Selbstreparierende DNA basierend auf einem reduktiven Elektronentransfer durch den Basenstapel

Schwögler, Anja; Burgdorf, Lars T.; Carell, Thomas

doi:10.1002/1521-3757(20001103)112:21<4082::aid-ange4082>3.0.co;2-k

Angewandte Chemie

2000

DOI: 10.1002/1521-3757(20001103)112:21<4082::aid-ange4082>3.0.co;2-k

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Selbstreparierende DNA basierend auf einem reduktiven Elektronentransfer durch den Basenstapel

Anja Schwögler

Lars T. Burgdorf

Thomas Carell

Abstract: Lichtinduzierte Selbstreparatur wird beobachtet in DNA‐Strängen, die mit einem Flavinrest und einem UV‐Licht‐induzierten Schaden modifiziert wurden. Die Grundlage für die Reparatur ist ein lichtgetriebener Elektronentransfer vom Flavinrest zum Dimerschaden (siehe schematische Darstellung). Dieser Prozess findet sogar statt, wenn der DNA‐Schaden und der Flavinrest durch ein Basenpaar getrennt sind.

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Cited by 15 publications

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“…The large excess of NADH with respect to the flavin (20:1-2000:1), as well as the lack of flavin fluorescence below 470 nm and the limited absorption of the flavin between 290-350 nm, prevents flavin absorption/fluorescence from interfering under the assay conditions used. [15,16] In Figure 2 is shown the effect on NADH oxidation at 25 8C of adding increasing amounts of 5, a 41-mer oligonucleotide containing a 20-mer sequence complementary to that of 3, to a 10 mm solution of conjugate 3. We determined a melting temperature (T m ) value of 62 8C for the 3:5 duplex under the conditions of the assay and verified that the presence of Fre has no effect on this value.…”

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DNA Detection through Signal Amplification by Using NADH:Flavin Oxidoreductase and Oligonucleotide–Flavin Conjugates as Cofactors

et al. 2005

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Sensitive detection of DNA on the basis of hybridization to a complementary DNA probe and complex-specific signal (for example, fluorescence) detection, may be improved by molecular amplification methods.[1] DNA target amplification is one of the most widely used methods and is mainly based on the polymerase chain reaction (PCR). Recently, signalamplification techniques based on catalytic reactions, which might be useful for PCR-independent detection of label-free DNA sequences, have also been investigated. [2][3][4][5] These methods allow each probe-hybridization event to be converted into many signal events because the catalyst (a chemical or an enzyme) turns over many copies of the sensing-reaction substrate. As a consequence, high sensitivity can be attained. For example, the insertion of ferrocene moieties or redox-active intercalators allows hybrids to catalyze electrochemical reactions that can be monitored either amperometrically or chemically. [2,3] Herein we propose an original and simple strategy that involves the cofactor of an enzyme as the catalytic species. In this system, the DNA probe is an oligonucleotide covalently attached to the cofactor, and the enzyme is selected on the basis of its ability to catalyze the cofactor-dependent conversion of a fluorogenic substrate into an optically silent product (Figure 1). If the enzyme is functional only with a single-stranded cofactoroligonucleotide conjugate, and not when the latter is hybridized to its complementary strand, enzymatic conversion of the substrate, monitored by fluorescence spectroscopy, can serve as a tool to differentiate whether the probe is hybridized or not (Figure 1). Since the enzyme turns over many copies of the fluorogenic substrate, the difference in the fluorescence signals obtained with the free and the hybridized probes can be greatly amplified enzymatically.This new concept is illustrated herein with an enzyme that catalyzes the oxidation of reduced pyridine nucleotides, either nicotinamide adenine dinucleotide phosphate (NADPH) or nicotinamide adenine dinucleotide (NADH), by molecular oxygen in the presence of a riboflavin, either flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD), as a cofactor. Such an enzyme is called an NAD(P)H:flavin oxidoreductase or flavin reductase.[6] We selected the flavin reductase from Escherichia coli, named Fre, which is soluble, monomeric, and very easy to purify in large amounts. [7,8] Structural, mechanistic, and substrate-specificity studies in our laboratory [9][10][11][12] have shown that Fre contains an active site which accomodates both the flavin and the reduced pyridine nucleotide and that the reaction proceeds in two steps (Scheme 1): first, a hydride transfer from NAD(P)H to the oxidized flavin and then an oxidation of the reduced flavin by molecular oxygen, thereby regenerating the cofactor for a new cycle. With small amounts of flavin it is thus possible to oxidize large excesses of NAD(P)H, a process that can be easily monitored spectrophotometrically since NAD(P)H ...

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DNA Detection through Signal Amplification by Using NADH:Flavin Oxidoreductase and Oligonucleotide–Flavin Conjugates as Cofactors

et al. 2005

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Weak Distance Dependence of Excess Electron Transfer in DNA

et al. 2002

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Reduktive Fernreparatur von Thymin‐Dimeren in einem DNA‐Duplex durch Transfer von Überschusselektronen über eine Entfernung bis zu ca. 24 Å (n=7) lässt sich auf thermisch aktiviertes Elektronen‐„Hüpfen“ zurückführen (siehe Schema). Eine mögliche Konsequenz: Die schädigende Wirkung von UV‐Strahlung als eine Ursache der Entstehung von Hautkrebs könnte durch Verbindungen unterdrückt werden, die an DNA binden und weit reichenden Elektronentransport ermöglichen.

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Weak Distance Dependence of Excess Electron Transfer in DNA

Behrens

Burgdorf

Schwögler

et al. 2002

Angew. Chem. Int. Ed.

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Remote reductive repair of thymine dimers in a DNA duplex by transfer of excess electrons over a distance of up to roughly 24 Å (n=7) has been attributed to thermally activated hopping (see scheme). Possible consequences for humans: the harmful effect of UV irradiation responsible for the development of skin cancer could potentially be reduced by compounds that bind to DNA and trigger long‐range electron transport.

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Selbstreparierende DNA basierend auf einem reduktiven Elektronentransfer durch den Basenstapel

Cited by 15 publications

References 36 publications

DNA Detection through Signal Amplification by Using NADH:Flavin Oxidoreductase and Oligonucleotide–Flavin Conjugates as Cofactors

DNA Detection through Signal Amplification by Using NADH:Flavin Oxidoreductase and Oligonucleotide–Flavin Conjugates as Cofactors

Weak Distance Dependence of Excess Electron Transfer in DNA

Weak Distance Dependence of Excess Electron Transfer in DNA

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