DNA‐Templated Synthesis of Trimethine Cyanine Dyes: A Versatile Fluorogenic Reaction for Sensing G‐Quadruplex Formation

Abstract

It has been known for several decades that G-rich nucleic acid sequences have a propensity to fold into highly stable four-stranded structures in vitro in the presence of physiological cations, notably K and Na. Such structures, termed quadruplexes, have had their biological significance demonstrated for a number of processes. For example, it has been shown that the single-stranded 3’-end of telomeric DNA could adopt a quadruplex conformation under near physiological conditions, which has implications on telomere maintenance mechanisms. More recently, a number of DNA Gquadruplex sequences have been identified in the promoter region of genes that have been proposed to act as regulatory elements for gene expression at the transcriptional level. Amongst the 43% of human genes that contain a putative quadruplex forming sequence in their promoter, specific oncogenes have received particular attention. These include the c-myc, bcl-2, K-ras, and c-kit genes. Although there exist increasing evidences for the formation of Gquadruplexes at telomere ends in vivo, the possible existence of promoter quadruplexes in vivo is still subject to debate. Recent studies using small molecule approaches have demonstrated that quadruplex formation within the nuclease hypersensitive element of the c-myc gene or within the promoter of the c-kit gene upstream were coupled to a significant inhibition of c-myc and c-kit expression at the transcriptional level in various cell lines. However, whilst the 3’-overhang of telomeric DNA is single-stranded, and therefore is free to adopt any stable secondary structure, quadruplex formation within a promoter would require at least a local and temporary opening of the DNA double-helix, despite the high stability of Watson-Crick G-C base pairs. Recent studies using fluorescence resonance energy transfer (FRET) or fluorescent probes have demonstrated that quadruplexes could potentially form, even when in competition with a thermodynamically more stable duplex form. Moreover, it is well established that doublestranded DNA transiently becomes single-stranded during key biological processes like DNA replication, transcription or even recombination, thus allowing the folding of each DNA strand into alternative (i.e. non B-DNA) structures. Herein, we were interested in designing sensitive fluorescent biosensors that would be highly specific for unique G-quadruplexes in the genome. The general strategy consists in targeting simultaneously the quadruplex structure itself but also its two flanking regions in a sequence specific manner. Briefly, two short peptide nucleic acids (PNAs) complementary to both quadruplex flanking regions are functionalised with two non-fluorescent components A and B of a fluorogenic reaction (i.e. reaction between non-fluorescent derivatives A with B leads to the formation of fluorescent entity C). The system can be designed in such a way that, upon hybridization of the PNA probes to their complementary DNA sequences through Watson-Crick base pairing, A and B will be in close enough proximity to react with each other when the DNA sequence between both PNAs is folded into a quadruplex structure only, while they will be kept separated if the DNA remains singlestranded (Figure 1).