Enzymatic Synthesis of Chemical Nuclease Triplex-Forming Oligonucleotides with Gene-Silencing Applications.
Journal
Nucleic acids research
ISSN: 1362-4962
Titre abrégé: Nucleic Acids Res
Pays: England
ID NLM: 0411011
Informations de publication
Date de publication:
10 06 2022
10 06 2022
Historique:
accepted:
09
05
2022
revised:
10
04
2022
received:
01
02
2022
pubmed:
1
6
2022
medline:
11
6
2022
entrez:
31
5
2022
Statut:
ppublish
Résumé
Triplex-forming oligonucleotides (TFOs) are short, single-stranded oligomers that hybridise to a specific sequence of duplex DNA. TFOs can block transcription and thereby inhibit protein production, making them highly appealing in the field of antigene therapeutics. In this work, a primer extension protocol was developed to enzymatically prepare chemical nuclease TFO hybrid constructs, with gene-silencing applications. Click chemistry was employed to generate novel artificial metallo-nuclease (AMN)-dNTPs, which were selectively incorporated into the TFO strand by a DNA polymerase. This purely enzymatic protocol was then extended to facilitate the construction of 5-methylcytosine (5mC) modified TFOs that displayed increased thermal stability. The utility of the enzymatically synthesised di-(2-picolyl)amine (DPA)-TFOs was assessed and compared to a specifically prepared solid-phase synthesis counterpart through gel electrophoresis, quantitative PCR, and Sanger sequencing, which revealed similar recognition and damage properties to target genes. The specificity was then enhanced through coordinated designer intercalators-DPQ and DPPZ-and high-precision DNA cleavage was achieved. To our knowledge, this is the first example of the enzymatic production of an AMN-TFO hybrid and is the largest base modification incorporated using this method. These results indicate how chemical nuclease-TFOs may overcome limitations associated with non-molecularly targeted metallodrugs and open new avenues for artificial gene-editing technology.
Identifiants
pubmed: 35640595
pii: 6595289
doi: 10.1093/nar/gkac438
pmc: PMC9177962
doi:
Substances chimiques
Oligonucleotides
0
DNA
9007-49-2
Endonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
5467-5481Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/R008655/1
Pays : United Kingdom
Informations de copyright
© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.
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