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Development of a 'clickable' non-natural nucleotide to visualize the replication of non-instructional DNA lesions.

Motea EA, Lee I, Berdis AJ - Nucleic Acids Res. (2011)

Bottom Line: The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health.This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions.In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using 'click' chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.

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Non-natural nucleotides as probes for TLS. (A) Comparison of the structures for an abasic site with that for a tetrahydrofuran moiety, the stable and non-reactive mimetic for an abasic site. (B) Structures of dATP and 5-NITP, a prototypical non-natural nucleotide that is selectively and efficiently incorporated opposite an abasic site. (C) Strategy for using ‘clickable’ nucleotides to monitor TLS. (D) Synthesis of 3-Eth-5-NITP using the following reagents and conditions: (a) I2, KOH, DMF (b) i. NaH, 1-α-chloro-3,5-di-(O-p-toluoyl)-2-deoxy-d-ribose, anhydrous ACN, RT, 16 h; ii. NaOMe, MeOH, pH > 12, RT, 16 h; (c) Pd(PPh3)2Cl2, CuI, triethylamine, trimethylsilylacetylene, anhydrous THF, RT, 3 h; (d) 1 M TBAF, THF, RT, 3 h; (e) i. POCl3, Proton Sponge®, trimethylphosphate, 0°C; ii. Tributylammonium pyrophosphate, DMF, tributylamine, RT, 15 min; iii. 1 M TEAB, RT, 2 h.
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gkr980-F1: Non-natural nucleotides as probes for TLS. (A) Comparison of the structures for an abasic site with that for a tetrahydrofuran moiety, the stable and non-reactive mimetic for an abasic site. (B) Structures of dATP and 5-NITP, a prototypical non-natural nucleotide that is selectively and efficiently incorporated opposite an abasic site. (C) Strategy for using ‘clickable’ nucleotides to monitor TLS. (D) Synthesis of 3-Eth-5-NITP using the following reagents and conditions: (a) I2, KOH, DMF (b) i. NaH, 1-α-chloro-3,5-di-(O-p-toluoyl)-2-deoxy-d-ribose, anhydrous ACN, RT, 16 h; ii. NaOMe, MeOH, pH > 12, RT, 16 h; (c) Pd(PPh3)2Cl2, CuI, triethylamine, trimethylsilylacetylene, anhydrous THF, RT, 3 h; (d) 1 M TBAF, THF, RT, 3 h; (e) i. POCl3, Proton Sponge®, trimethylphosphate, 0°C; ii. Tributylammonium pyrophosphate, DMF, tributylamine, RT, 15 min; iii. 1 M TEAB, RT, 2 h.

Mentions: To investigate the molecular basis for the ‘A-rule’ catalyzed by high-fidelity polymerases, we developed a series of non-natural nucleotides that mimic dATP and characterized their incorporation opposite DNA containing a tetrahydrofuran moiety which is a stable and non-reactive mimetic for an abasic site (Figure 1A) (13–17). One particular analog, designated 5-nitroindolyl-2′-deoxynucleoside triphosphate (5-NITP) (Figure 1B), is noteworthy as it is incorporated opposite an abasic site with an incredibly high catalytic efficiency (kpol/Kd) of 106 M−1 s−1 by the high-fidelity bacteriophage T4 DNA polymerase (13,14). This kpol/Kd value is ~1000-fold higher than dATP, the preferred natural nucleotide substrate (18). Subsequent crystal structures of an exonuclease deficient variant of analogous DNA polymerase from bacteriophage RB69 showed that the non-natural nucleotide existed in an interhelical configuration when paired opposite the lesion (19). This thermodynamically favored configuration explains the enhanced kinetics of 5-NITP as the nucleotide is stabilized by dipole-induced stacking interactions between the 5-nitro group and the nucleobase that is 3′ to the lesion. Equally important, this nucleotide analog is poorly inserted opposite any of the four natural nucleobases, displaying low catalytic efficiencies of >103 M−1s−1 (13,14). In fact, 5-nitroindole was initially reported as a ‘universal nucleobase’ due to its ability to indiscriminately pair with each of the four natural nucleobases as defined by duplex melting experiments (20). However, while 5-nitroindole can indiscriminately pair with natural nucleobases, the corresponding nucleoside triphosphate was poorly incorporated opposite unmodified DNA (21,22). Indeed, our results showing that 5-NITP is incorporated opposite non-instructional DNA lesions with very high efficiencies (14) suggested that it could be further developed as a chemical probe to quantify the replication of this lesion. In addition, the high selectivity of this analog for damaged DNA could be used to differentiate replication of the lesion from that of normal DNA synthesis. This report provides a detailed analysis for the conversion of 5-NITP into a chemical probe that can visualize and quantify TLS. This was achieved using ‘click’ chemistry (23,24) to selectively introduce an ethynyl moiety at the 3-position of 5-NITP so that it can be tagged with a fluorogenic reporter after it is incorporated opposite a DNA lesion (Figure 1C).Figure 1.


Development of a 'clickable' non-natural nucleotide to visualize the replication of non-instructional DNA lesions.

Motea EA, Lee I, Berdis AJ - Nucleic Acids Res. (2011)

Non-natural nucleotides as probes for TLS. (A) Comparison of the structures for an abasic site with that for a tetrahydrofuran moiety, the stable and non-reactive mimetic for an abasic site. (B) Structures of dATP and 5-NITP, a prototypical non-natural nucleotide that is selectively and efficiently incorporated opposite an abasic site. (C) Strategy for using ‘clickable’ nucleotides to monitor TLS. (D) Synthesis of 3-Eth-5-NITP using the following reagents and conditions: (a) I2, KOH, DMF (b) i. NaH, 1-α-chloro-3,5-di-(O-p-toluoyl)-2-deoxy-d-ribose, anhydrous ACN, RT, 16 h; ii. NaOMe, MeOH, pH > 12, RT, 16 h; (c) Pd(PPh3)2Cl2, CuI, triethylamine, trimethylsilylacetylene, anhydrous THF, RT, 3 h; (d) 1 M TBAF, THF, RT, 3 h; (e) i. POCl3, Proton Sponge®, trimethylphosphate, 0°C; ii. Tributylammonium pyrophosphate, DMF, tributylamine, RT, 15 min; iii. 1 M TEAB, RT, 2 h.
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Related In: Results  -  Collection

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gkr980-F1: Non-natural nucleotides as probes for TLS. (A) Comparison of the structures for an abasic site with that for a tetrahydrofuran moiety, the stable and non-reactive mimetic for an abasic site. (B) Structures of dATP and 5-NITP, a prototypical non-natural nucleotide that is selectively and efficiently incorporated opposite an abasic site. (C) Strategy for using ‘clickable’ nucleotides to monitor TLS. (D) Synthesis of 3-Eth-5-NITP using the following reagents and conditions: (a) I2, KOH, DMF (b) i. NaH, 1-α-chloro-3,5-di-(O-p-toluoyl)-2-deoxy-d-ribose, anhydrous ACN, RT, 16 h; ii. NaOMe, MeOH, pH > 12, RT, 16 h; (c) Pd(PPh3)2Cl2, CuI, triethylamine, trimethylsilylacetylene, anhydrous THF, RT, 3 h; (d) 1 M TBAF, THF, RT, 3 h; (e) i. POCl3, Proton Sponge®, trimethylphosphate, 0°C; ii. Tributylammonium pyrophosphate, DMF, tributylamine, RT, 15 min; iii. 1 M TEAB, RT, 2 h.
Mentions: To investigate the molecular basis for the ‘A-rule’ catalyzed by high-fidelity polymerases, we developed a series of non-natural nucleotides that mimic dATP and characterized their incorporation opposite DNA containing a tetrahydrofuran moiety which is a stable and non-reactive mimetic for an abasic site (Figure 1A) (13–17). One particular analog, designated 5-nitroindolyl-2′-deoxynucleoside triphosphate (5-NITP) (Figure 1B), is noteworthy as it is incorporated opposite an abasic site with an incredibly high catalytic efficiency (kpol/Kd) of 106 M−1 s−1 by the high-fidelity bacteriophage T4 DNA polymerase (13,14). This kpol/Kd value is ~1000-fold higher than dATP, the preferred natural nucleotide substrate (18). Subsequent crystal structures of an exonuclease deficient variant of analogous DNA polymerase from bacteriophage RB69 showed that the non-natural nucleotide existed in an interhelical configuration when paired opposite the lesion (19). This thermodynamically favored configuration explains the enhanced kinetics of 5-NITP as the nucleotide is stabilized by dipole-induced stacking interactions between the 5-nitro group and the nucleobase that is 3′ to the lesion. Equally important, this nucleotide analog is poorly inserted opposite any of the four natural nucleobases, displaying low catalytic efficiencies of >103 M−1s−1 (13,14). In fact, 5-nitroindole was initially reported as a ‘universal nucleobase’ due to its ability to indiscriminately pair with each of the four natural nucleobases as defined by duplex melting experiments (20). However, while 5-nitroindole can indiscriminately pair with natural nucleobases, the corresponding nucleoside triphosphate was poorly incorporated opposite unmodified DNA (21,22). Indeed, our results showing that 5-NITP is incorporated opposite non-instructional DNA lesions with very high efficiencies (14) suggested that it could be further developed as a chemical probe to quantify the replication of this lesion. In addition, the high selectivity of this analog for damaged DNA could be used to differentiate replication of the lesion from that of normal DNA synthesis. This report provides a detailed analysis for the conversion of 5-NITP into a chemical probe that can visualize and quantify TLS. This was achieved using ‘click’ chemistry (23,24) to selectively introduce an ethynyl moiety at the 3-position of 5-NITP so that it can be tagged with a fluorogenic reporter after it is incorporated opposite a DNA lesion (Figure 1C).Figure 1.

Bottom Line: The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health.This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions.In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, USA.

ABSTRACT
The misreplication of damaged DNA is an important biological process that produces numerous adverse effects on human health. This report describes the synthesis and characterization of a non-natural nucleotide, designated 3-ethynyl-5-nitroindolyl-2'-deoxyriboside triphosphate (3-Eth-5-NITP), as a novel chemical reagent that can probe and quantify the misreplication of damaged DNA. We demonstrate that this non-natural nucleotide is efficiently inserted opposite an abasic site, a commonly formed and potentially mutagenic non-instructional DNA lesion. The strategic placement of the ethynyl moiety allows the incorporated nucleoside triphosphate to be selectively tagged with an azide-containing fluorophore using 'click' chemistry. This reaction provides a facile way to quantify the extent of nucleotide incorporation opposite non-instructional DNA lesions. In addition, the incorporation of 3-Eth-5-NITP is highly selective for an abasic site, and occurs even in the presence of a 50-fold molar excess of natural nucleotides. The biological applications of using 3-Eth-5-NITP as a chemical probe to monitor and quantify the misreplication of non-instructional DNA lesions are discussed.

Show MeSH