In vitro selection of structure-switching signaling aptamers.

Signaling aptamers are aptamer probes that couple target binding to fluorescent-signal generation. These molecular sensors have potential applications in biosensing, proteomics, and drug discovery. Here we describe a new in vitro selection approach for generating unmodified DNA aptamers that can be immediately transformed into effective signaling probes without the need for further optimization. Standard aptamers can be easily generated by the technique of in vitro selection. However, for biosensing applications, postselection modifications must be carried out in order to convert the aptamer concerned into a signaling probe. Although several conversion strategies have been reported, the modification processes often require lengthy optimization steps to ensure that the affinity and specificity of the original aptamer is not lost upon labeling and that the modified signaling aptamer is able to significantly modulate the fluorescent signal upon target binding. An alternative method is to create signaling aptamers directly by in vitro selection. Jhaveri et al. have reported the only signaling-aptamer selection method by which signaling aptamers labeled with a single fluorophore can be created. However, their results showed that even if the selected aptamers were capable of target binding, a significant number of them failed to induce a concomitant fluorescence change upon target binding, presumably due to the insubstantial alteration of fluorescence properties of the attached fluorophore. More recently, the same group published a selection method for the optimization of signaling properties of molecular beacons containing a fluorophore–quencher pair. It is conceivable, as the authors have already suggested in their report, that this method could be adapted to the selection of signaling-aptamer probes that detect nonnucleic acid targets. Encouraged by their work, we conducted an in vitro selection study for deriving signaling aptamers that function by the structure-switching mechanism previously described by us for the rational design of signaling aptamers. The structure-switching idea takes advantage of the universal ability of any aptamer to adopt two different structures: a duplex structure with an antisense DNA and a complex structure with the cognate target. We have shown that an aptamer can switch structures from duplex to complex upon target addition and, if the aptamer is labeled with a fluorophore and the antisense DNA is labeled with a quencher (denoted QDNA), the structure-switching process can be synchronized to fluorescence signaling. However, fine-tuning of the QDNA represents a major challenge in the design of structure-switching signaling aptamers, as many QDNA sequences must be tested before a suitable signalingaptamer system can be established. Therefore, we sought to overcome this problem by utilizing the power of in vitro selection. The strategy described in this report permits the creation of standard aptamers that are encoded with a duplexto-complex switching capability and can be converted into signaling probes immediately upon their isolation. The selection strategy is shown in Figure 1. A special DNA library (Figure 1a) was used that contained a central 15nucleotide fixed-sequence domain (red) flanked by two random-sequence domains (green) of 10 and 20 nucleotides, each further flanked by primer-binding sequences (black). The central fixed-sequence domain was designed to be complementary to an antisense oligonucleotide biotinylated at its 5’-end (denoted BDNA). This arrangement permitted immobilization of the DNA library onto avidin-coated beads through DNA hybridization (step 1 of Figure 1 b; see the Supporting Information for more details regarding the library design). We also added two short oligonucleotides, P1 and P2, to prevent the involvement of the primer-binding sequences in the tertiary folding of eventual aptamers. To demonstrate our concept, we used four standard NTPs (nucleoside triphosphates; blue star) as the potential aptamer targets. The immobilized DNA assembly was then exposed to a solution of a mixture of NTPs, each at 0.1 mm (step 2). Aptamers able to form the DNA/target complex should switch from the bead-bound state to the solution. These molecules were then collected and amplified by PCR (step 3). The reverse primer contained a ribonucleotide (R) at its 3’end to create a chimeric antisense strand prone to NaOHmediated cleavage, which permitted the isolation of the sense strand by gel electrophoresis (step 4). The recovered sense DNA was reannealed to BDNA, P1, and P2 and used for the [*] R. Nutiu, Prof. Dr. Y. Li Department of Biochemistry and Department of Chemistry McMaster University Hamilton, ON, L8N3Z5 (Canada) Fax: (+ 1)905-522-9033 E-mail: liying@mcmaster.ca