Synthesis, oligonucleotide incorporation and fluorescence properties in DNA of a bicyclic thymine analogue

Christopher P Lawson,L Marcus Wilhelmsson,Nicolai K Andersen,Tristan Giraud,Tom Brown,Afaf H El-Sagheer,Morten Grøtli,Moa S Wranne,Anders F Füchtbauer,Henrik Gradén,Blaise Dumat,Thomas Floyd

doi:10.1038/s41598-018-31897-2

Abstract

Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids. In our overall mission to develop bright and useful FBAs for all natural nucleobases, herein we describe the synthesis and thorough characterization of bicyclic thymidine (bT), both as a monomer and when incorporated into DNA. We have developed a robust synthetic route for the preparation of the bT DNA monomer and the corresponding protected phosphoramidite for solid-phase DNA synthesis. The bT deoxyribonucleoside has a brightness value of 790 M−1cm−1 in water, which is comparable or higher than most fluorescent thymine analogues reported. When incorporated into DNA, bT pairs selectively with adenine without perturbing the B-form structure, keeping the melting thermodynamics of the B-form duplex DNA virtually unchanged. As for most fluorescent base analogues, the emission of bT is reduced inside DNA (4.5- and 13-fold in single- and double-stranded DNA, respectively). Overall, these properties make bT an interesting thymine analogue for studying DNA and an excellent starting point for the development of brighter bT derivatives.

Highlights

Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids
In reports by Eldrup et al, a series of 1,8-naphthyridin-2(1 H)-ones were presented as novel bicyclic and tricyclic analogues of thymine, some of which were found to be more efficient than thymine in the recognition of adenine in peptide nucleic acids (PNAs) duplex and triplex structures[11,12]
Unlike the PNA monomer of bicyclic thymidine (bT) reported by Eldrup et al.[11], a deoxyribose bT analogue (1, Fig. 2a) has no linker to the naphthyridinone core

Summary

Introduction

Fluorescent base analogues (FBAs) have emerged as a powerful class of molecular reporters of location and environment for nucleic acids. Over the last two decades, a multitude of fluorescent nucleobase moieties have been synthesized[6,7,8] This involved the attachment of fluorescent labels to native nucleosides via a non-emissive linker, which allowed them to report on e.g. changes in the microenvironment around nucleic acids[9]. An alternative approach involves the design and synthesis of modified nucleosides with intrinsic fluorescence These FBAs can mimic the shape and hydrogen-bonding ability of the natural nucleobases and can be incorporated directly into DNA- or RNA-strands, chemically or enzymatically, often causing minimal perturbation of the nucleic acid structure[6,7,8]. We envision that the bT scaffold will serve as an excellent starting point for the development of bright thymine analogues with interesting photophysical properties

Methods

Results

Conclusion