Error correction of microchip synthesized genes using Surveyor nuclease.

The development of economical and high-throughput gene synthesis technology has been hampered by the high occurrence of errors in the synthesized products, which requires expensive labor and time to correct. Here, we describe an error correction reaction (ECR), which employs Surveyor, a mismatch-specific DNA endonuclease, to remove errors from synthetic genes. In ECR reactions, errors are revealed as mismatches by re-annealing of the synthetic gene products. Mismatches are recognized and excised by a combination of mismatch-specific endonuclease and 3'→5' exonuclease activities in the reaction mixture. Finally, overlap extension polymerase chain reaction (OE-PCR) re-assembles the resulting fragments into intact genes. The process can be iterated for increased fidelity. With two iterations, we were able to reduce errors in synthetic genes by >16-fold, yielding a final error rate of ∼1 in 8700 bp.

Identification of RNA binding protein interacting with circular RNA and hub candidate network for hepatocellular carcinoma.

The interaction between RNA binding protein (RBP) and circular RNA (circRNA) is important for the regulation of tumor progression. This study aimed to identify the RBP-circRNA network in hepatocellular carcinoma (HCC). 22 differentially expressed (DE) circRNAs in HCC were screened out from Gene Expression Omnibus (GEO) database and their binding RBPs were predicted by Circular RNA Interactome. Among them, 17 DERBPs, which were commonly dysregulated in HCC from The Clinical Proteomic Tumor Analysis Consortium (CPTAC), The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) projects, were utilized to construct the RBP-circRNA network. Through survival analysis, we found TARDBP was the only prognostic RBP for HCC in CPTAC, TCGA and ICGC projects. High expression of TARDBP was correlated with high grade, advanced stage and low macrophage infiltration of HCC. Additionally, gene set enrichment analysis showed that dysregulated TARDBP might be involved in some pathways related to the HCC pathogenesis. Therefore, a hub RBP-circRNA network was generated based on TARDBP. RNA immunoprecipitation and RNA pull-down confirmed that hsa_circ_0004913 binds to TARDBP. These findings indicated certain RBP-circRNA regulatory network potentially involved in the pathogenesis of HCC, which provides novel insights into the mechanism study and biomarker identification for HCC.

Accurate multiplex gene synthesis from programmable DNA microchips.

Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of 'construction' oligonucleotides and tagged complementary 'selection' oligonucleotides are synthesized on photo-programmable microfluidic chips, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.

Circular polymerase extension cloning.

High-throughput genomics, proteomics, and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries, and synthetic pathways. Here, we describe an extremely simple, efficient, and cost-effective cloning method, circular polymerase extension cloning (CPEC), for complex, combinatorial, or multi-fragment assembly as well as routine cloning. This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required.

[Progress in gene synthesis technology].

Gene synthesis is the most fundamental and widely used technique in biological research. The synthesis of DNA encoding regulatory elements, genes, pathways and entire genomes provides powerful ways to both test biological hypotheses and harness biology for our use. The emerging field of synthetic biology is generating insatiable demands for synthetic genes. And the past couple of years witnessed exciting new developments in microchip-based gene synthesis technologies. This review discusses the current methods of chemical DNA synthesis and gene assembly, as well as the latest engineering tools, technologies and trends which could potentially lead to breakthroughs in the development of accurate, low-cost and high-throughput gene synthesis technology. These new technologies are leading the field of synthetic biology to a higher level.

One-pot assembly of a hetero-dimeric DNA origami from chip-derived staples and double-stranded scaffold.

Although structural DNA nanotechnology, and especially scaffolded DNA origami, hold great promise for bottom-up fabrication of novel nanoscale materials and devices, concerns about scalability have tempered widespread enthusiasm. Here we report a single-pot reaction where both strands of double-stranded M13-bacteriophage DNA are simultaneously folded into two distinct shapes that then heterodimerize with high yield. The fully addressable, two-dimensional heterodimer DNA origami, with twice the surface area of standard M13 origami, formed in high yield (81% of the well-formed monomers undergo dimerization). We also report the concurrent production of entire sets of staple strands by a unique, nicking strand-displacement amplification (nSDA) involving reusable surface-bound template strands that were synthesized in situ using a custom piezoelectric inkjet system. The combination of chip-based staple strand production, double-sized origami, and high-yield one-pot assembly markedly increases the useful scale of DNA origami.

Error correction in gene synthesis technology.

Accurate, economical and high-throughput gene and genome synthesis is essential to the development of synthetic biology and biotechnology. New large-scale gene synthesis methods harnessing the power of DNA microchips have recently been demonstrated. Yet, the technology is still compromised by a high occurrence of errors in the synthesized products. These errors still require substantial effort to correct. To solve this bottleneck, novel approaches based on new chemistry, enzymology or next generation sequencing have emerged. This review discusses these new trends and promising strategies of error filtration, correction and prevention in de novo gene and genome synthesis. Continued innovation in error correction technologies will enable affordable and large-scale gene and genome synthesis in the near future.

DNA synthesis, assembly and applications in synthetic biology.

The past couple of years saw exciting new developments in microchip-based gene synthesis technologies. Such technologies hold the potential for significantly increasing the throughput and decreasing the cost of gene synthesis. Together with more efficient enzymatic error correction and genome assembly methods, these new technologies are pushing the field of synthetic biology to a higher level.

Circular polymerase extension cloning for high-throughput cloning of complex and combinatorial DNA libraries.

High-throughput genomics, proteomics and synthetic biology studies require ever more efficient and economical strategies to clone complex DNA libraries or variants of biological modules. In this paper, we provide a protocol for a sequence-independent approach for cloning complex individual or combinatorial DNA libraries, and routine or high-throughput cloning of single or multiple DNA fragments. The strategy, called circular polymerase extension cloning (CPEC), is based on polymerase overlap extension and is therefore free of restriction digestion, ligation or single-stranded homologous recombination. CPEC is highly efficient, accurate and user friendly. Once the inserts and the linear vector have been prepared, the CPEC reaction can be completed in 10 min to 3 h, depending on the complexity of the gene libraries.

Parallel on-chip gene synthesis and application to optimization of protein expression.

Low-cost, high-throughput gene synthesis and precise control of protein expression are of critical importance to synthetic biology and biotechnology. Here we describe the development of an on-chip gene synthesis technology, which integrates on a single microchip the synthesis of DNA oligonucleotides using inkjet printing, isothermal oligonucleotide amplification and parallel gene assembly. Use of a mismatch-specific endonuclease for error correction results in an error rate of ~0.19 errors per kb. We applied this approach to synthesize pools of thousands of codon-usage variants of lacZα and 74 challenging Drosophila protein antigens, which were then screened for expression in Escherichia coli. In one round of synthesis and screening, we obtained DNA sequences that were expressed at a wide range of levels, from zero to almost 60% of the total cell protein mass. This technology may facilitate systematic investigation of the molecular mechanisms of protein translation and the design, construction and evolution of macromolecular machines, metabolic networks and synthetic cells.

In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

Circular polymerase extension cloning of complex gene libraries and pathways.

High-throughput genomics and the emerging field of synthetic biology demand ever more convenient, economical, and efficient technologies to assemble and clone genes, gene libraries and synthetic pathways. Here, we describe the development of a novel and extremely simple cloning method, circular polymerase extension cloning (CPEC). This method uses a single polymerase to assemble and clone multiple inserts with any vector in a one-step reaction in vitro. No restriction digestion, ligation, or single-stranded homologous recombination is required. In this study, we elucidate the CPEC reaction mechanism and demonstrate its usage in demanding synthetic biology applications such as one-step assembly and cloning of complex combinatorial libraries and multi-component pathways.

Advancing high-throughput gene synthesis technology.

The emerging field of synthetic biology is generating insatiable demands for synthetic genes, which far exceed existing gene synthesis capabilities. This review discusses the current methods of chemical DNA synthesis and gene assembly, as well as the latest engineering tools, technologies and trends which could potentially lead to breakthroughs in the development of accurate, low-cost and high-throughput gene synthesis technology. The capability of generating unlimited supplies of DNA molecules of any sequence or size will transform biomedical research in the near future.

Protein interfacial pocket engineering via coupled computational filtering and biological focusing criterion.

To engineer bio-macromolecular systems, protein-substrate interactions and their configurations need to be understood, harnessed, and utilized. Due to the inherent large numbers of combinatorial configurations and conformational complexity, methods that rely on heuristics or stochastics, such as practical computational filtering (CF) or biological focusing (BF) criterions, when used alone rarely yield insights into these complexes or successes in (re)designing them. Here we use a coupled CF-BF criterion upon an amenable interfacial pocket (IP) of a protein scaffold complexed with its substrate to undergo residue replacement and R-group refinement (R4) to filter out energetically unfavorable residues and R-group conformations, and focus in on those that are evolutionarily favorable. We show that this coupled filtering and focusing can efficiently provide a putative engineered IP candidate and validate it computationally and empirically. The CF-BF criterion may permit holistic understanding of the nuances of existing protein IPs and their scaffolds and facilitate bioengineering efforts to alter substrate specificity. Such approach may contribute to accelerated elucidation of engineering principles of bio-macromolecular systems.