Rationalisation of a mechanism for sensing single point variants in target DNA using anthracene-tagged base discriminating probes

Abstract

The labelling of DNA oligonucleotides with signalling groups that give a unique response to duplex formation depending on the target sequence is a highly effective strategy in the design of DNA-based hybridisation sensors. A key challenge in the design of these so-called base discriminating probes (BDPs) is to understand how the local environment of the signalling group affects the sensing response. The work herein describes a comprehensive study involving a variety of photophysical techniques, NMR studies and molecular dynamics simulations, on anthracene-tagged oligonucleotide probes that can sense single base changes (point variants) in target DNA strands. A detailed analysis of the fluorescence sensing mechanism is provided, with a particular focus on rationalising the high dependence of this process on not only the linker stereochemistry but also the site of nucleobase variation within the target strand. The work highlights the various factors and techniques used to respectively underpin and rationalise the BDP approach to point variant sensing, which relies on different responses to duplex formation rather than different duplex binding strengths.

Probing of Nucleic Acid Structures, Dynamics, and Interactions With Environment-Sensitive Fluorescent Labels

“…Notably, anthracene is sensitive enough to report single point variants (Zhao Z.-Y. et al, 2012;Duprey et al, 2018) as well as to discriminate cytosine from 5-methylcytosine and even the challenging 5hydroxymethylcytosine (C vs. 5 mC and 5 hmC; Duprey et al, 2011Duprey et al, , 2016. The quencher-free molecular beacon developed by Kim et al proposes the reverse strategy to distinguish a fully complementary strand from a mismatched target (Hwang et al, 2004;Ryu et al, 2007).…”

Photoreactivity of an Exemplary Anthracene Mixture Revealed by NMR Studies, including a Kinetic Approach

“…This includes various polymeric materials [30,31], such as intelligent shape memory polymers [32], as well as soft materials, such as photoresponsive hydrogels [16] and liquid crystals [33]. Hence, the main applications cover a vast number of fields, including tissue engineering [30], optoelectronic devices [33] such as organic light-emitting diodes or transistors [34], sensors for metal ion detection [35], molecular containers for catalysis or drug delivery [36] and photomedicine, where these compounds can be used as intracellular pH probes [37], DNA-based hybridization sensors [38] and other biosensors [39]. According to the latest results, anthracene derivatives have even been suggested as potential candidates for applications in laser photonics [40] and as smart optical materials [41].…”

Direct Detection and Discrimination of Nucleotide Polymorphisms Using Anthraquinone Labeled DNA Probes

“…This amplification step is then followed by the determination of the homology of the DNA to specific sequences through hybridization to probes within a biosensor to provide identification of the causative pathogen. DNA hybridization can be detected using a range of approaches, including a number of optical techniques such as colorimetric, fluorescent, luminescent, and Raman spectroscopy (Järvinen et al, 2009;Tomlinson et al, 2014;Papadopoulou et al, 2015;Stulz, 2015;Boynton et al, 2017;Nano et al, 2017;Duprey et al, 2018).…”