DNA Melting in the Presence of Fluorescent Intercalating Oxazole Yellow Dyes Measured with a Gel-Based Assay Michael T. Bjorndal D. Kuchnir Fygenson Physics Department, University of California, Santa Barbara, CA 93106 Received 6 March 2002; accepted 2 June 2002 Published online 00 Month, 2002 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.10220 Abstract: We measured the effect of the intercalating oxazole yellow DNA dye quinolinium,4-[(3- methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(trimethylammonio)propyl]-,diiodide (YO-PRO) and its homodimer (YOYO) on the melting of self-complementary DNA duplexes using a gel-based assay. The assay, which requires a self-complementary DNA sequence, is independent of the optical properties of the molecules in solution. The melting temperature of the DNA is observed to increase in direct proportion to the number of occupied intercalation sites on the DNA, irrespective of whether the dye molecules are in monomer or dimer form. The increase is  2.5°C for each intercalation site occupied in the presence of 38 mM [Na  ], for dye/duplex ratios in which less than 1/5 of the available intercalation sites are occupied. © 2002 Wiley Periodicals, Inc. Biopolymers 65: 40 – 44, 2002 Keywords: DNA melting; fluorescent intercalating oxazole yellow dyes; hairpin; gel-based assay INTRODUCTION Major advances in single molecule research have been made with fluorescently labeled DNA. 1–10 The cyanine nucleic acid dyes YO-PRO and YOYO have a number of features especially desirable for such studies including high affinity for nucleic ac- ids, low affinity for other biopolymers, low fluo- rescence when not bound to nucleic acids and large fluorescence enhancement upon binding to nucleic acids. However, the effect of these dyes on DNA melting has apparently gone unmeasured, perhaps because these dyes have absorption characteristics that complicate conventional uv measurements of DNA melting. In this article, we report measurement of the effect of YO-PRO and YOYO on the melting temperature of duplex DNA with a new method, developed in the laboratory of G. Zocchi (UCLA). This method mon- itors the melting of double stranded “duplex” DNA by trapping melted (single-stranded) DNA in a well- defined “hairpin” secondary structure and measuring the proportion of duplex and hairpin populations by gel electrophoresis. The method was designed to be complementary to uv absorption measurements in that it reports the number of melted duplexes, whereas uv absorption indicates the number of melted base pairs. 11 Besides this fundamental distinction, there are two major dif- ferences between the gel-based method used here and Correspondence to: D. Kuchnir Fygenson; email: deborah@ physics.ucsb.edu Contract grant sponsor: National Science Foundation Biopolymers, Vol. 65, 40 – 44 (2002) © 2002 Wiley Periodicals, Inc. 40