepa- by a fully synthetic approach: constructing rate biopolymer systems: , RNA and simple cells comprising a limited number of DNA. If we can step back from this para- Philipp Holliger and James Attwater are at the essential components derived from biology. digm of cooperating biopolymers, simple, Council Laboratory of , Cambridge, UK. A number of strategies have recapitulated more streamlined forms of life might be e-mail: [email protected] transcription, translation and metabolic feasible.

nature methods | VOL.11 NO.5 | MAY 2014 | 495 commentary FOCUS ON SYNTHETIC BIOLOGY

26 abGenome: ~1 Mbp ~100 kbp, 150 genes c 2 genes template ), demonstrating the synthetic potential of such . One might ask if relatively complicated Synthetic DNA DNA DNA ‘replicases’ are needed to implement simple Whole- forms of self-replication and evolution. genome RNA RNA RNA assembly Work on replication of RNA using a purely

In silico Protein non-enzymatic, chemical strategy was 27 initiated in the 1970s and has seen sig- nificant recent progress through the use of non-native linkage and base chemistries28, stepwise removal of hydrolysis products29 and discovery of cofactors enabling some Figure 1 | Synthetic biological systems of increasing simplicity. (a) DNA as software: a bacterium reprogrammed by transformation with a synthetic genome5. Changes are transmitted through the template-directed RNA synthesis inside a 30 central dogma, implementing a new by influencing informational systems, metabolism and model . Non-enzymatic replica- the (black arrows represent information transfer; orange arrows show catalysis). tion systems, though, remain limited in rate (b) A proposed minimal heterotrophic cell, lacking metabolism and comprising components dedicated and fidelity and, consequently, the genome solely to maintaining DNA replication, transcription and translation17. (c) A putative maximally simple size that they would support. RNA organism: a synthetic protocell founded on a single biopolymer (RNA) inhabiting a dynamic Furthermore, both non-enzymatic and membranous vesicle20. An RNA replicase copies both itself and a metabolic (synthase) that provides building blocks (by activating or trapping permeable precursors). The synthase is dispensable enzymatic approaches must still overcome a if activated building blocks are membrane permeable; alternatively, the replicase is dispensable if set of formidable challenges before a robust activated building blocks are capable of non-enzymatic RNA synthesis. replicating and evolving RNA system can be achieved31. Among the most pressing issues are the inhibitory effects of template One might begin by asking which critical able to evolve. To access and explore such a secondary and the related need requirements these must satisfy. A unique scenario, synthetic strategies must gener- for strand separation. These require the property of life is its capacity for self- ate and integrate novel components: for definition of either enzymatic activities or replication and open-ended improvement, instance, a replicating RNA genome. physicochemical regimes able to unfold through evolution enabled by a heritable Can self-replication be implemented template RNA duplex structures while sup- molecular memory. In the search for plau- using RNA as the sole informational com- porting RNA folding and base-pairing for sible molecular strategies to implement ponent? Dramatic exponential growth has synthesis. A counterintuitive yet elegant this capability in a synthetic system, one already been observed for a pair of engi- concept that has emerged from recent work conceptually attractive approach is to take neered cross-catalytic RNA ligases wherein is that replication (as well as inspiration from research into abiogenesis, each ribozyme catalyzes the other’s assembly assembly and division of protocellular the of life on the early Earth, from two pieces, allowing emergence of new membranes32) may be aided by a degree of 22 33

Nature America, Inc. All rights reserved. America, Inc. © 201 4 Nature which provides useful constraints on both ligase phenotypes through recombination . ‘helpful heterogeneity’ (Fig. 2). Chemical chemical and biophysical parameters18,19. Cooperative networks of self-assembly have heterogeneity in the form of sporadic incor- The generation of a simple evolving system also been described developing from pools poration of transient 2′ modifications34 or guided by these parameters in turn would of diversified variants of the Azoarcus self- alternative RNA backbone linkages (2′-5′ vs. npg have clear implications for the plausibility splicing intron23. Such life-like ‘growth’ of 3′-5′)35 would destabilize product duplex- of proposed transitions from inanimate to raises hopes for RNA replication, but es. Compositional heterogeneity in the animate matter at the origin of life; thus, the evolutionary potential of these systems substrate pool—moving beyond the para- advances in either field might drive prog- remains circumscribed by the sequence digm of controlled monomer extensions to ress in the other. constraints of the prefabricated RNA com- dynamic multisubstrate systems, as initially Extant biology is thought to have been ponents required for assembly. explored in the Azoarcus system23—could preceded by a primordial biology lacking The capacity of RNA systems to evolve yield helpful emergent properties through DNA and encoded proteins, where RNAs freely could be enhanced by harnessing oligomer interactions33. Indeed, such ‘sys- served as both genetic material and meta- near-complete information transfer from tems ’ approaches have driven bolic . Inspired by this ‘RNA world’ a template via base-pairing, enabling RNA fundamental advances in the prebiotic syn- conjecture, a simple synthetic ‘protocell’ has self-replication from monomer build- thesis of RNA building blocks36 and may be been proposed comprising a self-replicating ing blocks. Perhaps the most remarkable the key to enabling RNA self-replication. RNA system (Fig. 1c) that also promotes replicative activity of this kind is an RNA If efficient RNA replication were to be growth and division of a dynamic mem- polymerase ribozyme evolved from realized in a compliant membrane, a simple brane envelope20, with faster replicating a random RNA sequence pool24. Initially protocellular system could be constructed driving protocell division and ran- able to copy up to 14 , it has from synthetic components that could dom assortment heredity not dissimilar to undergone numerous cycles of engineering exhibit many life-like properties such as L-form bacterial growth21. This concept and evolution to yield variants able to syn- growth, division, heredity and evolution20. provides a framework to address a funda- thesize other enzymatically active RNAs25 Great progress has been made in the past mental parameter of life: the minimal level or RNAs longer than the polymerase ribo- decade in the study of such potential mem- of complexity needed for a system to be zyme itself (>200 nucleotides on a favorable branous with regard to protocell

496 | VOL.11 NO.5 | MAY 2014 | nature methods FOCUS ON SYNTHETIC BIOLOGY commentary

B makeup compatible with both essential O 9,46 O nucleic acid and protein function , and O O emergent properties of simple protocellu- PO 32 22,23 –O lar membranes , replication systems O Bilayer and replicases48. Indeed, in vitro evolution membrane 2′-acetyl RNA methodologies developed to isolate synthet- RNA ic components, together with the advent of B B high-throughput technologies, O O promise a better understanding of evolution O OH HO O itself, offering unprecedented insight into PO PO –O –O adaptive landscapes, routes to the O O emergence of functional phenotypes, con- 3′-5′ RNA 2′-5′ RNA nectedness of adaptive peaks, and the roles of recombination and drift49,50. Compositional heterogeneity Chemical heterogeneity The RNA world conjecture represents an Figure 2 | Helpful heterogeneity in an RNA-based protocell. Clockwise from top left: attractive framework for the assembly of a incorporation can facilitate –based membrane growth and competition33. 2’-acetylation of synthetic cell owing to RNA’s tractability to ribonucleotide building blocks can aid RNA synthesis by enhancing regiospecificity and yields product evolutionary refinement and its likely rel- duplexes that are easier to melt34. This is also promoted by sporadic incorporation of 2’-5’ linkages, evance to early biology. However, success and with more such linkages could serve as better templates35. Initiation of RNA synthesis in the assembly of a simple synthetic system in both directions at multiple points on a template, instead of from a single primer, might yield faster, with life-like properties such as heterotro- 33 more robust replication . phic growth, self-replication, heredity and evolution will be instrumental in revealing growth and division32, permeability, stabil- discourages spread of inactive and parasitic fundamental concepts of biology and abio- ity37 and compatibility with non-enzymatic molecules39. However, a range of alternative genesis, regardless of its precise molecular nucleic acid replication30. An RNA rep- physical compartmentalization strategies architecture. licase in a protocell could also transcribe (e.g., minerals40, ice41 and coacervates42) 25 ACKNOWLEDGMENTS other ribozymes , facilitating a primitive might fulfill this minimal ‘container’ func- This work was supported by a Homerton College, metabolism—such as synthesis of RNA tion even in the absence of membranes. Cambridge, Junior Research Fellowship (J.A.) and by building blocks38—that in turn promotes Indeed, although membranous protocells the Medical Research Council (programme number RNA replication (Fig. 1c). Such a protocell are attractive conceptually owing to their U105178804). would provide a powerful framework for analogy with extant biology, on occasion COMPETING FINANCIAL INTERESTS evaluation and evolutionary optimization some organisms can cope without mem- The authors declare no competing financial interests. of system-level properties of all interacting branes43. 1. Woese, C. Proc. Natl. Acad. Sci. USA 95, 6854–

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