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X. Fang, X. Liu, and Sheldon Schuster, W. Tan
Published 1999 · Chemistry

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Xiaohong Fang, Xiaojing Liu, Sheldon Schuster, and Weihong Tan* Department of Chemistry and UF Brain Institute UniVersity of Florida, GainesVille, Florida 32601 ReceiVed October 29, 1998 We have designed a biotinylated ssDNA molecular beacon for DNA hybridization studies at a solid interface. DNA hybridization and molecular interaction studies are major tools for the diagnosis of genetic disease, in which the clinical symptoms are linked to alterations in DNA. Identifying these mutations in human genome has become the focus of many research efforts. One recent new development is a novel class of oligonucleotide probes, molecular beacons (MBs). Molecular beacons, first developed by Tyagi and Kramer in 1996,1 are single stranded oligonucleotide probes that possess a stem-and-loop structure. The loop portion of the molecule can report the presence of a specific complementary nucleic acid.1-5 The five bases at the two ends of the MB are complementary to each other, forming the stem. A fluorophore and a quencher are linked to the two ends of the stem, as shown in Figure 1. The stem keeps these two moieties in close proximity to each other, causing the fluorescence of the fluorophore to be quenched by energy transfer. When the probe encounters a target DNA molecule, it forms a hybrid that is longer and more stable than the stem, and its rigidity and length preclude the simultaneous existence of the stem hybrid. Thus, the MB undergoes a spontaneous conformational reorganization that forces the stem apart and causes the fluorophore and the quencher to move away from each other, leading to the restoration of fluorescence. Therefore, at room temperature, the MBs emit an intense fluorescent signal only when hybridized to their target molecules.1-8 The size of the loop and its content can be varied by designing different MBs. Also, the quencher and the fluorophores can be changed according to the problem studied. There have been a variety of applications of MBs,1-8 including the real-time monitoring of polymerase chain reactions,1 and even the investigation of HIV-1 disease progression.4,5 MBs have extremely high selectivity with single base pair mismatch identification capability. They hold great promise for studies in genetics, disease mechanisms, and molecular interactions, for applications in disease diagnostics, and in new drug development. It is expected that there will be many interesting applications for surface-immobilized molecular beacons. So far, MBs have only been used in a homogeneous liquid solution. This limits the applications of MBs in in vivo biomedical studies and in DNA biosensor development. To fully explore the potentials of MBs, we have designed a biotinylated ssDNA MB, shown in Figure 1, which is intended for immobilization onto a silica surface for a variety of applications. The MB has a total of 28 bases, of which 18 bases are the sequence of interest and 5 base pairs form the stem. The biotinylated ssDNA molecular beacon has been synthesized with tetramethylrhodamine (TMR) as the fluorophore and DABCYL (dimethylaminoazobenzen aminoexal-3-acryinido) as the quencher. DABCYL, a nonfluorescent chromophore, serves as a universal quencher for any fluorophore in MBs.2 There are five important considerations in MB design. First is the functional group for surface immobilization. One of the most common ways for biomolecule immobilization onto a solid surface is through biotin-avidin binding.9,10 The biotin-avidin linkage to a surface is suitable for DNA hybridization. Since the 5′and 3′ends are linked to a fluorophore and a quencher, respectively, adding a biotin functional group to the MB is the easiest strategy to attach the MB to a surface. Second is the position for biotin binding. We tried different positions to link biotin: the loop sequence, the second base pair position of the fluorophore side of the stem, and the same position on the quencher side of the stem. We chose to link biotin to the quencher side of the stem to minimize the effects biotin might have on fluorescence, quenching, and hybridization of the MB. Third is the length of the stem and the loop sequence. Most MB studies1-8 indicate that a 15-25 base sequence together with a 5 base pair stem is an excellent balance. We chose an 18 base sequence. The 5 base pair stem is strong enough to form the hairpin structure for efficient fluorescence quenching, while it is still weak enough to be dissociated when a complementary DNA hybridizes with the 18 base loop of the MB. Fourth is a spacer between biotin and the sequence. We used a biotin-dT to provide an easy access for target DNA molecules to efficiently interact with the loop sequence and an adequate separation to minimize potential interactions between avidin and the DNA sequence. Fifth is the fluorophores. So far most of the MB are based on fluorescein.1 It is known that rhodamine dyes have higher quantum yields and are much more photostable than fluorescein in fluorescence detection. If bulky samples are used, photobleaching of fluorescein may not be critical. However, when an ultratrace amount of MB is used or the MBs are immobilized on a surface, photobleaching will become a more severe problem. This is the reason most of the single-molecule fluorescence detections have been carried out on rhodamine dye molecules.11 In addition, fluorescence intensity of fluorescein is highly dependent on the pH used in a sample matrix. There is one (1) Tyagi, S.; Kramer, F. R. Nature Biotech. 1996, 14, 303-308. (2) Tyagi, S.; Bratu, D.; Kramer, F. R. Nature Biotech. 1998, 16, 49-53. (3) Piatek, A. S.; Tyagi, S.; Pol, A. C.; Telenti, A.; Miller, L. P.; Kramer, F. R.; Alland, D. Nature Biotech. 1998, 16, 359-363. (4) Kostrikis, L. G.; Tyagi, S.; Mhlanga, M. M.; Ho, D. D.; Kramer, F. R. Science 1998, 279, 1228-1229. (5) Kostrikis, L. G.; Huang, Y.; Moore, J. P.; Wolinsky, S. M.; Zhang, L. Q.; Guo, Y.; Deutsch, L.; Phair, J.; Neumann, A. U.; Ho, D. D. Nat. Med. 1998, 4, 350-353. (6) Giesendorf, B. A. J.; Vet, J. A. M.; Tyagi, S.; Mensink, E. J. M. G.; Trijbels, F. J. M.; Blom, H. J. Clin. Chem. 1998, 44, 482-486. (7) Ehricht, R.; Kirner, T.; Ellinger, T.; Foerster, P.; McCaskill. J. S. Nucleic Acids. Res. 1997, 25, 4697-4699. (8) Gao, W.; Tyagi, S.; Kramer, F. R. Mol. Microbiol. 1997, 25, 707716. (9) Anzai, J.; Hoshi, T.; Osa, T. Trends Anal. Chem. 1994, 13, 205-210. (10) Narasaiah, D.; Nowall, W. B.; Kuhr, W. G. Anal. Chem. 1997, 69, 2619-2625, 1997. (11) Xu, X.; Yeung, E. Science 1997, 275, 1106-1109; Ambrose, W. P.; Goodwin, P. M.; Martin, J. C.; Keller, R. A. Science 1994, 265, 364-7. (12) Cordek, J.; Wang, X.; Tan, W. Anal. Chem. 1999, in press. (13) Kleinjung, F.; Klussman, S.; Erdmann, V. A.; Scheller, F. W.; Furste, J. P.; Bier, F. F. Anal.Chem. 1998, 70, 328-331. (14) Tan, W.; Shi, Z -Y.; Smith, S.; Birnbaum, D.; Kopelman, R. Science 1992, 258, 778-781. Figure 1. Schematic of the operation of a biotinylated MB immobilized on a solid surface. Biotin is added to the stem of the molecular beacon for surface immobilization with avidin. The MB is nonfluorescent since the stem hybrid keeps the fluorophore (F) close to the quencher (Q). When the probe sequence in the loop hybridizes with its target, forming a rigid double helix, a conformational reorganization occurs that separates the quencher from the fluorophore, restoring the fluorescence of the fluorophore. In our MB design, TMR is used as the fluorophore and DABCYL as the quencher. 2921 J. Am. Chem. Soc. 1999, 121, 2921-2922

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