The Expanded Central Dogma: Genome Resynthesis, Orthogonal Biosystems, Synthetic Genetics

Abstract

Synthetic biology seeks to probe fundamental aspects of biological form and function by construction [i.e., (re)synthesis] rather than deconstruction (analysis). In this sense, biological sciences now follow the lead given by the chemical sciences. Synthesis can complement analytic studies but also allows novel approaches to answering fundamental biological questions and opens up vast opportunities for the exploitation of biological processes to provide solutions for global problems. In this review, we explore aspects of this synthesis paradigm as applied to the chemistry and function of nucleic acids in biological systems and beyond, specifically, in genome resynthesis, synthetic genetics (i.e., the expansion of the genetic alphabet, of the genetic code, and of the chemical make-up of genetic systems), and the elaboration of orthogonal biosystems and components.

Keywords: XNA; genome synthesis; orthogonality; synthetic biology; synthetic genetics; unnatural base pairs.

Figure 1

Synthetic genomes: design, construction, and repurposing. Three main genome resynthesis projects have been applied in Mycoplasma mycoides, Escherichia coli, and Saccharomyces cerevisiae, respectively, with sizes ranging from 1 Mbp to 12 Mbp. Work on Syn1.0 established the hierarchical construction workflow to synthesize the M. mycoides genome, from oligonucleotides to larger fragments that were eventually assembled into the 1 Mbp genome. With the bottom-up method, a genome of half the size—Syn3.0—was constructed to study what genes are essential for maintaining a near-minimal cell. In the synthetic genome for E. coli, two serine codons and a stop codon were removed and replaced by their isoforms, resulting in the Syn61 strain with 61 codons. Further deletion of the corresponding tRNAs enabled resistance to viral infection and provided orthogonal tRNAs with space for heteropolymer synthesis. The synthetic yeast project includes the swap of a stop codon, removal of repetitive elements, and addition of recombinase recognition sites. Various rearrangements and genome diversity can be generated by SCRaMbLE, enabling directed evolution of the synthetic strain for downstream applications. Abbreviations: RF1, release factor 1; tRNA, transfer RNA.

Figure 2

Semantic versus alphabetic orthogonality. In semantic orthogonality, after genome resynthesis with code compression, an orphan codon (TCG, previously coding for Ser) is reassigned to an unnatural amino acid (uAA) (left) (unnatural or engineered components are designated with *). Alphabetic orthogonality requires genetic alphabet expansion by genomic insertion, replication, and transcription of a UBP X:Y (creating the new codon dGXC) and a CYG anticodon transfer RNA (tRNA). This also requires import of (d)XTP [(d)YTP] for replication (transcription), as well as potentially (engineered, marked by *) DNA and RNA polymerases and ribosome (right). Both require an (engineered) orthogonal tRNA synthetase (uaaRS*):tRNA pair assigned to the new codon and uAA import.

Figure 3

Unnatural base pair (UBP) chemistries. The UBP design concepts shown are H-bond reassignment (Z:P and S:B; left), shape complementarity (Ds:Px; right), and hydrophobic interactions (NaM:5SICS and NaM:TPT3; bottom).

Figure 4

Orthogonality concepts. Genetic containment, i.e., inability to transfer genes to and from natural E. coli cells (center), may be achieved via semantic orthogonality (code reassignment; top left), alphabetic orthogonality (genetic alphabet expansion; bottom left), or (stereo)chemical orthogonality [(stereo)chemical divergence of genetic polymers; right].

Figure 5

Xeno-nucleic acids (XNAs) with potential for chemical orthogonality. (a,b) Two different approaches to steric orthogonality either by (a) expansion of natural bases or (b) new base pairs between either pyrimidine-like (skinny) or purine-like (fat) DNA bases. (c) Chemical orthogonality.