Dual pyrene-labeled pyrrolidinyl peptide nucleic acid as an excimer- to-monomer switching probe for DNA sequence detection Nattapon Maneelun, Tirayut Vilaivan * Organic Synthesis Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand article info Article history: Received 6 September 2013 Received in revised form 15 October 2013 Accepted 28 October 2013 Available online 5 November 2013 Keywords: PNA Pyrene Excimer Hybridization probe Molecular becon abstract The unique ability of pyrene to form excimers with distinct emission characteristic from monomer offers an attractive means to signal the interactions between biomolecules. In this work, dual pyrene-labeled pyrrolidinyl peptide nucleic acid probe with a D-prolyl-2-aminocyclopetanecarboxylic acid a,b-di- peptide backbone (acpcPNA) was designed as an excimer-to-monomer switching probe for DNA se- quence detection. In single stranded state, the excimer emission at 470 nm was mainly observed in the fluorescence spectrum. In the presence of DNA target, the hybridization resulted in separation of the two pyrene units, therefore the spectrum displayed increased monomer emission at 380 nm with concom- itant decreased excimer emission. Switching ratio, which is defined as the ratio of the monomer to excimer in the double stranded form [F 380 /F 470 (ds)] divided by the same value obtained from the single stranded form [F 380 /F 470 (ss)], was used to describe the performance of the probes. Switching ratios in the range of 5e30 were observed with various dual pyrene-labeled acpcPNA probes bearing pyrenebutyryl label attached five-base apart. Practically no excimer-to-monomer switching behavior was observed with DNA targets carrying a single mismatched base as shown by the small switching ratios of w1. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Pyrene-functionalized oligonucleotide and analogues are in- teresting molecules because they combine unique optical proper- ties of the pyrene chromophore and the ability of nucleic acids to form predictable secondary and higher-order structures. 1 In addi- tion to the sensitivity of pyrene fluorescence to solvent polarity, pyrene can interact with nucleobases and other aromatic molecules including other pyrene chromophores, which may result in fluo- rescence quenching and/or formation of exciplex or excimer. 2 The regular structure of DNA allows placement of the pyrene chromo- phores in a precisely controlled fashion, which can lead to several interesting structures with potential applications in various re- search areas. 3 The monomereexcimer switching of pyrene chro- mophores is attractive for sensing applications because the excimer emission exhibits large Stokes shift and long fluorescence lifetime. In addition, the fluorescence change can be measured at two wavelengths (monomer vs excimer) therefore should be less de- pendent on concentration. One such application is the use of pyr- ene monomer/excimer switching to signal a binding event between pyrene-labeled probes and targets, such as DNA or proteins. 4 In one design, two pieces of singly pyrene-labeled probes are assembled onto the same target, which results in excimer formation. 5 In an- other equally popular design, two or more pyrene labels are in- corporated in the same probe. Binding to the target causes a conformational change that results in monomer/excimer switching. The multiple pyrene-labeled probes according to this design may have a stem-loop 6 or completely open or ‘stemless’ 7 structures. The stemless design is more attractive in terms of the synthetic simplicity, although the generality of such design is rather unpredictable due to the variable interactions between pyrene and neighboring base sequences. 8 In addition, the ability of pyrene to intercalate into the base stacks in DNA double helices 9 makes some of these probes applicable only for detection of RNA, 7d or DNA with a special feature, such as base insertion. 10 Peptide nucleic acid (PNA) is an electrostatically neutral ana- logue DNA designed by replacing the deoxyribose phosphate backbone of DNA with a polyamide chain. 11 PNA binds to DNA by the usual Watson-Crick base pairing with extraordinary high af- finity and sequence specificity. 12 These properties make PNA an ideal candidate as a probe for DNA/RNA sequence determination. In recent years, our group developed a new conformationally con- strained pyrrolidinyl PNA deriving from D-proline and a cyclic b- amino acid, 13 such as 2-aminocyclopentane carboxylic acid (acpcPNA). 14 Some of these new PNA systems show higher se- quence and directional specificity than the original PNA. 15 Several applications of acpcPNA as probes for discrimination between * Corresponding author. E-mail address: vtirayut@chula.ac.th (T. Vilaivan). Contents lists available at ScienceDirect Tetrahedron journal homepage: www.elsevier.com/locate/tet 0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tet.2013.10.096 Tetrahedron 69 (2013) 10805e10810