Fluorescent Properties of Oligonucleotide-conjugated Thiazole Orange Probes¶

Abstract

The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca‐2′‐deoxyriboadenylate (dA15), 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 (Φfl) 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′[CACT15CAC3′]); Φfl values of between 0.06–0.35 were determined for the double‐stranded conjugates. The Φfl 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: dA15·d5′[CACT15CAC3′] (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 Φfl 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 Φfl and the greatest fraction of the longest‐lived fluorescent thiazole orange species (in the hybrid form).

Fluorescent DNA Nanotags Featuring Covalently Attached Intercalating Dyes: Synthesis, Antibody Conjugation, and Intracellular Imaging

“…Kubista and Svanvik reported similar properties for PNA “light-up” probes in which TO was attached to the PNA terminus . A variety of other TO conjugates with DNA and PNA have been reported in which the TO was attached to internal or terminal positions. ,− …”

“…We relied on a Cu(I)-mediated Huisgen cyclization (“click”) reaction to covalently attach the dye molecules to the DNA. , As shown in Scheme , an alkyne group linked through a flexible spacer to C5 of a uracil residue can react with an azide-substituted intercalator dye (TO) to covalently attach the two via a newly formed triazole linkage. A variety of methods have been reported for conjugating TO to DNA, ,− ,,− but we chose this reaction because Carell and co-workers recently reported that it can be used to incorporate six consecutive fluorescein dyes onto a DNA single strand in high yield. , Seela and Pujari have reported successful conjugation of coumarin dyes to alkyne-functionalized DNA. , …”