Improving the Design of Synthetic Oligonucleotide Probes by Fluorescence Melting Assay

Chembiochem : a European Journal of Chemical Biology
Ilaria FurlanKira Astakhova

Abstract

Nucleic acid hybridisation plays a key role in many biological processes, including transcription, translation, and regulation of gene expression. Several sophisticated applications rely on this fundamental interaction, including the polymerase chain reaction, sequencing, and gene therapy. To target a nucleic acid sequence specifically, synthetic oligonucleotides with a suitable affinity and specificity towards the target need to be designed. The affinity of potential probes or therapeutic agents to their target sequence is generally investigated by melting experiments, which break the hydrogen-bonding and stacking interactions that stabilise the double helix resulting in the formation of two single strands. In this paper, we report a comparative study of hybridisation for short fluorescent oligonucleotides labelled with cyanine and ATTO dyes, performed by the currently used UV melting assay and by a more sensitive fluorescence melting experiment. Using different oligonucleotide sequences in the concentration range of 5 nm to 2 μm, we observed a stabilising effect of the fluorophores on the duplexes, especially at low concentrations. We paid particular attention to the effect of polycations and to molecular crowding as major pa...Continue Reading

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