Fluorophore modified nucleic acids have been frequently utilized in various biological applications, including in the detection and quantification of desired sequences. Often the fluorophore is covalently attached on the end of the oligomer, thereby limiting the number of fluorophores and ultimately the brightness of the probe. By attaching the fluorophore to a modified nucleobase, multiple fluorescent dyes can be incorporated into the interior of a hybridization probe . In this study we have synthesized PNA probes with multiple internal fluorescent dyes using the Huisgen-Meldal Sharpless �Click� reaction between an azide-modified fluorescent dye and alkynyl-uracil-containing PNA. A rigid triazole linker restricts dye movement and minimizes quenching. As a proof of concept we have recently synthesized a dye-modified PNA monomer (coumarin-uracil) and have incorporated it into a series of oligomers. It was determined that the PNA:DNA duplex stability was not affected by the presence of the dye and only a marginal decrease in fluorescence due to quenching was observed, even when the two dyes were placed in adjacent positions. We have demonstrated the feasibility of this approach which suggests that several fluorescent dyes can be introduced into a PNA probe at internal positions in order to enhance the brightness of DNA/RNA-targeting PNA probes.

Click PNA: A versatile tool for high density fluorescent labeling of peptide nucleic acids

248th National Meeting (2014)

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