Fluorescent Properties of Oligonucleotide-conjugated Thiazole Orange Probes¶

Abstract

The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophillic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2'-deoxyriboadenylate (dA(15)), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (Phi(fl)) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5'[CACT(15)CAC(3')]); Phi(fl) values of between 0.06-0.35 were determined for the double-stranded conjugates. The Phi(fl) value (0.17) of thiazole orange, 1-(N,N'-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)- 2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA(15)(.)d(5')[CACT(15)CAC(3')] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide-thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher Phi(fl) values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest Phi(fl) and the greatest fraction of the longest-lived fluorescent thiazole orange species (in the hybrid form).