Combined nucleobase and backbone modifications enhance DNA duplex stability and preserve biocompatibility

Afaf H. El-Sagheer,Tom Brown

doi:10.1039/c3sc51753e

Abstract

DNA strands containing a triazole linkage flanked on its 3′-side by an aminoethylphenoxazine nucleobase analogue (G-clamp) have been prepared by solid-phase synthesis followed by CuAAC-mediated click oligonucleotide ligation. The stability of the doubly modified DNA duplexes and DNA–RNA hybrids is greatly increased, whereas a single base pair mismatch located at or adjacent to the modifications is strongly destabilising, making triazole G-clamp a potent mismatch/point mutation sensor. A DNA strand containing this unnatural combination was successfully amplified by PCR to produce unmodified copies of the original template, with deoxyguanosine inserted opposite to the G-clamp-triazole nucleotide analogue. This study shows for the first time that a polymerase enzyme can read through a combined backbone/nucleobase modification surprisingly well. These favourable properties suggest new applications for oligonucleotides containing the G-clamp triazole modification in biotechnology, nanotechnology, diagnostics and therapeutics.

Highlights

Templated chemical ligation of alkyne and azide-functionalised oligonucleotides using the CuAAC reaction[1,2] has recently been used to assemble long DNA strands up to 300 bases in length containing a triazole mimic of a DNA phosphodiester linkage.[3]
It was apparent to us that this might be achieved using G-clamp phosphoramidite monomer[18] combined with a variation of the methodology we have previously developed to synthesise triazolelinked DNA.[3]
The G-clamp is introduced by standard solid-phase oligonucleotide synthesis and the triazole linkage is inserted in a CuAAC reaction between 30-propargyl and 50-azido G-clamp oligonucleotides.[1,2]

Summary

Introduction

Edge Article oligonucleotides, protecting them against degradation in vivo.[12]. Other triazole backbones have been synthesised,[12,13,14] but none give rise to duplexes of the same stability as canonical DNA. This is achieved by the incorporation of the aminoethylphenoxazine analogue of 20-deoxycytidine (Gclamp) adjacent to the triazole linkage This cytosine analogue increases duplex stability by a combination of increased intrahelical base stacking and additional hydrogen bonding to guanine (Fig. 1).[16] It has been shown to be effective in a DNA17,18 and PNA19 context, and analogues have been developed to recognise 8-oxoguanine in DNA.[20] G-clamp is essentially deoxycytidine with an additional aminoethoxyphenoxy group protruding into the major groove. We reasoned that this structural change might be accommodated by polymerase enzymes without causing mutagenesis, and we have investigated this concept

Results and discussion

Conclusions