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Enzyme-free synthesis of cyclic single-stranded DNA constructs containing a single triazole, amide or phosphoramidate backbone linkage and their use as templates for rolling circle amplification and nanoflower formation

Enzyme-free synthesis of cyclic single-stranded DNA constructs containing a single triazole, amide or phosphoramidate backbone linkage and their use as templates for rolling circle amplification and nanoflower formation
Enzyme-free synthesis of cyclic single-stranded DNA constructs containing a single triazole, amide or phosphoramidate backbone linkage and their use as templates for rolling circle amplification and nanoflower formation
Cyclic oligonucleotides are valuable targets with a broad range of potential applications spanning molecular biology and nanotechnology. Of particular importance is their role as templates in the rolling circle amplification (RCA) reaction. We describe three different chemical cyclisation methods for the preparation of single-stranded cyclic DNA constructs. These chemical cyclisation reactions are cheaper to carry out than the enzymatic reaction, and more amenable to preparative scale purification and characterisation of the cyclic product. They can also be performed under denaturing conditions and are therefore particularly valuable for cyclic DNA templates that contain secondary structures. The resulting single-stranded cyclic DNA constructs contain a single non-canonical backbone linkage at the ligation point (triazole, amide or phosphoramidate). They were compared to unmodified cyclic DNA in rolling circle amplification reactions using ϕ-29 and Bst 2.0 DNA polymerase enzymes. The cyclic templates containing a phosphoramidate linkage were particularly well tolerated by ϕ-29 polymerase, consistently performing as well in RCA as the unmodified DNA controls. Moreover, these phosphoramidate-modified cyclic constructs can be readily produced in oligonucleotide synthesis facilities from commercially available precursors. Phosphoramidate ligation therefore holds promise as a practical, scalable method for the synthesis of fully biocompatible cyclic RCA templates. The triazole-modified cyclic templates generally gave lower and more variable yields of RCA products, a significant proportion of which were double-stranded, while the performances of the templates containing an amide linkage lie in between those of the phosphoramidate- and triazole-containing templates.
1478-6524
8110-8120
Chen, Jinfeng
1be70334-2c20-485f-8b22-467053f130cb
Baker, Ysobel
4fceec1f-89ed-4a32-a753-8967daf6763a
Brown, Asha
a5a87d94-5857-487a-801b-3a918cb7542c
El-Sagheer, Afaf H
05b8295a-64ad-4fdf-ad57-c34934a46c04
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89
Chen, Jinfeng
1be70334-2c20-485f-8b22-467053f130cb
Baker, Ysobel
4fceec1f-89ed-4a32-a753-8967daf6763a
Brown, Asha
a5a87d94-5857-487a-801b-3a918cb7542c
El-Sagheer, Afaf H
05b8295a-64ad-4fdf-ad57-c34934a46c04
Brown, Tom
a64aae36-bb30-42df-88a2-11be394e8c89

Chen, Jinfeng, Baker, Ysobel, Brown, Asha, El-Sagheer, Afaf H and Brown, Tom (2018) Enzyme-free synthesis of cyclic single-stranded DNA constructs containing a single triazole, amide or phosphoramidate backbone linkage and their use as templates for rolling circle amplification and nanoflower formation. Chemical Science, 2018 (9), 8110-8120. (doi:10.1039/C8SC02952K).

Record type: Article

Abstract

Cyclic oligonucleotides are valuable targets with a broad range of potential applications spanning molecular biology and nanotechnology. Of particular importance is their role as templates in the rolling circle amplification (RCA) reaction. We describe three different chemical cyclisation methods for the preparation of single-stranded cyclic DNA constructs. These chemical cyclisation reactions are cheaper to carry out than the enzymatic reaction, and more amenable to preparative scale purification and characterisation of the cyclic product. They can also be performed under denaturing conditions and are therefore particularly valuable for cyclic DNA templates that contain secondary structures. The resulting single-stranded cyclic DNA constructs contain a single non-canonical backbone linkage at the ligation point (triazole, amide or phosphoramidate). They were compared to unmodified cyclic DNA in rolling circle amplification reactions using ϕ-29 and Bst 2.0 DNA polymerase enzymes. The cyclic templates containing a phosphoramidate linkage were particularly well tolerated by ϕ-29 polymerase, consistently performing as well in RCA as the unmodified DNA controls. Moreover, these phosphoramidate-modified cyclic constructs can be readily produced in oligonucleotide synthesis facilities from commercially available precursors. Phosphoramidate ligation therefore holds promise as a practical, scalable method for the synthesis of fully biocompatible cyclic RCA templates. The triazole-modified cyclic templates generally gave lower and more variable yields of RCA products, a significant proportion of which were double-stranded, while the performances of the templates containing an amide linkage lie in between those of the phosphoramidate- and triazole-containing templates.

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More information

Accepted/In Press date: 23 August 2018
e-pub ahead of print date: 24 August 2018

Identifiers

Local EPrints ID: 473685
URI: http://eprints.soton.ac.uk/id/eprint/473685
ISSN: 1478-6524
PURE UUID: 94725af1-8833-4216-a828-f5060a14262d
ORCID for Ysobel Baker: ORCID iD orcid.org/0000-0002-0266-771X
ORCID for Afaf H El-Sagheer: ORCID iD orcid.org/0000-0001-8706-1292

Catalogue record

Date deposited: 27 Jan 2023 17:48
Last modified: 17 Mar 2024 04:17

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Contributors

Author: Jinfeng Chen
Author: Ysobel Baker ORCID iD
Author: Asha Brown
Author: Afaf H El-Sagheer ORCID iD
Author: Tom Brown

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