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Enzymatic synthesis of chemical nuclease triplex-forming oligonucleotides with gene-silencing applications

Enzymatic synthesis of chemical nuclease triplex-forming oligonucleotides with gene-silencing applications
Enzymatic synthesis of chemical nuclease triplex-forming oligonucleotides with gene-silencing applications
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.
0305-1048
5467-5481
McGorman, Bríonna
69a336b1-4e70-488d-abc9-1f180acb55dc
Zuin Fantoni, Nicolò
fe40bd5c-cf93-40da-893c-1fec71845020
O’Carroll, Sinéad
31fce886-2d44-41ca-8077-12505c91dcf5
Ziemele, Anna
e452b188-32fa-4a25-b47e-c9e0c2632a16
El-Sagheer, Afaf H.
05b8295a-64ad-4fdf-ad57-c34934a46c04
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Kellett, Andrew
1de5738f-a91a-410c-96b6-c6b016b4a1c2
McGorman, Bríonna
69a336b1-4e70-488d-abc9-1f180acb55dc
Zuin Fantoni, Nicolò
fe40bd5c-cf93-40da-893c-1fec71845020
O’Carroll, Sinéad
31fce886-2d44-41ca-8077-12505c91dcf5
Ziemele, Anna
e452b188-32fa-4a25-b47e-c9e0c2632a16
El-Sagheer, Afaf H.
05b8295a-64ad-4fdf-ad57-c34934a46c04
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Kellett, Andrew
1de5738f-a91a-410c-96b6-c6b016b4a1c2

McGorman, Bríonna, Zuin Fantoni, Nicolò, O’Carroll, Sinéad, Ziemele, Anna, El-Sagheer, Afaf H., Brown, Tom and Kellett, Andrew (2022) Enzymatic synthesis of chemical nuclease triplex-forming oligonucleotides with gene-silencing applications. Nucleic Acids Research, 50 (10), 5467-5481. (doi:10.1093/nar/gkac438).

Record type: Article

Abstract

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.

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Accepted/In Press date: 9 May 2022
e-pub ahead of print date: 30 May 2022
Published date: 10 June 2022

Identifiers

Local EPrints ID: 478406
URI: http://eprints.soton.ac.uk/id/eprint/478406
ISSN: 0305-1048
PURE UUID: 3b7603ae-db7e-4aab-b3a6-b43d52749c04
ORCID for Afaf H. El-Sagheer: ORCID iD orcid.org/0000-0001-8706-1292

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Date deposited: 30 Jun 2023 16:33
Last modified: 17 Mar 2024 03:04

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Contributors

Author: Bríonna McGorman
Author: Nicolò Zuin Fantoni
Author: Sinéad O’Carroll
Author: Anna Ziemele
Author: Afaf H. El-Sagheer ORCID iD
Author: Tom Brown
Author: Andrew Kellett

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