Quick Click: The DNA-Templated Ligation of 3'-O-Propargyl- and 5'-Azide-Modified Strands Is as Rapid as and More Selective than Ligase.
Chembiochem
; 19(19): 2081-2087, 2018 10 04.
Article
en En
| MEDLINE
| ID: mdl-30059599
ABSTRACT
The copper(I)-mediated azide-alkyne cycloaddition (CuAAC) of 3'-propargyl ether and 5'-azide oligonucleotides is a particularly promising ligation system because it results in triazole linkages that effectively mimic the phosphate-sugar backbone of DNA, leading to unprecedented tolerance of the ligated strands by polymerases. However, for a chemical ligation strategy to be a viable alternative to enzymatic systems, it must be equally as rapid, as discriminating, and as easy to use. We found that the DNA-templated reaction with these modifications was rapid under aerobic conditions, with nearly quantitative conversion in 5â
min, resulting in a kobs value of 1.1â
min-1 , comparable with that measured in an enzymatic ligation system by using the highest commercially available concentration of T4 DNA ligase. Moreover, the CuAAC reaction also exhibited greater selectivity in discriminating CA or CT mismatches from the CG match than that of T4 DNA ligase at 29 °C; a temperature slightly below the perfect nicked duplex dissociation temperature, but above that of the mismatched duplexes. These results suggest that the CuAAC reaction of 3'-propargyl ether and 5'-azide-terminated oligonucleotides represents a complementary alternative to T4 DNA ligase, with similar reaction rates, ease of setup and even enhanced selectivity for certain mismatches.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Oligonucleótidos
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Azidas
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ADN
/
Alquinos
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Éteres
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Química Clic
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Reacción de Cicloadición
Idioma:
En
Revista:
Chembiochem
Asunto de la revista:
BIOQUIMICA
Año:
2018
Tipo del documento:
Article
País de afiliación:
Canadá
Pais de publicación:
ALEMANHA
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ALEMANIA
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DE
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DEUSTCHLAND
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GERMANY