Harnessing intrinsic fluorescence for typing of secondary structures of DNA

Abstract

DNA is polymorphic since, despite its ubiquitous presence as a double-stranded helix, it is able to fold into a plethora of other secondary structures both in vitro and in cells. Despite the considerable advances that have been made in understanding this structural diversity, its high-throughput investigation still faces severe limitations. This mainly stems from the lack of suitable label-free methods, combining a fast and cheap experimental workflow with high information content. Here, we explore the use of intrinsic fluorescence emitted by nucleic acids for this scope. After a preliminary assessment of the suitability of this phenomenon for tracking the conformational changes of DNA, we examined the intrinsic steady-state emission spectra of an 89-membered set of synthetic oligonucleotides with reported conformation (G-quadruplexes, i-motifs, single- and double stranded DNA) by means of multivariate analysis. Specifically, principal component analysis of emission spectra resulted in successful clustering of oligonucleotides into three corresponding conformational groups, albeit without discrimination between single- and double-stranded structures. Linear discriminant analysis of the same training set was exploited for the assessment of new sequences, allowing the evaluation of their G4-forming propensity. Our method does not require any labelling agent or dye, avoiding the related intrinsic bias, and can be utilized to screen novel sequences of interest in a high-throughput and cost-effective manner. In addition, we observed that left-handed (Z-) G4 structures were systematically more fluorescent than most other G4 structures, almost reaching the quantum yield of 5'-d[(G3T)3G3]-3' (G3T), the most fluorescent G4 structure reported to date. This property is likely to arise from the similar base-stacking geometry in both types of structures.