title: 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 RIalpha 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 (RIalpha+), 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 RIalpha 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 (RIalpha+) cells incubated at 37 degrees C with the anti-RIalpha 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.
authors.affiliation: Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
authors.forename: Minmin
authors.initials: M
authors.lastname: Liang
authors.forename: Xinrong
authors.initials: X
authors.lastname: Liu
authors.forename: Kayoko
authors.initials: K
authors.lastname: Nakamura
authors.forename: Xiangji
authors.initials: X
authors.lastname: Chen
authors.forename: Dengfeng
authors.initials: D
authors.lastname: Cheng
authors.forename: Guozheng
authors.initials: G
authors.lastname: Liu
authors.forename: Shuping
authors.initials: S
authors.lastname: Dou
authors.forename: Yi
authors.initials: Y
authors.lastname: Wang
authors.forename: Mary
authors.initials: M
authors.lastname: Rusckowski
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Hnatowich
mesh_terms: D000818:Animals; D052160:Benzothiazoles / Q000737:chemistry*; D001681:Biological Assay; D045744:Cell Line, Tumor; D016373:DNA, Antisense / Q000737:chemistry* / Q000235:genetics*; D005456:Fluorescent Dyes / Q000737:chemistry*; D051379:Mice; D008961:Models, Structural; D009693:Nucleic Acid Hybridization / Q000235:genetics; D011804:Quinolines / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors
chemical_list: D052160:Benzothiazoles; D016373:DNA, Antisense; D005456:Fluorescent Dyes; D011804:Quinolines; D012333:RNA, Messenger; C051000:thiazole orange