Biochemical properties of oligo[(+)]-carbocyclic-thymidylates] and their complexes

Sági, János; Szemzö, Attila; Szécsi, Judit; Ötvös, László

doi:10.1093/nar/18.8.2133

Cited by 30 publications

(9 citation statements)

References 40 publications

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“…There has been very little investigation of the role played by the sugar moiety in such recognition processes usually for the very good reason that any significant change in the sugar moiety leads to a structure incompatible with naturally occurring polymers and hence makes their study less interesting. Any modifications to the sugar unit have been made primarily to increase the stability of the phosphodiester bond toward exonucleases thus enhancing the ability of the oligodeoxynucleotides to act as antisense inhibitors of gene expression (9)(10)(11)(12)(13)(14). A large variety of nucleoside analogues containing modified glycones have been prepared as potential chemotherapeutic agents (15) but most are unsuitable for incorporation into oligodeoxynucleotides for the reason described above.…”

Section: Introductionmentioning

confidence: 99%

Synthesis and properties of oligodeoxynucleotides containing the analogue 2′-deoxy-4′-thiothemidine

Hancox

Connolly

Walker³

1993

Nucl Acids Res

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The 2'-deoxythymidine analogue 2'-deoxy-4'-thiothymidine has been incorporated, using standard methodology, into a series of dodecadeoxynucleotides containing the EcoRV restriction endonuclease recognition site (GATATC). The stability of these oligodeoxynucleotides and their ability to act as substrates for the restriction endonuclease and associated methylase have been compared with a normal unmodified oligodeoxynucleotide. No problems were encountered in the synthesis despite the presence of a potentially oxidisable sulfur atom in the sugar ring. The analogue had very little effect on the melting temperature of the self-complementary oligoeoxynucleotides so synthesised and all had a CD spectrum compatible with a B-DNA structure. The oligodeoxynucleotide containing one analogue in each strand within the recognition site, adjacent to the bond to be cleaved (i.e. GAXATC, where X is 2'-deoxy-4'-thiothymidine), was neither a substrate for the endonuclease nor was recognized by the associated methylase. When still within the recognition hexanucleotide but two further residues removed from the site of cleavage (i.e. GATAXC), the oligodeoxynucleotide was a poor substrate for both the endonuclease and methylase. Binding of the oligodeoxynucleotide to the endonuclease was unaffected but the kcat value was only 0.03% of the value obtained for the parent oligodeoxynucleotide. These results show that the incorporation of 2'-deoxy-4'-thionucleosides into synthetic oligodeoxynucleotides may shed light on subtle interactions between proteins and their normal substrates and may also show why 2'-deoxy-4'-thiothymidine itself is so toxic in cell culture.

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Section: Introductionmentioning

confidence: 99%

Synthesis and properties of oligodeoxynucleotides containing the analogue 2′-deoxy-4′-thiothemidine

Hancox

Connolly

Walker³

1993

Nucl Acids Res

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“…Such nucleosides are easily accessible by synthesis, [24] are structurally isomorphous to natural nucleosides, and are known to base-pair with complementary nucleosides much in the same way as their natural congeners. [25,26] At the same time this is a way to determine which anomeric form is responsible for the observed affinities.…”

mentioning

confidence: 99%

A Parallel Screen for the Discovery of Novel DNA Base Pairs

2011

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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...

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“…b To avoid potential N‐glycosidic cleavage (analogous to depurination), our synthetic design started with the elaboration of the J AT heterocycle onto a carbocyclic deoxyribose analogue. In addition, this carbocycle was also chosen because it provides enhanced stability of duplexes and triplexes in other contexts 16. Thus, herein, we report an efficient gram‐scale synthesis of the first carbocyclic J AT phosphoramidite and its incorporation into oligonucleotides.…”

Section: Introductionmentioning

confidence: 99%

Base‐Pairing Behavior of a Carbocyclic Janus‐AT Nucleoside Analogue Capable of Recognizing A and T within a DNA Duplex

et al. 2013

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Janus-type nucleosides are heterocycles with two faces, each of which is designed to complement the H-bonding interactions of natural nucleosides comprising a canonical Watson-Crick base pair. By intercepting all of the hydrogen bonds contained within the base pair, oligomeric Janus nucleosides are expected to achieve sequence-specific DNA recognition through the formation of J-loops that will be more stable than D-loops, which simply replaces one base-pair with another. Herein, we report the synthesis of a novel Janus-AT nucleoside analogue, JAT , affixed on a carbocyclic analogue of deoxyribose that was converted to the corresponding phosphoramidite. A single JAT was successfully incorporated into a DNA strand by solid phase for targeting both A and T bases, and characterized through biophysical and computational methods. Experimental UV-melting and circular dichroism data demonstrated that within the context of a standard duplex, JAT associates preferentially with T over A, and much more poorly with C and G. Density functional theory calculations confirm that the JAT structure is well suited to associate only with A and T thereby highlighting the importance of the electronic structure in terms of H-bonding. Finally, molecular dynamics simulations validated the observation that JAT can substitute more effectively as an A-analogue than as a T-analogue without substantial distortion of the B-helix. Overall, this new Janus nucleotide is a promising tool for the targeting of A-T base pairs in DNA, and will lead to the development of oligo-Janus-nucleotide strands for sequence-specific DNA recognition.

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Biochemical properties of oligo[(+)]-carbocyclic-thymidylates] and their complexes

Cited by 30 publications

References 40 publications

Synthesis and properties of oligodeoxynucleotides containing the analogue 2′-deoxy-4′-thiothemidine

Synthesis and properties of oligodeoxynucleotides containing the analogue 2′-deoxy-4′-thiothemidine

A Parallel Screen for the Discovery of Novel DNA Base Pairs

Base‐Pairing Behavior of a Carbocyclic Janus‐AT Nucleoside Analogue Capable of Recognizing A and T within a DNA Duplex

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