The Experts below are selected from a list of 174 Experts worldwide ranked by ideXlab platform
Ashutosh Chilkoti - One of the best experts on this subject based on the ideXlab platform.
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direct fluorescence detection of rna on microarrays by surface initiated enzymatic polymerization
Analytical Chemistry, 2013Co-Authors: Vinalia Tjong, Angus Hucknall, Hua Yu, Ashutosh ChilkotiAbstract:We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly(A) polymerase (PaP) to incorporate Deoxyadenosine Triphosphate (dATP) at the 3′–OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3′–OH of an RNA–DNA hybrid. We found that the 3′-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection ...
Michal Hocek - One of the best experts on this subject based on the ideXlab platform.
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2‐Formyl‐dATP as Substrate for Polymerase Synthesis of Reactive DNA Bearing an Aldehyde Group in the Minor Groove
ChemPlusChem, 2020Co-Authors: Matous Kromer, Maria Brunderova, Ivana Ivancova, Lenka Postova Slavětinska, Michal HocekAbstract:2-Formyl-2'-Deoxyadenosine Triphosphate (dCHO ATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of dCHO ATP was based on the preparation of protected 2-dihydroxyethyl-2'-Deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHO ATP Triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31-mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single-nucleotide extension followed by PEX was used for site-specific incorporation of one aldehyde-linked adenosine into a longer 49-mer sequence. The reactive formyl group was used for cross-linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647-linked hydroxylamine.
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2 formyl datp as substrate for polymerase synthesis of reactive dna bearing an aldehyde group in the minor groove
ChemPlusChem, 2020Co-Authors: Matous Kromer, Maria Brunderova, Ivana Ivancova, Lenka Postova Slavětinska, Michal HocekAbstract:2-Formyl-2'-Deoxyadenosine Triphosphate (dCHO ATP) was synthesized and tested as a substrate in enzymatic synthesis of DNA modified in the minor groove with a reactive aldehyde group. The multistep synthesis of dCHO ATP was based on the preparation of protected 2-dihydroxyethyl-2'-Deoxyadenosine intemediate, which was triphosphorylated and converted to aldehyde through oxidative cleavage. The dCHO ATP Triphosphate was a moderate substrate for KOD XL DNA polymerase, and was used for enzymatic synthesis of some sequences using primer extension (PEX). On the other hand, longer sequences (31-mer) with higher number of modifications, or sequences with modifications at adjacent positions did not give full extension. Single-nucleotide extension followed by PEX was used for site-specific incorporation of one aldehyde-linked adenosine into a longer 49-mer sequence. The reactive formyl group was used for cross-linking with peptides and proteins using reductive amination and for fluorescent labelling through oxime formation with an AlexaFluor647-linked hydroxylamine.
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2-Substituted dATP Derivatives as Building Blocks for Polymerase-Catalyzed Synthesis of DNA Modified in the Minor Groove.
Angewandte Chemie (International ed. in English), 2016Co-Authors: Ján Matyašovský, Pavla Perlíková, Vincent Malnuit, Radek Pohl, Michal HocekAbstract:2′-Deoxyadenosine Triphosphate (dATP) derivatives bearing diverse substituents (Cl, NH2, CH3, vinyl, ethynyl, and phenyl) at position 2 were prepared and tested as substrates for DNA polymerases. The 2-phenyl-dATP was not a substrate for DNA polymerases, but the dATPs bearing smaller substituents were good substrates in primer-extension experiments, producing DNA substituted in the minor groove. The vinyl-modified DNA was applied in thiol–ene addition and the ethynyl-modified DNA was applied in a CuAAC click reaction to form DNA labelled with fluorescent dyes in the minor groove
Hans-achim Wagenknecht - One of the best experts on this subject based on the ideXlab platform.
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Triazine-Modified 7-Deaza-2′-Deoxyadenosines: Better Suited for Bioorthogonal Labeling of DNA by PCR than 2′-Deoxyuridines
Bioconjugate chemistry, 2019Co-Authors: Ulrike Reisacher, Gergely B. Cserép, Péter Kele, Ralf Strasser, Bastian Groitl, Hans-achim WagenknechtAbstract:6-Ethynyl-1,2,4-triazine is a small bioorthogonally reactive group we applied for fluorescent labeling of oligonucleotides by Diels-Alder reactions with inverse electron demand. We synthetically attached this functional group to the 7-position of 7-deaza-2'-Deoxyadenosine Triphosphate and to the 5-position of 2'-deoxyuridine Triphosphate. Both modified nucleotide Triphosphates were used in comparison for primer extension experiments (PEX) and PCR amplification to finally yield multilabeled oligonucleotides by the postsynthetic reaction with a highly reactive bicyclo[6.1.0]nonyne-rhodamine conjugate. These experiments show that 6-ethynyl-1,2,4-triazine is much better tolerated by the DNA polymerase when attached to the 7-position of 7-deaza-2'-Deoxyadenosine in comparison to the attachment at the 5-position of 2'-deoxyuridine. This became evident both by PAGE analysis of the PCR products and real-time kinetic observation of DNA polymerase activity during primer extension using switchSENSE. Generally, our results imply that bioorthogonal labeling strategies are better suited for 7-deaza-2'-adenosines than conventional and available 2'-deoxyuridines.
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Copper-free dual labeling of DNA by triazines and cyclopropenes as minimal orthogonal and bioorthogonal functions.
Chemical science, 2019Co-Authors: Ulrike Reisacher, Damian Ploschik, Franziska Rönicke, Gergely B. Cserép, Péter Kele, Hans-achim WagenknechtAbstract:Two different and small functions for inverse electron demand Diels–Alder reactions were applied for dual labeling of DNA: the 1,2,4-triazine was attached to the 5-position of 2′-deoxyuridine Triphosphate, and the 1-methylcyclopropene to the 7-position of 7-deaza-2′-Deoxyadenosine Triphosphate. These two modified nucleotides were sequence-selectively incorporated into oligonucleotides by DNA polymerases. These products were labeled by two different fluorescent dyes using postsynthetic reactions that are not only bioorthogonal in general, but also mutually orthogonal.
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DNA Primer Extension with Cyclopropenylated 7‐Deaza‐2′‐Deoxyadenosine and Efficient Bioorthogonal Labeling in Vitro and in Living Cells
Chembiochem : a European journal of chemical biology, 2018Co-Authors: Damian Ploschik, Franziska Rönicke, Hanna Beike, Ralf Strasser, Hans-achim WagenknechtAbstract:A Deoxyadenosine Triphosphate (dATP) analogue for DNA labeling was synthesized with the 1-methylcyclopropene (1MCP) group at the 7-position of 7-deaza-2'-Deoxyadenosine and applied for primer extension experiments. The real-time kinetic data reveals that this 1MCP-modified dATP analogue is incorporated into DNA much faster than that of the similarly 1MCP-modified deoxyuridine Triphosphate (dUTP) analogue. The postsynthetic fluorescent labeling of these oligonucleotides works efficiently according to PAGE analysis, and can be applied for immobilization of a functional antibody on a surface. Site-specific labeling at two different positions in DNA was achieved and the bioorthogonality of the postsynthetic fluorescent labeling was demonstrated in living HeLa cells.
Piet Herdewijn - One of the best experts on this subject based on the ideXlab platform.
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3-Phosphono-L-alanine as pyrophosphate mimic for DNA synthesis using HIV-1 reverse transcriptase.
Organic & biomolecular chemistry, 2010Co-Authors: Shi-qiong Yang, Mathy Froeyen, Philippe Marlière, Eveline Lescrinier, Piet HerdewijnAbstract:A series of sulf(on)ate and phosph(on)ate amino acid phosphoramidate analogues of deoxynucleotides were synthesized as potential substrates for HIV-1 reverse transcriptase. Taurine, L-cysteic acid, 3-phosphono-L-alanine, O-sulfonato-L-serine, and O-phospho-L-serine were investigated as leaving groups in an enzyme catalyzed DNA synthesis protocol. Among these analogues, the phosphonate congener performed best and 3-phosphono-L-alanine can be considered as an excellent mimic of the pyrophosphate (PPi) moiety of Deoxyadenosine Triphosphate, to be used in enzymatic synthesis of nucleic acids. During a single nucleotide incorporation assay the use of 3-phosphono-L-Ala-dAMP as substrate resulted in 95% conversion to a P + 1 strand in 60 min at 50 μM (a concentration 10 times less than found for L-Asp-dAMP) and with improved incorporation kinetics and less stalling. For the sequences investigated, the efficiency of the incorporation is base dependent and decreases in the order (A ≥ T = G > C). In all cases, the incorporation follows Watson–Crick rules.
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Polymerase-catalyzed synthesis of DNA from phosphoramidate conjugates of deoxynucleotides and amino acids
Nucleic acids research, 2007Co-Authors: Olga Adelfinskaya, Montserat Terrazas, Mathy Froeyen, Philippe Marlière, Koen Nauwelaerts, Piet HerdewijnAbstract:Some selected amino acids, in particular L-aspartic acid (L-Asp) and L-histidine (L-His), can function as leaving group during polymerase-catalyzed incorporation of Deoxyadenosine monophosphate (dAMP) in DNA. Although L-Asp-dAMP and L-His-dAMP bind, most probably, in a different way in the active site of the enzyme, aspartic acid and histidine can be considered as mimics of the pyrophosphate moiety of Deoxyadenosine Triphosphate. L-Aspartic acid is more efficient than D-aspartic acid as leaving group. Such P-N conjugates of amino acids and deoxynucleotides provide a novel experimental ground for diversifying nucleic acid metabolism in the field of synthetic biology.
Vinalia Tjong - One of the best experts on this subject based on the ideXlab platform.
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direct fluorescence detection of rna on microarrays by surface initiated enzymatic polymerization
Analytical Chemistry, 2013Co-Authors: Vinalia Tjong, Angus Hucknall, Hua Yu, Ashutosh ChilkotiAbstract:We report the first demonstration of surface-initiated enzymatic polymerization (SIEP) for the direct detection of RNA in a fluorescence microarray format. This new method incorporates multiple fluorophores into an RNA strand using the two-step sequential and complementary reactions catalyzed by yeast poly(A) polymerase (PaP) to incorporate Deoxyadenosine Triphosphate (dATP) at the 3′–OH of an RNA molecule, followed by terminal deoxynucleotidyl transferase (TdT) to catalyze the sequential addition of a mixture of natural and fluorescent deoxynucleotides (dNTPs) at the 3′–OH of an RNA–DNA hybrid. We found that the 3′-end of RNA can be efficiently converted into DNA (∼50% conversion) by polymerization of dATP using yeast PaP, and the short DNA strand appended to the end of the RNA by PaP acts as the initiator for the TdT-catalyzed polymerization of longer DNA strands from a mixture of natural and fluorescent dNTPs that contain up to ∼45 Cy3 fluorophores per 1 kb DNA. We obtained an ∼2 pM limit of detection ...