Enzymatic Synthesis of DNA with an Expanded Genetic Alphabet Using Terminal Deoxynucleotidyl Transferase.
ACS Synth Biol
; 11(12): 4142-4155, 2022 12 16.
Article
em En
| MEDLINE
| ID: mdl-36455255
ABSTRACT
Development of unnatural base pairs (UBPs) has significantly expanded the genetic alphabet both in vitro and in vivo and led to numerous potential applications in the biotechnology and biopharmaceutical industry. Efficient synthesis of oligonucleotides containing unnatural nucleobases is undoubtedly an essential prerequisite for making full use of the UBPs, and de novo synthesis of oligonucleotides with terminal deoxynucleotidyl transferases (TdTs) has emerged as a method of great potential to overcome limitations of traditional solid-phase synthesis. Herein, we report the efficient template-independent incorporation of nucleotides of unnatural nucleobases dTPT3 and dNaM, which have been designed to make one of the most successful UBPs to date, dTPT3-dNaM, into DNA oligonucleotides with a TdT enzyme under optimized conditions. We also demonstrate the efficient TdT incorporation of dTPT3 derivatives with different functional linkers into oligonucleotides for orthogonal labeling of nucleic acids and applications thereof. The development of a method for the daily laboratory preparation of DNAs with UBPs at arbitrary sites with the assistance of TdT is also described.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA Nucleotidilexotransferase
/
DNA Polimerase Dirigida por DNA
Idioma:
En
Revista:
ACS Synth Biol
Ano de publicação:
2022
Tipo de documento:
Article