Controlled Orientation of Active Sites in a Nanostructured Multienzyme Complex

Multistep cascade reactions in nature maximize reaction efficiency by co-assembling related enzymes. Such organization facilitates the processing of intermediates by downstream enzymes. Previously, the studies on multienzyme nanocomplexes assembled on DNA scaffolds demonstrated that closer interenzyme distance enhances the overall reaction efficiency. However, it remains unknown how the active site orientation controlled at nanoscale can have an effect on multienzyme reaction. Here, we show that controlled alignment of active sites promotes the multienzyme reaction efficiency. By genetic incorporation of a non-natural amino acid and two compatible bioorthogonal chemistries, we conjugated mannitol dehydrogenase to formate dehydrogenase with the defined active site arrangement with the residue-level accuracy. The study revealed that the multienzyme complex with the active sites directed towards each other exhibits four-fold higher relative efficiency enhancement in the cascade reaction and produces 60% more D-mannitol than the other complex with active sites directed away from each other.ope

Barcoding cells using cell-surface programmable DNA-binding domains

We report an approach to barcode cells through cell-surface expression of programmable zinc-finger DNA-binding domains (surface zinc fingers, sZFs). We show that sZFs enable sequence-specific labeling of living cells by dsDNA, and we develop a sequential labeling approach to image more than three cell types in mixed populations using three fluorophores. We demonstrate the versatility of sZFs through applications in which they serve as surrogate reporters, function as selective cell capture reagents and facilitate targeted cellular delivery of viruses

DNA Nanotechnology

Cite this entry as: Yaradoddi J.S. et al. (2019) DNA Nanotechnology. In: Martínez L., Kharissova O., Kharisov B. (eds) Handbook of Ecomaterials. Springer, Cham DOI: https://doi.org/10.1007/978-3-319-68255-6_191 First Online: 14 February 2019 Online ISBN: 978-3-319-68255-6 Print ISBN: 978-3-319-68254-9Since from the past few decades DNA appeared as an excellent molecular building block for the synthesis of nanostructures because of its probable encoded and confirmation intra- and intermolecular base pairing, various case strategies and consistent assembly techniques have been established to manipulate DNA nanostructures to at higher complexity. The capability to develop DNA construction with precise special control has permitted scientists to discover novel applications in many ways, such as scaffold development, sensing applications, nanodevices, computational applications, nanorobotics, nanoelectronics, biomolecular catalysis, disease diagnosis, and drug delivery. The present chapter emphasizes to brief the opportunities, challenges, and future prospective on DNA nanotechnology and its advancements.Peer reviewe

Redirection of pyruvate flux toward desired metabolic pathways through substrate channeling between pyruvate kinase and pyruvate-converting enzymes in Saccharomyces cerevisiae

Spatial organization of metabolic enzymes allows substrate channeling, which accelerates processing of intermediates. Here, we investigated the effect of substrate channeling on the flux partitioning at a metabolic branch point, focusing on pyruvate metabolism in Saccharomyces cerevisiae. As a platform strain for the channeling of pyruvate flux, PYK1-Coh-Myc strain was constructed in which PYK1 gene encoding pyruvate kinase is tagged with cohesin domain. By using high-affinity cohesin-dockerin interaction, the pyruvate-forming enzyme Pyk1 was tethered to heterologous pyruvate-converting enzymes, lactate dehydrogenase and α-acetolactate synthase, to produce lactic acid and 2,3-butanediol, respectively. Pyruvate flux was successfully redirected toward desired pathways, with a concomitant decrease in ethanol production even without genetic attenuation of the ethanol-producing pathway. This pyruvate channeling strategy led to an improvement of 2,3-butanediol production by 38%, while showing a limitation in improving lactic acid production due to a reduced activity of lactate dehydrogenase by dockerin tagging

Computation of ratios using chemical reactions and DNA strand displacements

Recent researches has focused on nucleic acids as a substrate for designing biomolecular circuits for in situ monitoring and control. A common approach is to express them by a set of idealised abstract chemical reaction networks (ACRNs). Here, we present new results on how abstract chemical reactions, viz., catalysis, annihilation and degradation, can be used to implement circuits that accurately compute ratio of two input signals. The input signals can be either constant-valued scalars or time-varying scalars or polynomials. We also characterise the robustness of our circuits to parameter variations, as would be encountered in wet-lab implementations