Approaches to chemical synthetic biology

FEBS Letters 586 (2012) 2138–2145

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Review

Approaches to chemical synthetic biology Cristiano Chiarabelli 1, Pasquale Stano 1, Fabrizio Anella, Paolo Carrara, Pier Luigi Luisi ⇑ Biology Department, University of Roma Tre, Viale Guglielmo Marconi 446, 00146 Rome, Italy

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Article history: Received 4 January 2012 Accepted 10 January 2012 Available online 17 January 2012 Edited by Thomas Reiss and Wilhelm Just Keywords: Minimal Cell Lipid vesicle Random sequence RNA stability Protein folding

a b s t r a c t Synthetic biology is first represented in terms of two complementary aspects, the bio-engineering one, based on the genetic manipulation of extant microbial forms in order to obtain forms of life which do not exist in nature; and the chemical synthetic biology, an approach mostly based on chemical manipulation for the laboratory synthesis of biological structures that do not exist in nature. The paper is mostly devoted to shortly review chemical synthetic biology projects currently carried out in our laboratory. In particular, we describe: the minimal cell project, then the ‘‘Never Born Proteins’’ and lastly the Never Born RNAs. We describe and critically analyze the main results, emphasizing the possible relevance of chemical synthetic biology for the progress in basic science and biotechnology. Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction The notion of synthetic biology (SB) is by now well accredited in the experimental life sciences, and is generally seen as the modern and most ambitious development of bioengineering and biotechnology in general. The term ambitious is appropriate, as one of the declared aims of SB is the laboratory construction of alternative forms of life, namely forms of life that do not exist in nature. The term life is till now still restricted to microorganisms, and various aspects of this design have been described in the literature about microbes capable of eventually producing fuels and energy for mankind, to the various genomic modifications of extant microorganisms to enrich their functionality [1–3]. Those are all manipulations of extant bacteria and it is fair to say at this point that ambition has been restricted to this – namely manipulation of extant forms of life. The claim to have synthesized new life ‘‘simply’’ by replacing a synthetic genome with the original one [4] cannot be really called new synthetic life – it is a kind of organ transplant – as skillful and admirable as it is. All this has to do with genetic manipulation of one sort or another, an enterprise that for some is not exempt from bioethical problems – particularly if in a non-distant future we begin to play with multicellular and higher organisms. Together with this bioengineering-genetic aspect, SB has another aspect, which is more oriented towards basic science. This has to do with the question ‘‘why nature did this and not that’’ – why there is ⇑ Corresponding author. Fax: +39 0657336329. 1

E-mail address: [email protected] (P.L. Luisi). These two authors have contributed equally to this review.

ribose in nucleic acids and not glucose; why we have proteins with 20 amino acids and not with 10 or 15; why we have the proteins we have, and not all the others. . . And the beauty of SB is the possibility to answer, or at least, tackle these questions with experimental laboratory means: just making ‘‘that’’ and comparing it with ‘‘this’’, with what we have. In this way, we may discover – this is the claim – why nature went into one avenue and discarded the others. Thus, nucleic acids with pyranose instead of ribose have been synthesized by Eschenmoser and his group [5], proteins with a reduced alphabet of amino acids have been prepared and studied by Doi [6]; and nucleic acid alphabet different from the extant ones have been also studied by Benner and collaborators [7]. In this kind of SB, chemistry more than genetic manipulation is the main tool, and in fact the term ‘‘chemical synthetic biology’’ has been coined to describe this particular field [8–10]. The epistemology, which is at the basis of the discrimination between these two sub-fields of SB, has been also the subject of study [11]. The present article is devoted to chemical SB, and in particular to the two research areas which we have been developing over the latest years, namely the project ‘‘minimal cell’’/‘‘minimal life’’; and the project ‘‘Never Born Proteins’’/‘‘Never Born RNAs’’. Introduction to these terms is given below. For the other aspects of chemical SB the reader is referred to a recent volume [10]. 2. The minimal cell: from origin of life to synthetic biology The notion of ‘‘minimal’’ cell can be approached from several viewpoints, with many overlapping concepts. Traditionally, in the origin of life community the interest is focused on the first primitive living cell [12,13]. It is reasonable to assume that such

0014-5793/$36.00 Ó 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2012.01.014

C. Chiarabelli et al. / FEBS Letters 586 (2012) 2138–2145

primitive cells were much simpler than modern cells, whose complexity has been shaped by the evolution. However, despite their simplicity when compared with modern cells, primitive living cells were still able to perform basic functions that are characteristic of living organisms, such as self-maintenance and self-reproduction, and they were also able to evolve into more complex cells. Thinking to their structure and functions, therefore, primitive living cells were also ‘‘minimal’’ in the sense that they were able to act as living systems, but with a minimal molecular complexity. From this viewpoint, an important question would be: how do we study the origin of cellular life in the absence of the ‘‘object’’ of the investigation? Primitive minimal cells do not exist anymore and for this reason a possible experimental approach involves synthetic (constructive) biology as a theoretical and experimental framework to develop our understanding on the origin of cellular life. The next question becomes: is it possible to observe experimentally the emergence of minimal primitive cells in reconstructed pre-biotic conditions and using only allegedly pre-biotic functional compounds? Unfortunately, no one knows the answer, but the research activities associated to this question are flourishing recently [14– 17]. The major limitation factor is – in this context – the unavailability of prebiotically plausible molecular components that are required for establishing an autopoietic (self-producing) dynamics [18,19]. For example, a very well known scenario proposed for the origin of life foresees the onset of the so-called RNA world [20], where RNA molecules could act simultaneously as storage of genetic information (in their primary sequence) as well as catalysts for primitive metabolic transformations (i.e., the case of RNA enzymes or ribozymes). A possible molecular system based on ribozymes inside primitive cell-like compartments has been proposed [21], but it lies outside our current experimental possibilities, due to the fact that the kinds of ribozymes required for this assembly have not been discovered yet, and – moreover – it is not very clear how long RNA strands could originate from the building blocks. A scenario for the emergence of long and possibly catalytic biopolymers, such as polypeptides and polynucleotides, might involve a fragment condensation (ligation) pathway, possibly helped by spontaneously formed small catalysts (organocatalysts); see a short discussion in Box 1. An alternative approach to the construction of minimal cells in the laboratory involves the use of ‘‘modern’’ macromolecules such as DNA, RNA, and proteins which are experimentally available and their assembly in form of a synthetic compartment, such as a lipid vesicle (liposome). This approach, which has been started in our research group in Zurich in the early Nineties, is generally known as semi-synthetic. In addition to be quite attractive for its feasibility, modularity and possible development into future biotechnological applications (making it quite well fitting with the SB philosophy of parts, devices and systems [27]), the semi-synthetic approach is also very useful for answering the question of the emergence of cellular life. In this context, in fact, the focus is shifted from the chemical nature of primitive cell components (need to be prebiotically plausible) to the design and construction of a minimal selforganizing autopoietic network within cell-like compartments, by means of modern molecules. Clearly, semi-synthetic minimal cells cannot be a realistic model of primitive cells, nevertheless this approach might help to understand the critical features of the dynamic organization of cells, and to demonstrate experimentally that non-living molecules can produce a living entity as soon as the minimal requirements for life are met. 2.1. The technology of semi-synthetic minimal cells The general idea behind semi-synthetic minimal cells is illustrated in Fig. 1a. The minimal number of DNA genes, RNAs, enzymes, and low molecular-weight compounds are entrapped

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within a lipid vesicles so that a compartment is formed, which might display some of the basic features of living cells. Which functions need to be reconstructed in semi-synthetic minimal cells? Clearly, this depends on the goals of the experimenters, as they can go from ‘‘simple’’ functions like the synthesis of one enzyme, to more complex ones as sensing the environment and trigger an internal response, or even to the synchronized core-and-shell reproduction of the whole construct (achieved by self-producing all molecules required for the internalized network as well as the lipid molecules that constitute the membrane boundary). In order to be defined as ‘‘alive’’ a semi-synthetic minimal cell should function as an autonomous living cell, based on what is known as minimal genome. Based on comparative genomics, several studies have pointed out that the minimal number of genes for a viable cell can be derived by looking at the genomes of the smallest microorganisms, which are parasites or endosymbionts. For example, the small obligate parasite Mycoplasma genitalium has a genome of 517 genes with only 470 predicted coding regions [32]. It has been recently proposed that the minimal genome would consist in 208 genes [33], half of which involved in protein synthesis and processing. The remaining ones are for energy and metabolic processes, DNA and membrane synthesis. To date, the construction of a synthetic cell containing about two hundred genes is out of reach. Also, it is not useful to try to construct a very complex system without understanding how works a simpler one. Actually, since one of the major advantages of using a SB approach consists in ‘‘learning by constructing’’ (as opposite to learning by analyzing – i.e. dissecting – existing objects), it turns that the stepwise construction of semi-synthetic systems of increasing complexity will reveal what are the criticalities in assembling a cell-like dynamic systems with an autopoietic design, and how these were solved during the course of pre-biotic and biological evolution. The current state-of-the-art of minimal cell research deals with the synthesis of proteins inside lipid vesicles. This step is considered as crucial for the whole project on synthetic cells because it will permit the in situ production of functions for all aspects of the cell self-maintenance and self-reproduction. To date, several enzymes have been produced inside lipid vesicles. The experimental approach derives from the joining of cell-free technology and liposome technology. It is foreseeable that in the near future this match will be further enriched by microfluidic technology, due to the very recent advancements in the field of giant vesicle preparation in microfluidic devices [34–38]. The present experimental approach generally involves the formation of liposomes in a solution containing all components required to be encapsulated and therefore needed for establishing the biochemical reactivity inside a minimal cell. At this aim, cell extracts or reconstituted cell-free systems are used. In particular, the PURE system (Protein synthesis Using Recombinant Elements), introduced by the group of Ueda in 2001 [28,29], represents the standard SB ‘‘module’’ for synthesizing proteins inside liposomes, being composed by 36 purified enzymes/translation factors, purified ribosomes and tRNAs (see Fig. 1a caption). The PURE system is composed by the minimal number of molecular components required for the synthesis of a functional protein. Among the various liposome preparation methods [39], the most used one are the classical film hydration method [40], the hydration of freeze-dried liposomes [41], and the ethanol injection method [31]. These methods have been applied to the construction of semi-synthetic minimal cells capable of producing several proteins and enzymes, such as the green fluorescent protein (GFP) [40–42], T7 RNA polymerase [43], a-hemolysin [44], Qb-replicase [17], b-galactosidase [45], and two membrane-bound acyltransferases [46]. Thanks to these studies, in addition to the quantitation of a functional protein synthesis inside lipid vesicles [47], several

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Fig. 1. Semi-synthetic minimal cells. (a) The semi-synthetic approach consists in the construction of a cell-like compartment based on the encapsulation of DNA, ribosomes, tRNAs, enzymes and translation factors inside a lipid vesicle (liposome) at the aim of reconstructing a living cells containing the minimal number of molecular components. From the technological viewpoint, this approach merges liposome- and cell-free-technologies. The PURE system represents today the standard tool for the semi-synthetic approach. It includes 20 amino-acyl-tRNA synthases, 10 translation factors, T7 RNA polymerase, methionine-tRNA transformilase, 4 enzymes for the energy recycling, purified ribosomes and tRNAs, and low molecular weight compounds such as amino acids, NTPs, salts and buffer. The 36 protein factors are over-expressed and purified in His-tagged form without a loss in activity. [28,29]. Reproduced from reference [9] with permission from Elsevier. (b) Details of the droplet transfer method. (1) A solution of lipids in oil (hydrocarbons) is stratified over an aqueous solution (outer-solution: ‘‘o-solution’’). In a second step (2), a lipid-stabilized water-in-oil emulsion, prepared by emulsifying a small aliquot of an aqueous solution (inner-solution: ‘‘i-solution’’) in lipid containing oil (the lipid can be different than that one used for preparing the oil-water interface in (1)), is poured above the oil/water interface prepared in (1). Water droplets tend to fall down toward the interface due to their higher density when compared to oil. By crossing the lipid-containing oil/water interface (1) a droplet is covered by a second lipid layer, being transformed into a vesicle. The vesicles are generally collected by centrifugation. Reproduced from [30] with minor modifications, with permission of Elsevier. (c) Protein synthesis inside GVs produced by the droplet transfer method, as visualized by confocal laser scanning microscopy (left: bright field; center: fluorescence; right: overlay). Enhanced green fluorescent protein (EGFP)-encoding DNA was mixed with the PURE system as described elsewhere [31]. Giant vesicles were prepared as it follows: 5 lL of DNA/PURE system mixture (including 100 mM sucrose) were emulsified in 500 lL of 0.5 mM POPC in mineral oil (from Sigma, code M5904) by pipetting. The obtained w/o emulsion was gently poured above 500 lL of 0.5 mM POPC in mineral oil, previously stratified over 500 lL of PURE system buffer (including 100 mM glucose). Collected by centrifugation (2000 rpm, 10 min), giant vesicles were then incubated at 37 °C for 2 h.

basic features and properties have been investigated, such as the RNA synthesis on RNA template [17], the control of genetic expression by a two-stages cascade reaction [43], the formation of pore on the liposome membrane, allowing the entrance of fresh ‘‘nutrients’’ into the synthetic cell [44], the minimal size of a proteinproducing liposome [31], and the first attempts to construct a lipid-synthesizing synthetic cell [46,48]. The interested reader can find several more detailed technical discussions on the subject of compartmentalized biochemical reactions – also including some philosophical aspects – in some recent publications [22,49–51]. The most intriguing aspect of the construction of minimal cells deals with the process of self-assembly of an ordered structure (the compartment and the solutes encapsulated therein) from a mixture of separated compounds and lipids. This process corresponds practically (and conceptually) to the emergence of a ‘‘unit’’ that is distinct from the environment, by virtue of its self-bounding property. Thus, it is not an overstatement to emphasize that this is the core step of minimal cell research. From the technical viewpoint, however, it has been realized that this critical passage is also the bottleneck of the semi-synthetic technology. In fact, it is well

known that the spontaneous formation of lipid vesicles in a solution containing, for example, the transcription/translation kit, only a fraction of the resulting compartment is viable, due to the difficulty of co-entrapping all different molecular components inside the same vesicle. This becomes a very critical factor when small compartments are used. In a recent study, we pointed out that the GFP synthesis inside 200 nm vesicles can only be explained by assuming that the expected average number of encapsulated molecules is actually exceeded due to local concentration effects [31]. To test this hypothesis, a cryo-transmission electronmicroscopy study on the encapsulation of ferritin [52] and ribosomes [53] was carried out, bringing about the striking discovery that the entrapment of these macromolecular solutes into conventional vesicles does not obey to the expected Poisson statistics, but it is rather shaped as in a power law distribution, with several empty vesicles and few exceptionally filled ones. This phenomenon, which is currently under investigation in our laboratory (aiming at extending its validity to other proteins and to larger vesicles, as well as at understanding its generative mechanism via stochastic simulations), helps to explain how functional primitive cells,

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rich of internalized solutes, might have been originate from a diluted solution. Note that, puzzled by the anomalous entrapment hypothesis (the ‘‘conundrum’’ of solute encapsulation [31]), we exploited the SB approach to discover a novel physical mechanism underlying the origin of cell and its metabolism. However, the issue of solute encapsulation also triggered our interest toward novel vesicle preparation methods. Inspired by the 2003 report of Weitz and co-workers [54,55], we started a through investigation of the so-called ‘‘droplet transfer’’ method for producing giant vesicles (GVs). In particular we were interested in this method because it brings about – with good reproducibility – the facile entrapment of solutes into vesicles and bypasses the (yet very interesting) problems associated to the statistics of multi-molecular co-entrapment. The first step of this method consists in the formation of stable (lipid-stabilized) water-in-oil droplets, which contain the solute(s) of interest. Due to the presence of oil around the droplets all solutes result confined into the aqueous droplet volume. The core of the droplet transfer technology lies on the passage of solute(s)-filled, lipid-stabilized water-in-oil droplets through a water/oil interface containing an oriented lipid layer (ideally, a monolayer) (Fig. 1b). The water-in-oil droplet acquires a second lipid layer when crossing the water/oil interface and becomes a vesicle, still containing all solutes previously confined into the droplet. It is evident the advantage of this preparation method when compared to others. The ‘‘all inside’’ geometry, typical of water-in-oil droplets, is topologically transferred to form a vesicle already filled with the solute(s) of interest. Details of the preparation method will be given in a next publication, however it can be anticipated that we have optimized the GVs production by using different kinds of phospholipids, fatty acids, synthetic surfactants and mixture of them. By tuning the volumes of emulsion, emulsified phase, oil/water interface and outer (collecting) solution, we were able to produce in reproducible way about 5000 GVs/lL. Consequently, we have extended out semi-synthetic cell technology to the reproducible preparation of cationic and anionic GVs which entrap a wide range of water-soluble solutes. As an example of the application of this method, we encapsulated all components of the PURE system and a gfp-encoding plasmid into GVs, and followed the production of GFP (Fig. 1c). As already reported by other groups [44,56], this preparation method is really advantageous when a complex liposome bioreactor needs to be built. Interestingly, GVs prepared by the droplet transfer method can also be sized down by extrusion. In this respect, we plan to further exploit this method for constructing semi-synthetic minimal cells designed to perform specific functions such as lipid production, DNA or RNA replication, and especially core-and-shell reproduction. 2.2. Future developments As it has been shortly commented above, the state-of-the-art of semi-synthetic minimal cell research is now focused to acquiring the control of protein synthesis inside liposomes and to improve the liposome preparation methods. Quite probably, this ‘‘transition period’’, that follows the pioneering era, will precedes the next phase of full development of complex functions in semi-synthetic minimal cells. Which are the most attractive goals? Certainly, if semisynthetic minimal cells are considered as model of primitive cells, the most important goal will be the synchronized ‘‘core-and-shell’’ reproduction, namely, the coordinated replication of genetic material inside the cell, together with all proteins and the ribosomes, and the production of lipid molecules, so that the surface growth will allow the cell growth. From this state, it might follow a cell

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division (see the studies on self-reproduction of fatty acid vesicles [57,58]). An interesting study on the replication of RNA as genetic polymer inside lipid vesicles has been recently reported [17]. By noting that current approaches are based on feeding semisynthetic minimal cells with high-energy compounds like ATP, another future development might involve the reconstruction of molecular machines that are able to produce chemical energy directly inside the synthetic cell. In this direction, the recent works of Kuruma et al. [59,60] are worth of mention. Additional research directions go toward the use of semi-synthetic minimal cell as biotechnological tool, for example as micro-bioreactors for advanced drug delivery technologies [61], taking advantage of their potential sensoring abilities, triggered metabolic transformations, and target recognition. 3. The Never Born Biopolymers: from the proteins to the RNA Our work on Never Born Biopolymers lays within the framework of the novel and unconventional approach dubbed ‘‘chemical synthetic biology’’ [8–10], which, as already mentioned, is concerned with the synthesis of chemical structures such as proteins, nucleic acids, vesicular forms and other which do not exist in nature. This research specifically addresses the question of the design, synthesis and analysis of completely de novo biopolymers (proteins and RNAs), exploring the sequence space for novel biochemical structures that do not exist in nature, to be exploited as novel functional scaffolds for synthetic biology rather then try to answer scientific basic questions on origin of life [9,10]. In particular, the aim of developing completely de novo biopolymers (Never Born Proteins (NBPs) and Never Born RNAs (NB-RNAs)) could unveil an immense reservoir of potentialities that could tremendously enrich our knowledge and biotechnology. The rationale behind these projects relies on the observation that the number of natural proteins and RNAs on our Earth, although apparently large, is only an infinitesimal fraction of the possible ones. This means that there are an astronomically large number of proteins and RNAs that have never been sampled by natural evolution on Earth awaiting human exploration and exploitation [62,63]. 3.1. Never Born Proteins As already mentioned, the rationality behind this project relies on the observation that the number of natural proteins on our Earth, although apparently large, is only a tiny fraction of all the possible ones. Indeed, speaking about proteins, there are thought to be roughly 1013 proteins of all sizes in extant organisms. This apparently huge number represents less than noise when compared to the number of all theoretically possible different proteins [64]. The discrepancy between the actual collection of proteins and all possible ones stands clear if one considers that the number of all possible 50 residue peptides that can be synthesized with the standard 20 amino acids is 2050, namely 1065. Moreover, the number of theoretically possible proteins increases with length, so that the related sequence space is beyond contemplation; in fact, if we take into account the living organisms, where the average length of proteins is much greater, the number of possible different proteins becomes even bigger [65]. The difference between the number of possible proteins and the number of those actually present in living organisms is comparable, in a figurative way, to the difference that exists between a drop of water and an entire Ocean [22]. This means that there is an astronomically large number of proteins that have never been subjected to the long pathway of natural evolution on Earth: the ‘‘Never Born Proteins’’ (NBPs). Furthermore,

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the question whether a functionality is a common feature or a rare result of natural selection is of the utmost importance to elucidate the role of proteins in the origin of life and to fully exploit its biological potential and find new scaffolds for biological activities [10,22]. The use of random sequences to create protein libraries to search for new active sequences in both, pharmaceutical application and basic science research is a relatively commonplace [66–67]. Respect to our approach the difference is based on the kind of randomization applied; whereas usually the protein libraries produced are only partially randomized (directed randomization [68]) in our case we use a total randomization approach [62]. In the directed randomization the researcher uses natural scaffolds to engraft short random peptides or to mutate restricted regions of a target protein, whereas the rest of the sequence is left unchanged. In contrast, our libraries are constituted by de novo totally random sequences [62] with no preconception as to what their properties might be. In the preliminary work we set up a method to test, as a first approximation threshold, the folding stability of the random library, introducing a very short sequence (a tripeptide) substrate for a protease into the random sequence. The proteins with a stable fold are much more resistant to proteolytic digestion than unfolded ones [69–72], resulting preferred during selection cycles (Fig. 2a). The work until now has been to set up an efficient protocol for the production and study of NBPs and, in particular, the production of large libraries (of the order of 108–9 clones) of totally random proteins (with 50 and 20 residues) by phage display (Fig. 2b) [9–10,62]. The NBPs analyzed by spectroscopic methods reveal a word of novel structured sequences with a great potential in terms of properties. Indeed, NBPs pose a series of challenging questions in basic and applied science: do they present novel principles of structure? Have they any, possibly novel, catalytic properties? Do they elicit

the formation of antibodies? Are they compatible with extant life forms? Do they represent novel therapeutic agents? 3.2. Never Born RNAs Likewise described for random protein, the study of random RNAs poses a lot of considerations to take into account. A single RNA sequence is only a point in a 4l space of all the possible sequences of a given length l [22,73]. This dimension 4l comes from the fact that for a given polymer of length l, 4l is the number of all the possible generable sequences, being each position of the sequence occupied by one of the four nucleobases. Several independent investigations have shown that for a fixedlength polynucleotide, the number of different generable sequences far exceeds the number of the possible structures. Schuster and colleagues [73], using an inverse folding algorithm to calculate the number of different sequences with the same secondary structure, found that the sequence-space structures is the type of many-toone (Fig. 2c) and that the different sequences that take the same shape are spread evenly in the space of sequences, showing a small or none homology degree [74]. These theoretical indications are experimentally confirmed by the observation that only seven nucleotides are strictly conserved in the intron self-splicing group I [75], although the secondary structure of the ribozyme and its function are preserved. This case clearly illustrates the fact that sequences with little or no homology can achieve the same structure and perform the same function in spite of the diversity of their sequences. The reasons behind the redundancy of the sequence/structure space of RNA can be found in the modular organization of the secondary structure of the RNA. Knight and colleagues have highlighted how RNA molecules are organized into independent functional domains connected by flexible spacers, whose sequences are irrelevant for the secondary and tertiary structures [76]. In this work they developed a method to calculate the abundance of these functional domains, in a pool of random RNA sequences. The

Fig. 2. Never Born Biopolymers. (a) Schematic representation of the NBPs selection system. The random peptide library is bound to the bacteriophage. In the middle of the random sequences is a tripeptide substrate for the protease. The tag serves to bind the specific antibody during the selection of the protease-resistant sequences. (b) Schematic representation of the three-dimensional structure of few selected random proteins using the ab-initio modelling program Rosetta as implemented in the Robetta web server (http://robetta.bakerlab.org). (c) Structural redundancy of the RNA sequence space. The gray sphere represents the potential diversity of molecular species, i.e. all the different sequences generable for a given length l. Different sequences of RNA (black spots inside the sphere), with little or no homology, could fold getting the same structure. This indicates that sequence/structure space is many-to-one. (d) Schematic representation of the RNA Foster assay. It combines the thermal incubation with nucleasic activity of nuclease S1, which is acted on single strand RNAs. In fewer words, the most stable sequences at high temperature will be those with a more stable secondary and eventually tertiary structure. (e) RNA Foster on a single Never Born RNA presenting a good stability at 70 °C. Plot showing the residual random RNA amount (in black) at the different temperatures. The fraction of folded RNA for each temperature is estimated by image analysis of the band intensity on PAGE urea gel, which corresponds to the amount of uncleaved RNA after S1 incubation. The random sequence stability is compared with the one of the natural tRNA (in gray) and the RNA population average (18 random sequences) (in light gray).

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results showed that the modularity of RNA sequences further increases the redundancy of the sequence/structure space, since the number of ways that can be achieved a specific fold increases markedly due to the modularity of the domains of RNA. In addition, the RNA secondary motifs, crucial for ribozymic activity, are due to the annealing of complementary bases that follow the Watson and Crick rules (A:U and C:G), increasing even more the probability of finding these domains in different regions of the sequences space and inside sequences no related to each other [77]. The simple consequence of these results is that the RNA sequence space is rich in folded RNAs. In addition, the RNA sequence/structure space seems to be characterized by a high redundancy of related sequences that share the same structure and probably the same function too. Starting from the considerations on the structural redundancy of the sequence space, i.e. sequences with low homology may take the same structure, a comprehensive research has been undertaken in our group to study the structural properties of random RNAs, that is the draft of the Never Born RNAs (NB-RNAs) project [22]. On this basis we decided to explore the RNA sequence space in search of random RNA molecules that have a stable folding at high temperatures, by an assay set up in our lab: the RNA Foster (RNA Folding Stability Test). This approach is based on the digestion of RNAs by S1 nuclease, a single-strand-specific nuclease, at several temperatures to determine the presence and thermal stability of secondary domains by coupling enzymatic digestion with temperature denaturation (Fig. 2d). The assay is capable to quantitatively determine the fraction of folded RNAs (ffold) as a function of the temperature [78]. The resistance to S1 nuclease digestion depends on the RNA fold, in fact, the unfolded RNAs are degraded faster than the folded ones. A temperature increment destabilizes the RNA fold, inducing either global or local unfolding. Using nuclease S1, capable to work over a broad range of temperatures, is possible to probe RNA secondary domain stability. Measuring the amount of RNA remaining after S1 digestion using electrophoresis and a suitable staining method, it is feasible to determine the fraction of folded RNA (ffold) and to assess the Tm of the RNA molecules at each experimental temperature. The Foster assay has several advantages: the simplicity and rapidity of execution, the requirement of simple instrumentation, and in particular the minimal RNA amount required. In general, this methodology has one most notable advantage: it is a timesaving technique. Therefore, it is suitable for the screening of large libraries and could be easily adapted for high-throughput studies, like in vitro selection experiments [79]. For our studies we have prepared a library of de novo RNAs, containing 60 totally random residues flanked by a pair of constant regions for operational reasons, asking the question on stability: whether or not, and to what extent these totally random RNA sequences are able to exhibit a stable compact fold. A stable and well-defined fold is a fundamental prerequisite for the biological activity of extant biopolymers, and this applies to RNAs as well. Accordingly, in order to exploit the potential of random RNA sequences – Never Born RNAs – as a scaffold for chemical synthetic biology one must determine whether and to what extend random RNAs adopt a stable and well-defined fold. Until now the most general result of our studies lies in the demonstration that RNAs have the capacity to fold into compact secondary structures, even in absence of selective pressure [63]. This confirm our hypothesis that molecules involved in nowadays life do not have exclusive features at far as the ability to adopt a stable fold is concerned. In detail, the RNA Foster was successfully used to evaluate the structural properties of 18 random RNAs. RNA molecules were

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tested individually at 4 different temperatures, in order to evaluate their ability to form internal double helices and, thus, to acquire secondary and tertiary structures. The results were used to assess the temperature of the main unfolding transition of individual RNAs. The experimental data show that almost all screened RNAs possess folded structures at 30 °C. Furthermore, half of the sampled RNAs maintain the fold up to 50 °C. Surprisingly, one sequence presents a good stability at 70 °C showing a melting temperature close to the same degree value (Fig. 2e). Moreover, two sequences are more stable than the standard tRNA at the same temperature (70 °C) [63]. In light of the results obtained from the studies on the stability of single Never Born RNAs by RNA Foster, we decided to extend our analysis to a larger number of RNAs, in order to investigate the percentage of stable sequences at high temperatures found in a random RNA population. With this aim we used the in vitro selection [80] to select the random RNA library. Beginning with a broad population of RNAs we applied a selective pressure, then we recovered, reversetranscripted and amplified those molecules that satisfy the established condition. We found that, according to our predictions, the diversity of the RNA population tends to decrease as the selection keeps on, indicating a skimming working on it. Moreover, one of the sequences analyzed is more stable than the tRNA, taken as control, at 70 °C, with an approximately melting temperature higher than 80 °C (details will be given in a next publication). Even though the limited number of sequences explored is not statistically significant, the most general result of our studies lies in the demonstration that RNAs have the capacity to fold into compact structures, even in the absence of selective pressure. In other words, folding capability is a relatively common property, almost an intrinsic feature, of RNAs, as already mentioned in our previous work for longer RNAs [81]. 3.3. Potential impact and future goals The potential impact of this research is twofold, spanning from basic to applied science. From the basic science standpoint, the study of the structure of biopolymers that do no exist in nature will permit us to establish whether additional principles of structure stability may be present in the folding of proteins and RNAs. If novel structural elements will be found, these could be used as scaffolds for a new kind of structural chemistry of proteins and RNAs, which can be useful also from the biotechnological standpoint. From the applied science standpoint, Never Born Biopolymers (NBBs) will represent completely novel scaffolds for synthetic biology applications. It is well known that enzymes find several applications in biotechnology and chemical industry as specific catalysts or in general as active therapeutic molecules. The search for catalytic properties in the NBBs can therefore be relevant for applied science. Notice that NBBs are completely orthogonal (insulated) to extant organisms and therefore may be implemented into synthetic biology chassis possibly reducing cross talk and parasitic effects. In addition, NBBs – unlike natural polymers – are not the results of any evolutionary pathway and can therefore be regarded as virgin polymers which functionality can be engineered without constrictions to meet the user’s requirements. To this regard, NBBs may pave the way for novel design paradigms in synthetic biology and applications in metabolic engineering and fine-chemical production as well as complex system engineering. It is also well known that proteins and polypeptides in general are therapeutical agents, for example several proteins are used as inhibitors or for example as anti-inflammatory drugs. On the basis of all this, the selected clones of the NBBs could be screened for a series of potential therapeutical activities.

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4. Concluding remarks The common notion of synthetic biology refers to new forms of microbial life obtained through genetic manipulation of the extant life forms – a classic bioengineering approach. The few pages of the present review make however clear that synthetic biology has an additional dimension, that related to the term ‘‘chemical synthetic biology’’. Here, the production of biological structures alternative to the natural ones is carried out by using chemical and biochemical technology: we have seen in this review application of the physico-chemistry of vesicles, the ribosomal protein synthetic apparatus, peptide catalysis, enzymatic assays, etc. It is apparent that this approach is yielding a rich variety of novel forms and corresponding novel ideas, which may be relevant for our understanding of basic science – but also potentially interesting from the biotechnological point of view. Thus, the minimal cell project can bring us to a better understanding of the emergence of life on simple chemical ground- but can also offer a novel form of device for enzyme delivery system, or of entire metabolic pathways. And the NBPs can help us understanding why nature has chosen that particular family of proteins and not another one- at the same time possibly providing us with new catalytic functions. We ascribe now particular interest in the NB-RNAs, as this can help solving some still intriguing obscure points in the RNA world. It is also worth of note that the research on chemical synthetic biology has brought us, as a consequence, to the discovery of new aspects of the chemistry of compartmentation – the overcrowding effect that we consider now a possible origin of cellular metabolism in the origin of life. The NBPs can provide novel structures and novel forms of catalysis – possibly even novel form of therapeutic agents. Also interesting is the discovery that randomly designed RNAs can assume structures that are resistant to relatively high temperatures without having being optimized by evolution. It is a large field in which basic science and applicative research combine together, and we are confident that significant progress is waiting ahead of us. 5. Box 1. Fragment condensation and organocatalysis Another problem that can be approached by chemical synthetic biology concerns the stepwise construction of polypeptides (or polynucleotides) from short segments [22–23]. This scenario is also linked to the emergence of a ‘‘dynamic chemical network’’ (the model of spontaneously arising self-catalytic sets, as suggested by Stuart Kauffman [24], is an example of such scenarios). Not many experimental studies deal with this or similar systems, which have, however, several intriguing conceptual aspects. The key idea is that short oligomers, formed spontaneously by random oligomerization processes, are joined together to give longer sequences, which in turn can condense again and produce a biopolymer of sufficient length to fold and possibly have a catalytic site. A selection process might operate to reduce the huge combinatorial diversity that can be generated by this mechanism, for example removing sequences that can be easily cleaved, or insoluble ones. Amphiphilic sequences can be adsorbed on membranes, if available. To make possible this pathway, that resembles the convergent synthesis of complex organic molecules, it is necessary to join together the reactive end groups of oligomers. In the case of polypeptide, this means that an amide bond should be formed between a terminal amino group and a terminal carboxylic group, possibly in activated form. Is it possible to find a catalyst – possible a simple one – that can help this reaction? An attractive answer deals with small molecule catalysis, i.e., ‘‘organocatalysis’’.

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