A Convenient Thiazole Orange Fluorescence Assay for the Evaluation of DNA Duplex Hybridization Stability

Abstract

Objective-A simple and rapid method for measuring the hybridization stability of duplexes of DNAs and other oligomers in different environments is described. When added to an oligomer duplex, the thiazole orange (TO) dye intercalates and in this state is fluorescent. Therefore, when duplex dissociation occurs, the release of TO results in a detectable change in fluorescence intensity. This assay was developed primarily to screen antisense oligomer duplexes that are stable in serum and in the cytoplasm but unstable in the presence of their target messenger RNA (mRNA).Methods-The two antisense oligomers of this investigation were both 25 mer phosphorothioate (PS) DNAs, one directed against the RIα mRNA and the other directed against the mdr1 mRNA. The former duplex was first used in the solution studies, in most cases duplexed with a 16 mer phosphodiester (PO) complementary DNA (i.e., PS-DNA25/PO-cDNA16). Both duplexes were then tested in a series of cell studies using SK-BR-3 (RIα+), KB-G2 (mdr1++), and KB-31 (mdr1+/−) cells.Results-Preliminary measurements in solution showed that maximum fluorescence was achieved when more than ten TO molecules were bound to each duplex. When a 25 mer PO-DNA or PO-RNA with the base sequence of the RIα mRNA was added, the dramatic change in fluorescence intensity that followed signified dissociation of the antisense DNA from the study duplex and reassociation with the target DNA. Kinetic measurements showed that this process was completed in about 3 min. Fluorescent measurements of SK-BR-3 (RIα+) cells incubated at 37°C with the anti-RIα study duplex over time showed a maximum at the point where the loss of fluorescence due to dissociation of the study duplex, probably by an antisense mechanism, began to dominate over the increasing fluorescence due to continuing cellular accumulation. A similar result was observed in the KB-G2 (mdr1+) cells incubated with the anti-mdr1 study duplex.Conclusions-When study duplexes shown to be stable in serum were incubated with their target cells, the assay successfully detected evidence of dissociation, most likely by an antisense mechanism. Thus, a TO fluorescence assay has been developed that is capable of detecting the dissociation of DNA duplexes.

Optical Antisense Tumor Targeting in Vivo with an Improved Fluorescent DNA Duplex Probe

“…The five native duplexes were screened for stability in serum using a simple TO assay developed in this laboratory (13). Each duplex was added to a 96 well microplate at 100 μ L per well and incubated at 500 nM in 70% normal mouse serum (Jackson Immuno Research Laboratories, Inc., West Grove, PA) at 37 °C for different times before the addition of the indicator TO at 6 μ M. Fluorescence intensities were measured at room temperature on a Fluorescence Microplate Reader (Safire, Tecan Group Ltd.).…”

“…Although fluorescence in the tumor was clearly evident, this duplex also provided a fluorescence background making obvious the need for duplex optimization. In a process of optimization, we now report that five DNA duplexes differing in the length of the complementary minor strand were compared for stability using a simple thiazole orange (TO) fluorescence assay (13). On the basis of the screening results, the shortest minor strand (10 mer) was selected and, after hybridization to the 25 mer PS antisense major strand, was studied in cells and in tumored animals in comparison to that of the 18 mer major strand used previously.…”

A Nanoplasmonic‐Fluorescent Ruler for Detection of Site‐Specific Protein Binding to Composite DNA of Multiple Sites

“…We have demonstrated the practical application of this nanoplasmonic‐fluorescent ruler, constructed using thiazole orange (TO) and spherical AuNPs (20 nm and 100 nm diameter) and AgNPs (80 nm diameter) as a profiler, for studying the binding of specificity protein 1 (Sp1) and estrogen receptor α (ERα) with their composite DNA, i.e., +571 ERE/Sp1 site in promoter A derived from the human PR gene, which contains a half‐site for ERα and two adjacent Sp1‐binding sites. Sp1 is known to be involved in regulating progesterone receptor gene expression, especially at these sites where the presence of Sp1‐binding site next to ½ ERE is typically 10–17 bp away.…”