Synthesis and Incorporation into DNA of a Chemically Stable, Functional Abasic Site Analogue

Mosimann, Markus; Küpfer, Pascal A.; Leumann, Christian J.

doi:10.1021/ol052045g

Cited by 7 publications

(10 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…X structurally deviates from a natural DNA abasic site by the replacement of O(3') by a CH 2 group which renders it chemically stable towards base-or heat-induced strand cleavage at the 3'-end. [17] These duplexes were then incubated with a library of heterocyclic amines in a parallel fashion. During this treatment the amines become covalently attached to X through the formation of a hemiaminal, resulting in exo-amino nucleosides that are structurally similar to the natural nucleosides.…”

mentioning

confidence: 99%

A Parallel Screen for the Discovery of Novel DNA Base Pairs

2011

Self Cite

View full text Add to dashboard Cite

The genetic information in DNA is encoded in the sequence of the four nucleobases, and its readout is based on the highly specific Watson-Crick pairing A-T and G-C. Over the years there has been growing interest in expanding this genetic alphabet by adding novel, orthogonal base pairs. Such additional base pairs could be used in biotechnology to introduce nonnatural amino acids into engineered proteins, [1][2][3] and to create novel tools for identifying aberrant or exogenous, disease-related nucleic acids. [4][5][6][7][8] The degree of freedom in designing novel base pairs, however, is limited by the structural scaffold of the double helix. The most logical approach is based on structurally isomorphic bases with an alternative Watson-Crick-like hydrogen-bonding network. [6,9,10] Alternatively, nonisomorphic aromatic heterocycles can serve as base replacements; they recognize each other by means other than classical hydrogen bonding and can be replicated and transcribed with high fidelity and similar kinetic properties. [11][12][13][14][15][16] This latter approach provides access to a larger structural space for potential base replacements. Unfortunately this approach is hampered by a lack of design rules which makes it necessary to screen a large number of potential candidates. From each potential base analogue the corresponding nucleoside must first be synthesized and incorporated into oligonucleotides before its coding properties can be evaluated. This is associated with multistep syntheses for each nucleoside candidate which is a timeconsuming procedure.To address these drawbacks we set out to design an assay for the rapid parallel screening of novel potential base candidates out of a library of heterocyclic amines. In a first step we identified selective, high-affinity binders to natural nucleobases. The assay is based on a dodecamer DNA duplex which carries an abasic site X in the center and a fluorescencequencher pair on one end of the duplex (Scheme 1). X structurally deviates from a natural DNA abasic site by the replacement of O(3') by a CH 2 group which renders it chemically stable towards base-or heat-induced strand cleavage at the 3'-end.[17] These duplexes were then incubated with a library of heterocyclic amines in a parallel fashion. During this treatment the amines become covalently attached to X through the formation of a hemiaminal, resulting in exoamino nucleosides that are structurally similar to the natural nucleosides. The thermal stabilities of these duplexes, which reflect the relative affinity of each amine to its target, were then determined by parallel fluorescence measurements of the melting temperature T m (see the Supporting Information).To validate the assay we searched a library of 34 randomly chosen, commercially available heterocyclic amines (Scheme 2) for individuals that bind selectively with high affinity to each of the four natural nucleobases. Duplexes (0.6 mm) were incubated with a 1000-fold excess of the amines in buffer at pH 8 for two days at 55 8C to form the hemiamin...

show abstract

mentioning

confidence: 99%

A Parallel Screen for the Discovery of Novel DNA Base Pairs

2011

Self Cite

View full text Add to dashboard Cite

show abstract

“…Oligonucleotides containing the synthetic 3CAPS AP-sites were synthesised as described previously ( 12 ). They were purified by ion exchange HPLC (Dionex DNA Pac PA 200; Thermo Fisher Scientific, Reinach, Switzerland), desalted on SepPak columns (Waters Corporation, Milford, MA, USA) and drop dialysed on Millipore MF-membrane filters.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, the hemiacetal is proposed to stabilise and optimise the orientation of the AP–site in the active-site pocket of the major mammalian AP-endonucleases APE1 ( 11 ). By contrast, another more recently introduced AP–site analogue, 3CAPS ( 3 ′– c arbon AP – s ite), combines the full natural functionality with complete stability at the 3′–centre, even under alkaline conditions ( 12 ). This was achieved by the conversion of the 3′–oxygen of an AP-site into a methylene unit (Figure 1A ).…”

Section: Introductionmentioning

confidence: 99%

“… The 3CAPS AP–site analogue is chemically stable in physiological buffers. ( A ) Chemical structures of the natural AP-site (AP) and the analogues 3CAPS ( 12 ) and tetrahydrofuran (THF) ( 9 ), included in the synthetic repair substrates with a 5′–fluorescein label (F). ( B ) DNA substrates containing either G•U mismatches (MM), enzymatically produced AP-sites or 3CAPS were incubated in a physiological buffer supplemented with 130 μg/ml protein (BSA) at 37°C.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair

et al. 2016

Self Cite

View full text Add to dashboard Cite

Abasic sites (AP-sites) are frequent DNA lesions, arising by spontaneous base hydrolysis or as intermediates of base excision repair (BER). The hemiacetal at the anomeric centre renders them chemically reactive, which presents a challenge to biochemical and structural investigation. Chemically more stable AP-site analogues have been used to avoid spontaneous decay, but these do not fully recapitulate the features of natural AP–sites. With its 3′–phosphate replaced by methylene, the abasic site analogue 3CAPS was suggested to circumvent some of these limitations. Here, we evaluated the properties of 3CAPS in biochemical BER assays with mammalian proteins. 3CAPS-containing DNA substrates were processed by APE1, albeit with comparably poor efficiency. APE1-cleaved 3CAPS can be extended by DNA polymerase β but repaired only by strand displacement as the 5′–deoxyribophosphate (dRP) cannot be removed. DNA glycosylases physically and functionally interact with 3CAPS substrates, underlining its structural integrity and biochemical reactivity. The AP lyase activity of bifunctional DNA glycosylases (NTH1, NEIL1, FPG), however, was fully inhibited. Notably, 3CAPS-containing DNA also effectively inhibited the activity of bifunctional glycosylases on authentic substrates. Hence, the chemically stable 3CAPS with its preserved hemiacetal functionality is a potent tool for BER research and a potential inhibitor of bifunctional DNA glycosylases.

show abstract

“…Dies verhindert einen basen-oder wärmeinduzierten Strangbruch am 3'-Ende. [17] Diese Duplexe wurden parallel mit einer Vielfalt an heterocyclischen Aminen inkubiert, wobei die Amine unter Bildung einer Halbaminalfunktion kovalent an X banden. Die daraus resultierenden exo-Aminonucleoside sind den natürlichen Nucleosiden strukturell ähnlich.…”

unclassified

Ein paralleles Testverfahren zur Entdeckung neuer DNA‐Basenpaare

2011

Self Cite

View full text Add to dashboard Cite

In der DNA ist der genetische Code durch die Abfolge der vier Nucleobasen festgelegt, und die Weitergabe der genetischen Information erfolgt über die hochspezifischen WatsonCrick-Paarungen A-T und G-C. In den letzten Jahren entstand ein reges Interesse, dem bereits bestehenden genetischen Alphabet zusätzliche, orthogonale Basenpaare hinzuzufügen. Solche zusätzlichen Basenpaare könnten in der Biotechnologie zur Einführung nichtnatürlicher Aminosäu-ren in Proteine herangezogen [1][2][3] oder zur Entwicklung neuer Werkzeuge zur zuverlässigen Erkennung fehlerhafter oder fremder Nucleinsäuren verwendet werden. [4][5][6][7][8] Allerdings sind die Freiheitsgrade beim Entwurf neuer Basenpaare durch das gegebene Strukturgerüst der Doppelhelix beschränkt. Ein naheliegender Ansatz basiert deshalb auf strukturell isomorphen Basen mit alternativem Watson-Crick-ähnlichem Wasserstoffbrückenmuster.[6, 9, 10] Doch auch nichtisomorphe aromatische Heterocyclen, die sich gegenseitig nicht durch Wasserstoffbrücken, sondern durch andere intermolekulare Phänomene erkennen, sind mit hoher Präzision und mit kinetischen Eigenschaften ähnlich denen der natürlichen Basen replizier-und transkribierbar. [11][12][13][14][15][16] Durch diesen Ansatz ist die Bandbreite an möglichen Strukturen erheblich größer geworden, doch es fehlen ihm leider Regeln für den Basenentwurf, was das Durchprüfen einer großen Zahl potenzieller Kandidaten nötig macht. Von jeder potenziellen Base muss dazu zuerst das entsprechende Nucleosid synthetisiert und in DNA eingebaut werden, und erst danach können dessen Eigenschaften getestet werden -ein zeitraubendes Unterfangen.Um hier Abhilfe zu schaffen, haben wir ein Verfahren entwickelt, mit dem eine Bibliothek von heterocyclischen Aminen im Parallelverfahren auf potenzielle Basenkandidaten getestet werden kann. In einem ersten Schritt suchten wir nach Molekülen, die selektiv und hoch effizient natürliche Nucleobasen erkennen. Der Test beruht auf DNA-Dodecamer-Duplexen mit einer abasischen Stelle X im Zentrum und einem Fluoreszenzlöscherpaar an einem Ende (Schema 1). X unterscheidet sich strukturell von einer natürlichen abasischen DNA-Stelle durch Ersatz des O(3') durch eine CH 2 -Gruppe. Dies verhindert einen basen-oder wärmeinduzierten Strangbruch am 3'-Ende.[17] Diese Duplexe wurden parallel mit einer Vielfalt an heterocyclischen Aminen inkubiert, wobei die Amine unter Bildung einer Halbaminalfunktion kovalent an X banden. Die daraus resultierenden exo-Aminonucleoside sind den natürlichen Nucleosiden strukturell ähnlich. Durch Messung der thermischen Stabilität der Duplexe mittels Fluoreszenzschmelzkurven kann die relative Affinität jedes Amins zu jeder der vier natürlichen Nucleobasen bestimmt werden (siehe die Hintergrundinformationen).Zur Validierung des Testverfahrens suchten wir in einer Bibliothek von 34 willkürlich gewählten, kommerziell erhältlichen heterocyclischen Aminen (Schema 2) nach Kandidaten, die selektiv und mit hoher Affinität an alle vier natürlichen Nucleobasen binden. Um den Gleichgewichtszustan...

show abstract

Synthesis and Incorporation into DNA of a Chemically Stable, Functional Abasic Site Analogue

Cited by 7 publications

References 13 publications

A Parallel Screen for the Discovery of Novel DNA Base Pairs

A Parallel Screen for the Discovery of Novel DNA Base Pairs

3CAPS – a structural AP–site analogue as a tool to investigate DNA base excision repair

Ein paralleles Testverfahren zur Entdeckung neuer DNA‐Basenpaare

Contact Info

Product

Resources

About