Ishtiaq Saaem

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.

Toward Larger DNA Origami

Structural DNA nanotechnology, and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7,249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Additionally, increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) production of the largest to-date biologically derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have experimentally demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addition, we have demonstrated two-dimensional, asymmetric origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by atomic force microscopy.

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.

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.

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.

Versatile surface functionalization of cyclic olefin copolymer (COC) with sputtered SiO2 thin film for potential BioMEMS applications

Cyclic olefin copolymer (COC) is a new class of thermoplastic polymers used for a variety of applications ranging from bio-sensing to optics. However, lack of functional groups and intense surface hydrophobicity hamper further development and application of this material. Here, we describe fabrication and characterization of SiO2–COC hybrid material (oxCOC), which provides a desirable substrate for microfluidic devices and subsequent surface modifications. The deposited SiO2 thin film on COC surface was characterized by X-ray photoelectron spectroscopy and Fourier transform infrared spectroscopy. The new SiO2–COC hybrid was found to have similar high optical transmission properties as that of pristine COC. Profilometric and AFM analysis revealed no dramatic change in morphology or surface roughness of functionalized COC. The SiO2–COC hybrid appeared to be stable in most of the solvents evaluated and could be further modified by other compounds, such as 3-aminopropyltriethoxy silane (APTES). The new SiO2–COC hybrid material and the robust fabrication method are expected to enable a variety of BioMEMS applications.

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.

Fabrication of plastic biochips

A versatile surface functionalization procedure based on rf magnetron sputtering of silica was performed on poly(methylmethacrylate), polycarbonate, polypropylene, and cyclic olefin copolymers (Topas 6015). The hybrid thermoplastic surfaces were characterized by x-ray photoelectron spectrometer analysis and contact angle measurements. The authors then used these hybrid materials to perform a sandwich assay targeting an HIV-1 antibody using fluorescent detection and biotinylated peptides immobilized using the bioaffinity of biotin-neutravidin. They found a limit of detection similar to arrays on glass surfaces and believed that this plastic biochip platform may be used for the development of disposable immunosensing and diagnostic applications.

Optimized in situ DNA synthesis on patterned glass

This paper describes studies of patterned arrays on glass surfaces and their use as spatially separated reactors for in situ synthesis of DNA using an inkjet synthesizer. Photolithographic methods were employed to fabricate arrays composed of homogenous circular features containing a hydroxyl-terminated silane coupled to the surface of the glass via a siloxane bond. Features are embedded within a background matrix composed of a fluorosilane attached to the glass. Due to the differential wettability of the two silanes, whereby the hydroxyl-terminated silane and fluorosilane are hydrophilic and hydrophobic respectively because of their head groups, the patterned circular features are able to constrain liquid within a defined site. The silanization result was analyzed using X-ray photoelectron spectroscopy (XPS) to optimize silanization time and solvent. Synthesis was then performed using a custom-built inkjet system using phosphoramidite chemistry. Base-by-base analysis using fluorescent labeling showed consistent coupling efficiency on synthesis of a 50-mer homopolymer.