Fluorescent Sensors Based on Aptamer Self-Assembly.

In vitro selection of oligonucleotides has made possible the isolation of aptamers with high binding affinity for small molecules, proteins, or even whole cells.1 Recently, aptamers were demonstrated to rival antibodies in analytical methods based on heterogeneous assays.2 Aptamers often undergo a conformational change upon ligand binding and, if so, can be converted to fluorescent sensors by either modification with fluorescent oligonucleotide analogs3 or double-end-labeling with donor and acceptor fluorophores.4 We describe herein a simple and potentially general approach to the rational construction of sensors5 based on fluorophore-labeled heterodimeric aptamers that assemble as a function of ligand concentration. Oligonucleotide aptamers generally consist of a single oligonucleotide chain comprised of two partially complementary domains. These domains are connected through loops, and very often these loops are not essential for ligand binding. We hypothesized that removal of such loop regions would convert the aptamer into subunits that could, nonetheless, reassemble to form the ligand-binding pocket. We were encouraged by a precedent in which an anti-adenosine ribonucleotide triphosphate rATP aptamer consisting of two subunits was still able to bind to an rATP affinity column.6 We further assumed that, by adjusting the complementarity, we would be able to tune the system to an equilibrium that favors individual subunits over assembled heterodimer but that shifts toward the latter upon the binding of ligand. If each subunit were labeled with a fluorophore, then proximity-dependent communication of the fluorophores could be used to assess the position of the equilibrium and hence the concentration of the ligand. Ligand-dependent self-assembly of aptamers is analogous to the assembly of heavy and light chains of an Fv antibody fragment through ligand binding that was used to develop an “open sandwich fluoroimmunoassay”. 7 However, in the case of aptamers, the subunit interactions can be adjusted easily by modifying the length of complementary regions, and therefore the subunit affinity required for ligand-dependent assembly can be attained more predictably. In the course of constructing sensors capable of reporting the activity of anticocaine catalytic antibodies,8 we developed oligodeoxynucleotide aptamers for cocaine (1) by standard methods.1 To construct a hetorodimeric aptamer, we began with a cocainebinding 39-mer with Kd ) 5 μM at c(Mg2+) ) 1 mM and, based on the suggested secondary structure, separated it at a predicted loop into two subunits9 (apparent Kd of the self-assembled aptamer, ∼200 μM by equilibrium gel filtration). 10 We labeled one subunit with a 5′-6-carboxy-fluorescein fluorophore (6-FAM, F in F-C1) and the other with a 3′-dabcyl quencher (C2-D) (Figure 1). Dabcyl is a universal, nonfluorescent, non-Förster quencher that is used in molecular beacons to follow the intramolecular hybridization of the 5′and 3′-ends.11 Dabcyl is characterized by an efficient π-overlap quenching in the hybridized state and little energy transfer quenching in the nonhybridized state. As predicted, in the selection buffer (c(TRIS) ) 20 mM, pH ) 7.4, c(NaCl) ) 140 mM) at the optimum chain concentrations of c(F-C1) ) 10 nM and c(C2-D) ) 60 nM and c(Mg2+) between 0 and 4 mM, the two subunits behaved as a self-assembling fluorescent cocaine sensor. The sensor reliably reported concentrations of cocaine in the range from 10 to 1250 μM with fluorescein emission at 518 nm (λex ) 472) that was quenched to 65% of the initial value (Figure 2). The concentration range can be shifted to 1 μM by employing higher concentrations of Mg2+ or by prolonging incubation times. This sensor showed excellent selectivity for cocaine over its metabolites benzoyl ecgonine (2) and ecgonine methyl ester (3) (Figure 2). Two important negative controls support a mechanism of action based on ligand-driven association of subunits. First, when C2 without dabcyl was substituted for C2-D, no fluorescence quenching was observed. Second, when C2-D was combined with an analogue of F-C1 incapable of binding cocaine due to substitution of the N12-N20 segment d(ATGAAGTGG) with d(AAAAAAAAA), no cocaine-dependent quenching was (1) Hermann, T. ; Patel, D. J. Science 2000, 287 (5454), 820-825 and references therein. (2) (a) Jayasena S. D. Clin. Chem. 1999, 45 (9), 1628-1650. For homogeneous competitive assays based on aptamers, see: (b) Wang, Y. ; Killian, J.; Hamasaki, K. ; Rando, R. R. Biochemistry 1996, 35 (38) 1233812346. (c) Jayasena, S. ; Gold, G. PCT Int. Appl. WO 9931276 A1, CAN: 131: 54725, 1999. (3) Jhaveri, S. D. ; Kirby, R. ; Conrad, R. ; Maglott, E. J.; Bowser, M. ; Kennedy, R. T. ; Glick, G. ; Ellington, A. D. J. Am. Chem. Soc. 2000, 122, 2469-2473. (4) Stojanovic, M. N. ; de Prada, P.; Landry, D. W. , manuscript in preparation. (5) De Silva, A. P.; Gunaratne, H. Q. N. ; Gunnlaugsson, T. ; Huxley, A. J. M. ; McCoy, C. P.; Rademacher, J. T. ; Rice, T. E. Chem. ReV. 1997, 97 (5), 1515-1566 and references therein. (6) Huizenga, D. E. ; Szostak, J. W. Biochemistry 1995, 34, 656-665. (7) (a) Ueda, H. ; Kubota, K. ; Wang, Y. ; Tsumoto, K. ; Mahoney, W. ; Kumagai, I. ; Nagamune, T. BioTechniques 1999, 27, 738-742. (b) Ueda, H. ; Tsumoto, K. ; Kubota, K. ; Suzuki, E. ; Nagamune, T. ; Nishimura, H. ; Schueler, P. A. ; Winter, G. ; Kumagai, I. ; Mahoney, W. C. Nat. Biotechnol. 1996, 14, 1714-1718. (8) Yang, G. ; Arakawa-Uramoto, H. ; Wang, X. ; Gawinowicz, M. A. ; Zhao, K; Landry, D. W. J Am. Chem. Soc. 1996, 118, 5881-5890. (9) All oligonucleotides were custom-made and purified by Integrated DNA Technologies (Coralville, IA) and were used as received. (10) Isolation and full characterization of cocaine-binding aptamers will be reported separately: Stojanovic, M. N. ; de Prada, P.; Landry, D. W. , manuscript in preparation. (11) Tyagi, S. ; Bratu, D. P.; Kramer, F. R. Nat. Biotechnol. 1998, 16 (1), 49-58. Figure 1. Self-assembly of two aptamer subunits F-C1 and C2-D in the presence of cocaine (1): D, dabcyl quencher; F, 6-FAM; smaller font F quenched 6-FAM. The secondary structure of the aptamer is based on mutational analysis.1