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 F Conlon,James P Hall,Christopher J Law,Olga Eguaogie,Jamie S T Sweet,James H R Tucker,Jordan J Wilson,Joseph S Vyle,Oliver G A Hancox,Klaudia Englert,Sarah A Allman,Julian Steinhoegl

doi:10.1039/c9sc04098f

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 Michaelis-Arbuzov (M-A) reaction of nucleoside H-phosphonate derivatives with 5'-deoxythymidine-5'-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 chain extended 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 to -3.1 °C per phosphoroselenolate) when introduced at the 5'-termini of A-form or mixed 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.

Highlights

Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites
The automated solid-phase synthesis of oligonucleotides using phosphoramidite chemistry[1] (Fig. 1A) is a platform technology which has been transformative in a wide range of applications[2]
Concurrent increased access to third generation synchrotron sources which generate more focussed, tunable X-rays of high brightness and stability coupled with automation of the screening of crystallisation conditions has engendered a considerable upsurge in protein crystal structure solution such that in the year 1990, 132 protein structures were deposited in the protein databank whereas in 2018 this number had risen to 10 306

Summary

Introduction

The automated solid-phase synthesis of oligonucleotides using phosphoramidite chemistry[1] (Fig. 1A) is a platform technology which has been transformative in a wide range of applications[2]Whilst simple Watson–Crick base-pairing rules can be used to programme both intramolecular and intermolecular assembly of three-dimensional structures from simple duplexes,[10,11] the tertiary interactions of folded nucleic acids derived from more complex assemblies and their recognition, especially by nucleic acid binding proteins or small molecule effectors, typically requires structural elucidation at the atomic level.[12,13] Characterisation of novel structural motifs including such complexes by X-ray crystallography is augmented by the introduction of an anomalous heavy atom scattering centre.[14,15]Anomalous scattering of X-rays at the Se–K edge has been of considerable value in facilitating structural biology studies of proteins following the pioneering work of Hendrickson and co-10948 | Chem. Oligodeoxynucleotides incorporating internucleotide phosphoroselenolate linkages have been prepared under solid-phase synthesis conditions using dimer phosphoramidites.

Results

Conclusion