New Light-Sensitive Nucleosides for Caged DNA Strand Breaks

Dussy, Adrian; Meyer, Christoph; Quennet, Edith; Bickle, Thomas A.; Giese, Bernd; Marx, Andreas

doi:10.1002/1439-7633(20020104)3:1<54::aid-cbic54>3.0.co;2-2

Cited by 37 publications

(32 citation statements)

References 24 publications

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“…The modified nucleotide 1 was incorporated as described in ref. [5]. DR1, transferred into the corresponding phosphoramidite, was coupled to the DNA (15 min).…”

Section: Methodsmentioning

confidence: 99%

“…In the matched case, the higher melting point prevents dehybridization.As the cleavage site we used the 5'-o-nitrophenyl thymidine (1, T*; Scheme 2), which selectively recognizes adenosine and quantitatively cleaves the DNA strand upon irradiation (360 nm) in solution, whereby the whole DNA strand is cleaved. [5] This modified nucleotide 1 was incorporated through standard phosphoramidite chemistry into the modified 21mer 2, which was immobilized on agarose (formation of 3; Scheme 3). [6] We used dispers red 1 (DR1) as a tag, [7] and directly observed the color changes of the beads after irradiation (10 8C, 10 min, l = 360 nm).…”

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confidence: 99%

“…[8] Irradiation of the immobilized single-strand 3 released 98 % of the cleavage product 4 into the solvent, and the beads became colorless. Thus, a quantitative cleavage of the modified strand occurs not only in solution [5] but also if the strand is immobilized on agarose. This result is in accord with other photolysis experiments with agarose as a solid support.…”

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confidence: 99%

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Site‐Specific DNA Cleavage on a Solid Support: A Method for Mismatch Detection

2007

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The growing insight into genetically caused diseases induced by single nucleotide polymorphisms (SNPs) calls for the development of analytical techniques to detect these SNPs. The techniques should be quick, reliable, and inexpensive. In recent years several strategies for genetic analyses were introduced, which are based on polymerase chain reactions (PCR), [1] enzymatic digestion, [2] electrochemistry, [3] or methods where target binding directly changes the fluorescence. [4] We now present a new concept for mismatch detection using chemical DNA cleavage on a solid support (Scheme 1): A single DNA strand, which carries a cleavage site at the central position and a detection tag at the 5'-end, is immobilized on the solid support through the 3'-end and hybridized with the target strand. Upon irradiation the immobilized single strand is cleaved and releases the tag-containing fragment into the solution if a mismatch is present. In the matched case, the higher melting point prevents dehybridization.As the cleavage site we used the 5'-o-nitrophenyl thymidine (1, T*; Scheme 2), which selectively recognizes adenosine and quantitatively cleaves the DNA strand upon irradiation (360 nm) in solution, whereby the whole DNA strand is cleaved.[5] This modified nucleotide 1 was incorporated through standard phosphoramidite chemistry into the modified 21mer 2, which was immobilized on agarose (formation of 3; Scheme 3).[6] We used dispers red 1 (DR1)as a tag, [7] and directly observed the color changes of the beads after irradiation (10 8C, 10 min, l = 360 nm). The results were quantified by HPLC analysis of the tagged fragment 4, which was released into the solutions by irradiation. Subsequent heating of the solid-phase system quantitatively released the remaining tag-containing fragment 4 into solution (Scheme 3, Experimental Section). [8] Irradiation of the immobilized single-strand 3 released 98 % of the cleavage product 4 into the solvent, and the beads became colorless. Thus, a quantitative cleavage of the modified strand occurs not only in solution [5] but also if the strand is immobilized on agarose. This result is in accord with other photolysis experiments with agarose as a solid support.[9] Double-strand formation was quantitative when the immobilized strands 3 were hybridized with a slight excess of target strands, even if one or two mismatches were introduced (Experimental Section). But in contrast to the single-strand experiment, irradiation of a matched double strand at 10 8C released only a small amount of 10mer 4 into the solution (15 %), and the beads remained red. Subsequent heating quantitatively released the cleaved strand 4 (the beads became colorless).[10] This behavior changed if mismatches were incorporated. Irradiation of double strands with two mismatches released 70-98 % of the 10mer 4 into solution at 10 8C (Table 1).The melting points of the photocleaved systems are decreased by two mismatches to such an extent that the tagcontaining fragment 4 is released into the solvent. Experiments in homogen...

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“…The modified nucleotide 1 was incorporated as described in ref. [5]. DR1, transferred into the corresponding phosphoramidite, was coupled to the DNA (15 min).…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Site‐Specific DNA Cleavage on a Solid Support: A Method for Mismatch Detection

2007

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“…However, only a limited number of photocleavable DNA ‘building blocs’ have been developed. Most consist of nucleoside analogs chemically inserted in DNA fragments and are based on the photochemical properties of the o -nitrobenzyl group (7–13). …”

Section: Introductionmentioning

confidence: 99%

Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog

Crey-Desbiolles

2005

Nucleic Acids Research

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Universal DNA base analogs having photocleavable properties would be of great interest for development of new nucleic acid fragmentation tools. The photocleavable 7-nitroindole 2′-deoxyribonucleoside d(7-Ni) was previously shown to furnish a highly efficient approach to photochemically trigger DNA backbone cleavage at preselected position when inserted in a DNA fragment. In the present report, we examine its potential use as universal DNA nucleoside, by analogy with the 5-nitroindole analog that is generally considered as universal base. The d(7-Ni) phosphoramidite was incorporated into oligonucleotides. Hybridization properties of resulting 11mer duplexes indicated a behavior close to that of the 5-nitroindole analog. Enzymatic recognition by Klenow fragment exonuclease-free using 40mers containing the unnatural bases as templates indicated notably a decrease of the polymerase activity with preferential incorporation of dAMP opposite both the 7-Ni and 5-Ni bases. Incorporation of the d(7-Ni) triphosphate was also studied indicating absence of significant differences between the incorporation kinetics opposite each natural base in the template. All the hybridization and enzymatic data indicate that 7-nitroindole can be considered as a cleavable base analog, although not strictly fulfilling, like the 5-nitro isomer, all properties required for a universal base.

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“…Dieses bildet mit Adenosin selektiv Basenpaare und wird bei Bestrahlung (360 nm) in Lösung quantitativ gespalten, wodurch auch der gesamte DNA-Strang gespalten wird. [5]…”

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Selektiver DNA‐Strangbruch an fester Phase – eine Methode zur Detektion von Basenfehlpaarungen

2007

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Die zurzeit intensive Erforschung genetisch bedingter Krankheiten, die durch einen Einzelnucleotidpolymorphismus (SNP) ausgelöst werden, bringt einen Bedarf an analytischen Methoden zur Detektion dieser SNPs mit sich. Diese sollen schnell, verlässlich und billig sein. Methoden, die in den vergangenen Jahren vorgestellt wurden, basieren auf der Polymerasekettenreaktion (PCR), [1] auf enzymatischem Verdau [2] und elektrochemischen Reaktionen [3] sowie auf Techniken, bei denen die Doppelstrangbildung mit der Zielsequenz direkt eine Fluoreszenzänderung bewirkt.[4] Wir präsentieren hier ein neues Konzept zur Detektion von Basenfehlpaarungen (mismatches), das auf der chemischen Spaltung von DNA an fester Phase beruht (Schema 1). Bei dieser Methode ist ein einzelner DNA-Strang, der eine Sollbruchstelle an der Strangmitte und einen Detektionsmarker am 5'-Ende enthält, über das 3'-Ende an eine Festphase angebracht. Der DNA-Strang bildet mit dem Zieloligonucleotid einen Doppelstrang. Bei Bestrahlung wird der DNA-Strang gespalten, und falls sich im Doppelstrang eine Basenfehlpaarung befindet, wird das Fragment mit der Markierung freigesetzt. Der höhere Schmelzpunkt im Fall korrekter Basenpaarung ("matched case") verhindert die Dehybridisierung des Doppelstrangs.Als Sollbruchstelle wird das modifizierte Nucleotid 5'-oNitrophenylthymidin (1, T*; Schema 2) in die DNA eingebaut. Dieses bildet mit Adenosin selektiv Basenpaare und wird bei Bestrahlung (360 nm) in Lösung quantitativ gespalten, wodurch auch der gesamte DNA-Strang gespalten wird.

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New Light-Sensitive Nucleosides for Caged DNA Strand Breaks

Cited by 37 publications

References 24 publications

Site‐Specific DNA Cleavage on a Solid Support: A Method for Mismatch Detection

Site‐Specific DNA Cleavage on a Solid Support: A Method for Mismatch Detection

Hybridization properties and enzymatic replication of oligonucleotides containing the photocleavable 7-nitroindole base analog

Selektiver DNA‐Strangbruch an fester Phase – eine Methode zur Detektion von Basenfehlpaarungen

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