Consideration of end effects of DNA hybridization in selection of fluorescent dyes for development of optical biosensors

Intercalating fluorescent dyes are in widespread use to detect the presence of double-stranded DNA. Applications include the development of biosensors that rely on the attachment ("tethering") of a dye molecule by a short hyd rocarbon chain to the terminus of a strand of DNA so that dye is continuous ly available and the biosensor is fully reversible. Double strands of DNA h ave end effects that limit the stability of hybridization and dye intercala tion near the termini of the duplexes. Therefore, the selection of the dye must be based on consideration of spectroscopic properties and also issues associated with tether length and the stoichiometry of the binding of the d ye with double- and single-stranded DNA. Ethidium bromide (EB) has been use d extensively to detect hybridization of DNA in applications such as electr ophoresis, gene chips, and biosensors. A number of dyes with greater quantu m efficiency than EB for detection of hybridization have been reported. Fur thermore, other practical spectroscopic advantages can be gained in terms o f improved S/N by use of dyes that have excitation that is red shifted rela tive to EB. Pyrilium iodide has been disclosed as an intercalator of high q uantum efficiency and long excitation wavelength. This work investigates py rilium iodide in comparison to EB as a candidate for preparation of a tethe red dye for detection of hybridization of DNA 20-mers.