Fabrication of Omniphobic‐Omniphilic Micropatterns using GPOSS‐PDMS Coating

Surfaces with special wettability properties, such as omniphobicity or omniphilicity, are essential for functional devices that use both aqueous and organic media. Micropatterning of omniphobic and omniphilic properties can provide a wide range of applications, including miniaturized experiments using both aqueous and organic media. Herein, an approach for creating omniphobic-omniphilic micropatterns based on selective photoacid polymerization of octa(3-glycidyloxypropyl) polyhedral oligomeric silsesquioxane modified with mono-aminopropyl-terminated polydimethylsiloxane is reported. The composition of the polymeric coatings using infrared spectroscopy; patterning accuracy using atomic force microscopy and scanning electron microscopy; wettability characteristics of the omniphobic, and omniphilic surfaces using contact angle measurements are studied. The proposed approach allows for single-step micropatterning (sub-10 µm) or macropatterning (3 mm). Liquids with surface tensions >22.8 mN m−1 can be confined to the omniphilic areas by the omniphobic borders. C2C12 cells are successfully cultivated in omniphilic areas, demonstrating their cell compatibility. The cells adhere to and grow on the entire surface of the pattern, without any signs of cytotoxicity. However, the strongest adhesion is observed in the omniphilic areas, making it possible to create cell micropatterns in a single step. The proposed method for the fabrication of omniphobic-omniphilic transparent, mechanically robust, biocompatible patterns can find applications in microfluidics, biotechnology or miniaturized biological screening experiments

Fabrication of Omniphobic‐Omniphilic Micropatterns using GPOSS‐PDMS Coating

Abstract Surfaces with special wettability properties, such as omniphobicity or omniphilicity, are essential for functional devices that use both aqueous and organic media. Micropatterning of omniphobic and omniphilic properties can provide a wide range of applications, including miniaturized experiments using both aqueous and organic media. Herein, an approach for creating omniphobic‐omniphilic micropatterns based on selective photoacid polymerization of octa(3‐glycidyloxypropyl) polyhedral oligomeric silsesquioxane modified with mono‐aminopropyl‐terminated polydimethylsiloxane is reported. The composition of the polymeric coatings using infrared spectroscopy; patterning accuracy using atomic force microscopy and scanning electron microscopy; wettability characteristics of the omniphobic, and omniphilic surfaces using contact angle measurements are studied. The proposed approach allows for single‐step micropatterning (sub‐10 µm) or macropatterning (3 mm). Liquids with surface tensions >22.8 mN m−1 can be confined to the omniphilic areas by the omniphobic borders. C2C12 cells are successfully cultivated in omniphilic areas, demonstrating their cell compatibility. The cells adhere to and grow on the entire surface of the pattern, without any signs of cytotoxicity. However, the strongest adhesion is observed in the omniphilic areas, making it possible to create cell micropatterns in a single step. The proposed method for the fabrication of omniphobic‐omniphilic transparent, mechanically robust, biocompatible patterns can find applications in microfluidics, biotechnology or miniaturized biological screening experiments

A Novel Method for SNP Detection Using a New Duplex-Specific Nuclease From Crab Hepatopancreas

We have characterized a novel nuclease from the Kamchatka crab, designated duplex-specific nuclease (DSN). DSN displays a strong preference for cleaving double-stranded DNA and DNA in DNA-RNA hybrid duplexes, compared to single-stranded DNA. Moreover, the cleavage rate of short, perfectly matched DNA duplexes by this enzyme is essentially higher than that for nonperfectly matched duplexes of the same length. Thus, DSN differentiates between one-nucleotide variations in DNA. We developed a novel assay for single nucleotide polymorphism (SNP) detection based on this unique property, termed “duplex-specific nuclease preference” (DSNP). In this innovative assay, the DNA region containing the SNP site is amplified and the PCR product mixed with signal probes (FRET-labeled short sequence-specific oligonucleotides) and DSN. During incubation, only perfectly matched duplexes between the DNA template and signal probe are cleaved by DSN to generate sequence-specific fluorescence. The use of FRET-labeled signal probes coupled with the specificity of DSN presents a simple and efficient method for detecting SNPs. We have employed the DSNP assay for the typing of SNPs in methyltetrahydrofolate reductase, prothrombin and p53 genes on homozygous and heterozygous genomic DNA. [Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GenBank/EMBL/Date Bank under accession nos. AF520591. The following individuals kindly provided reagents, samples, or unpublished information as indicated in the paper: N.K. Yankovsky, A.V. Polyakov, and G.N. Rudenskaya.

Conformationally Locked Chromophores as Models of Excited-State Proton Transfer in Fluorescent Proteins

Members of the green fluorescent protein (GFP) family form chromophores by modifications of three internal amino acid residues. Previously, many key characteristics of chromophores were studied using model compounds. However, no studies of intermolecular excited-state proton transfer (ESPT) with GFP-like synthetic chromophores have been performed because they either are nonfluorescent or lack an ionizable OH group. In this paper we report the synthesis and photochemical study of two highly fluorescent GFP chromophore analogues: <i>p</i>-HOBDI-BF2 and <i>p</i>-HOPyDI:Zn. Among known fluorescent compounds, <i>p</i>-HOBDI-BF₂ is the closest analogue of the native GFP chromophore. These irrreversibly (<i>p</i>-HOBDI-BF₂) and reversibly (<i>p</i>-HOPyDI:Zn) locked compounds are the first examples of fully planar GFP chromophores, in which photoisomerization-induced deactivation is suppressed and protolytic photodissociation is observed. The photophysical behavior of <i>p</i>-HOBDI-BF2 and <i>p</i>-HOPyDI:Zn (excited state p<i>K</i>_a’s, solvatochromism, kinetics, and thermodynamics of proton transfer) reveals their high photoacidity, which makes them good models of intermolecular ESPT in fluorescent proteins. Moreover, <i>p</i>-HOPyDI:Zn is a first example of “super” photoacidity in metal–organic complexes

Genetically encodable bioluminescent system from fungi

Bioluminescence is found across the entire tree of life, conferring a spectacular set of visually oriented functions from attracting mates to scaring off predators. Half a dozen different luciferins, molecules that emit light when enzymatically oxidized, are known. However, just one biochemical pathway for luciferin biosynthesis has been described in full, which is found only in bacteria. Here, we report identification of the fungal luciferase and three other key enzymes that together form the biosynthetic cycle of the fungal luciferin from caffeic acid, a simple and widespread metabolite. Introduction of the identified genes into the genome of the yeast Pichia pastoris along with caffeic acid biosynthesis genes resulted in a strain that is autoluminescent in standard media. We analyzed evolution of the enzymes of the luciferin biosynthesis cycle and found that fungal bioluminescence emerged through a series of events that included two independent gene duplications. The retention of the duplicated enzymes of the luciferin pathway in nonluminescent fungi shows that the gene duplication was followed by functional sequence divergence of enzymes of at least one gene in the biosynthetic pathway and suggests that the evolution of fungal bioluminescence proceeded through several closely related stepping stone nonluminescent biochemical reactions with adaptive roles. The availability of a complete eukaryotic luciferin biosynthesis pathway provides several applications in biomedicine and bioengineering.This research was supported by Planta LLC and Evrogen JSC. IVIS imaging and animal experiments were carried out using the equipment of the Center for Collective Usage “Medical Nanobiotechologies” located in the Russian National Research Medical University. Experiments were partially carried out using the equipment provided by the Institute of Bioorganic Chemistry of the Russian Academy of Sciences Сore Facility (CKP IBCH; supported by Russian Ministry of Education and Science Grant RFMEFI62117X0018). T.G. and M.M.-H. acknowledge support from Spanish Ministry of Economy and Competitiveness Grant BFU2015-67107 cofounded by the European Regional Development Fund, European Research Council (ERC) Grant ERC-2012-StG-310325 under the European Union’s Seventh Framework Programme FP7/2007-2013, and the European Union’s Horizon 2020 Research and Innovation Programme under Marie Sklodowska-Curie Grant H2020-MSCA-ITN-2014-642095. F.A.K. acknowledges the support of HHMI International Early Career Scientist Program 55007424, the Spanish Ministry of Economy and Competitiveness (MINECO) Grants BFU2012-31329 and BFU2015-68723-P, MINECO Centro de Excelencia Severo Ochoa 2013-2017 Grant SEV-2012-0208, Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat’s Agency for Management of University and Research Grants Program 2014 SGR 0974, the Centres de Recerca de Catalunya Programme of the Generalitat de Catalunya, and ERC Grant 335980_EinME under the European Union’s Seventh Framework Programme FP7/2007-2013. H.E.W., A.G.O., and C.V.S. acknowledge support from São Paulo Research Foundation Fundação de Amparo à Pesquisa do Estado de São Paulo Grants 11/10507-0 (to H.E.W.), 10/11578-5 (to A.G.O.), and 13/16885-1 (to C.V.S.)