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

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.

A two-residue nascent-strand steric gate controls synthesis of 2'-O-methyl- and 2'-O-(2-methoxyethyl)-RNA.

Steric exclusion is a key element of enzyme substrate specificity, including in polymerases. Such substrate specificity restricts the enzymatic synthesis of 2'-modified nucleic acids, which are of interest in nucleic-acid-based drug development. Here we describe the discovery of a two-residue, nascent-strand, steric control 'gate' in an archaeal DNA polymerase. We show that engineering of the gate to reduce steric bulk in the context of a previously described RNA polymerase activity unlocks the synthesis of 2'-modified RNA oligomers, specifically the efficient synthesis of both defined and random-sequence 2'-O-methyl-RNA (2'OMe-RNA) and 2'-O-(2-methoxyethyl)-RNA (MOE-RNA) oligomers up to 750 nt. This enabled the discovery of RNA endonuclease catalysts entirely composed of 2'OMe-RNA (2'OMezymes) for the allele-specific cleavage of oncogenic KRAS (G12D) and ß-catenin CTNNB1 (S33Y) mRNAs, and the elaboration of mixed 2'OMe-/MOE-RNA aptamers with high affinity for vascular endothelial growth factor. Our results open up these 2'-modified RNAs-used in several approved nucleic acid therapeutics-for enzymatic synthesis and a wider exploration in directed evolution and nanotechnology.

Efficient synthesis and replication of diverse sequence libraries composed of biostable nucleic acid analogues.

Functional nucleic acids can be evolved in vitro using cycles of selection and amplification, starting from diverse-sequence libraries, which are typically restricted to natural or partially-modified polymer chemistries. Here, we describe the efficient DNA-templated synthesis and reverse transcription of libraries entirely composed of serum nuclease resistant alternative nucleic acid chemistries validated in nucleic acid therapeutics; locked nucleic acid (LNA), 2'-O-methyl-RNA (2'OMe-RNA), or mixtures of the two. We evaluate yield and diversity of synthesised libraries and measure the aggregate error rate of a selection cycle. We find that in addition to pure 2'-O-methyl-RNA and LNA, several 2'OMe-RNA/LNA blends seem suitable and promising for discovery of biostable functional nucleic acids for biomedical applications.

New chemistries and enzymes for synthetic genetics.

Beyond the natural nucleic acids DNA and RNA, nucleic acid chemistry has unlocked a whole universe of modifications to their canonical chemical structure, which can in various ways modify and enhance nucleic acid function and utility for applications in biotechnology and medicine. Unlike the natural modifications of tRNA and rRNA or the epigenetic modifications in mRNA and genomic DNA, these altered chemistries are not found in nature and therefore these molecules are referred to as xeno-nucleic acids (XNAs). In this review we aim to focus specifically on recent progress in a subsection of this vast field-synthetic genetics-concerned with encoded synthesis, reverse transcription, and evolution of XNAs.

Large Particle 3D Concrete Printing-A Green and Viable Solution.

The Large Particle 3D Concrete Printing (LP3DCP) process presented in this paper is based on the particle bed 3D printing method; here, the integration of significantly larger particles (up to 36 mm) for selective binding using the shotcrete technique is presented. In the LP3DCP process, the integration of large particles, i.e., naturally coarse, crushed or recycled aggregates, reduces the cement volume fraction by more than 50% compared to structures conventionally printed with mortar. Hence, with LP3DCP, the global warming potential, the acidification potential and the total non-renewable primary energy of 3D printed structures can be reduced by approximately 30%. Additionally, the increased proportion of aggregates enables higher compressive strengths than without the coarse aggregates, ranging up to 65 MPa. This article presents fundamental material investigations on particle packing and matrix penetration as well as compressive strength tests and geometry studies. The results of this systematic investigation are presented, and the best set is applied to produce a large-scale demonstrator of one cubic meter of size and complex geometry. Moreover, the demonstrator features reinforcement and subtractive surface processing strategies. Further improvements of the LP3DCP technology as well as construction applications and architectural design potentials are discussed thereafter.

The Effect of Accelerator Dosage on Fresh Concrete Properties and on Interlayer Strength in Shotcrete 3D Printing.

Recently, the progress in 3D concrete printing has developed enormously. However, for the techniques available, there is still a severe lack of knowledge of the functional interaction of processing technology, concrete rheology and admixture usage. For shotcrete 3D printing technology, we present the effect of accelerator dosages (0%, 2%, 4% and 6%) on fresh concrete properties and on interlayer strength. Therefore, early yield stress development up to 90 min is measured with penetration resistance measurements. Deformation of layers under loading is investigated with digital image correlation and a mechanical testing machine. One point in time (10 min after deposition) is examined to quantify vertical buildability of elements depending on the accelerator dosage. Four different interlayer times (0, 2, 5 and 30 min), which occur for the production of small and large elements as well as due to delay during production, are investigated mechanically as well as quantitatively with computed tomography regarding the formation of cold joints. With increased accelerator dosage, an instantaneous increase in early age yield stress and yield stress evolution was observed. An increase in interlayer time leads to a reduced strength. This is mainly attributed to the observed reduced mechanical interlocking effect of the strands. Finally, a model to describe interlayer quality is presented. In the end, advantages as well as limitations of the findings are discussed.