Fluorescence Probes DOI: 10.1002/anie.200700289 Triplex Molecular Beacons as Modular Probes for DNA Detection** Tom N. Grossmann, Lars Röglin, and Oliver Seitz* Detecting nucleic acid sequences is of central importance in modern life sciences. Given the rapid growth of the available sequence data, the diagnosis of DNA and RNA is likely to increaseinsignificanceoverthenextdecade.Themostuseful methods employ probes that allow quantitative measure- mentsinhomogeneoussolution,therebyomittingtheneedto separate bound from unbound probes. [1] Molecular beacons (MBs) [2] havebecomeaclassofwidelyusedprobesfornucleic acid analysis. These probes carry a fluorophore and a quencherattheirtermini.Intheabsenceofacomplementary target sequence, MBs form a stem–loop structure (Fig- ure 1A), which results in quenching of the fluorescence. Upon binding to the complementary target, the hairpin is opened, the labels are separated, and fluorescence occurs. MBs have been used in quantitative polymerase chain reactions (qPCR) to monitor the formation of the amplicon inasequence-specificmannerandintheimagingofpathogen RNA in living cells. [2] It is a useful feature that conformationally constrained oligonucleotide probes, such as MBs, recognize their targets with higher specificity than linear probes. [3] This property is desired in the analysis of single-nucleotide alterations. The enhancement of specificity observed with structured probes implies that the selectivity of probe–target hybridization can be modulated by altering the degree of conformational constraint. For molecular beacons, this can be achieved by variationofthestemlengthandsequence.However,thistype ofmodulationrequiresthesynthesisofseveraldifferentMBs. Among various conceptional modifications, replacement of the original DNA Watson–Crick base-paired stem by other pairing systems is rare, but examples include homo-DNA, [4a] LNA [4b] and G-quadruplex stems, [4c] as well as “stemless” peptide nucleic acid (PNA) molecular beacons. [4d–f] The cation-dependent G quadruplex stability was used to tune theconformationalconstraintofthecorrespondingbeacon, [4c] butinapplicationssuchasreal-timeqPCRandmeasurements in vivo, the cation concentration is difficult to address. Herein, we show that triplex-based molecular beacons (tMBs; Figure1B) enable a precise modulation of the conformational constraint by using a single DNA probe (triplex-forming molecular beacon, tfMB) labeled with a fluorophore and a quencher in combination with several stem-forming oligonucleotides (sfOs). In this setup, only one dual-labeled probe is needed and it is the sfO that allows tuning of the selectivity and sensitivity of the tMB towards targetDNAsequences.Furthermore,themodularassembly [5] facilitates the additional incorporation of functionalities that can be easily introduced via the sfO. In an example of this concept,weprepared“superquenched” [6] tMBsbyappending additional quencher moieties to the sfO. The tMB is designed in a way that the stem region is formed by a poly(a)·2poly(T) (a is the PNA base) structure (Figure1C). [7] The sfO poly(a) segment is composed of peptide nucleic acid (PNA), a DNA analogue bearing an uncharged N-(2-aminoethyl)glycine backbone. PNA is capa- ble of forming more-stable duplexes and triplexes with complementary DNA than corresponding DNA. [8] The sfO was designed to bind two poly(T) arm segments of the tfMB through Watson–Crick and Hoogsteen base pairing. We decided to investigate two tfMB systems (8T and 11T , Figure 1D). Both tfMBs consist of a central DNA sequence that is complementary to the matched target sequence (Ma). This central sequence is flanked by poly(T) regions labeled with 6-FAM/Dabcyl as the fluorphor/quencher. The sfO lengths varied from six to 11 adenine residues. In initial UV denaturationexperiments,westudiedthestabilityofduplexes formed between tfMB and the target (tfMB·target) in the absenceofsfOsaswellasthestabilityoftriplexes(tfMB·sfO) Figure 1. Comparison of a molecular beacon (MB; A) and a triplex molecular beacon (tMB, B) for the detection of complementary DNA oligonucleotides. C) Poly(a)·2poly(T) structure forming the triplex stem region. D) Sequences of DNA (target, tfMB) and PNA (sfO). Ac = ace- tyl, Dabcyl = 4-(4’-dimethylaminophenylazo)benzoic acid, FAM = 6-car- boxyfluorescein. [*] Dipl.-Chem. T.N. Grossmann, [+] Dipl.-Chem. L. Röglin, [+] Prof. Dr. O. Seitz Institut für Chemie der Humboldt-Universität zu Berlin Brook-Taylor-Strasse 2, 12489 Berlin (Germany) Fax: (+ 49)30-2093-7266 E-mail: oliver.seitz@chemie.hu-berlin.de [ + ] Both authors contributed equally to this work. [**] We acknowledge support from Schering AG. T.N.G. is grateful for a fellowship from the Studienstiftung des deutschen Volkes. Supporting information for this article is available on the WWW under http://www.angewandte.org or from the author. Angewandte Chemie 5223 Angew. Chem. Int. Ed. 2007, 46, 5223 –5225 # 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim