Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography

Patrick Conlon, Olga Eguaogie, Jordan Wilson, Jamie Sweet, Julian Steinhoegl, Klaudia Englert , Oliver Hancox, Christopher Law, Sarah Allman, James H.R. Tucker, James Hall, Joseph Vyle

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Abstract

Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michealis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5ʹ-deoxythymidine-5ʹ-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then after chain extension using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 ̶ -3.1 °C per phosphoroselenolate) when introduced at the 5′-termini of A-form duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 Å. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.
Original languageEnglish
Article numberDOI: 10.1039/C9SC04098F
Number of pages12
JournalChemical Science
DOIs
Publication statusPublished - 11 Oct 2019

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X ray crystallography
Dimers
Organophosphonates
Oligodeoxyribonucleotides
DNA
Selenium
Crystallization
Nucleosides
Nucleic Acids
Purification
Mass spectrometry
Demonstrations
Derivatives
Substrates
phosphoramidite

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@article{0f2939a249354b7c8e9db49afacab28b,
title = "Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography",
abstract = "Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michealis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5ʹ-deoxythymidine-5ʹ-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then after chain extension using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 ̶ -3.1 °C per phosphoroselenolate) when introduced at the 5′-termini of A-form duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 {\AA}. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.",
author = "Patrick Conlon and Olga Eguaogie and Jordan Wilson and Jamie Sweet and Julian Steinhoegl and Klaudia Englert and Oliver Hancox and Christopher Law and Sarah Allman and Tucker, {James H.R.} and James Hall and Joseph Vyle",
year = "2019",
month = "10",
day = "11",
doi = "10.1039/C9SC04098F",
language = "English",
journal = "Chemical Science",
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publisher = "Royal Society of Chemistry",

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Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography. / Conlon, Patrick; Eguaogie, Olga; Wilson, Jordan; Sweet, Jamie; Steinhoegl, Julian; Englert , Klaudia; Hancox, Oliver; Law, Christopher; Allman, Sarah; Tucker, James H.R.; Hall, James; Vyle, Joseph.

In: Chemical Science, 11.10.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography

AU - Conlon, Patrick

AU - Eguaogie, Olga

AU - Wilson, Jordan

AU - Sweet, Jamie

AU - Steinhoegl, Julian

AU - Englert , Klaudia

AU - Hancox, Oliver

AU - Law, Christopher

AU - Allman, Sarah

AU - Tucker, James H.R.

AU - Hall, James

AU - Vyle, Joseph

PY - 2019/10/11

Y1 - 2019/10/11

N2 - Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michealis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5ʹ-deoxythymidine-5ʹ-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then after chain extension using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 ̶ -3.1 °C per phosphoroselenolate) when introduced at the 5′-termini of A-form duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 Å. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.

AB - Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites. These dimers were constructed following the high yielding Michealis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5ʹ-deoxythymidine-5ʹ-selenocyanate and subsequent phosphitylation. Efficient coupling of the dimer phosphoramidites to solid-supported substrates was observed under both manual and automated conditions and required only minor modifications to the standard DNA synthesis cycle. In a further demonstration of the utility of M-A chemistry, the support-bound selenonucleoside was reacted with an H-phosphonate and then after chain extension using phosphoramidite chemistry. Following initial unmasking of methyl-protected phosphoroselenolate diesters, pure oligodeoxynucleotides were isolated using standard deprotection and purification procedures and subsequently characterised by mass spectrometry and circular dichroism. The CD spectra of both modified and native duplexes derived from self-complementary sequences with A-form, B-form or mixed conformational preferences were essentially superimposable. These sequences were also used to study the effect of the modification upon duplex stability which showed context-dependent destabilisation (-0.4 ̶ -3.1 °C per phosphoroselenolate) when introduced at the 5′-termini of A-form duplexes or at juxtaposed central loci within a B-form duplex (-1.0 °C per modification). As found with other nucleic acids incorporating selenium, expeditious crystallisation of a modified decanucleotide A-form duplex was observed and the structure solved to a resolution of 1.45 Å. The DNA structure adjacent to the modification was not significantly perturbed. The phosphoroselenolate linkage was found to impart resistance to nuclease activity.

U2 - 10.1039/C9SC04098F

DO - 10.1039/C9SC04098F

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JO - Chemical Science

JF - Chemical Science

SN - 2041-6520

M1 - DOI: 10.1039/C9SC04098F

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