Signaling Aptamers for Monitoring Enzymatic Activity and for Inhibitor Screening

We examined fluorescence-signaling aptamers as real-time reporters for quantifying enzyme activities and identifying enzyme inhibitors. The conversion of adenosine 5’-monophosphate (AMP) into adenosine by alkaline phosphatase (ALP) was used as a model reaction, and a previously described structure-switching signaling DNA aptamer was employed as a model reporter. The signaling aptamer, which has a higher affinity for adenosine than for AMP, is able to generate a twoleg signaling profile. The first leg of the signal is produced upon addition of AMP and indicates the formation of a reactant±aptamer complex. The second leg is produced upon addition of ALP and reports the enzymatic conversion of the reactant into the product. Aptamers are single-stranded nucleic acids with ligand-binding capabilities that can be isolated from random-sequence nucleic acid pools. Several reports have been published that describe the use of fluorescence-based signaling aptamers or signaling ribozymes and deoxyribozymes to detect small molecules and proteins in solution. We recently described a strategy for preparing signaling DNA aptamers that function by a mechanism involving coupled structure switching and fluorescence dequenching. Signaling aptamers usually display signals of great magnitude (more than tenfold fluorescence increase upon target binding) and are capable of real-time reporting at low temperatures (15±37 8C). These properties led us to speculate that structure-switching signaling aptamers might be used as sensitive probes to report enzymemediated reactions in real time. Take a simple chemical reaction A!B as an example. For a signaling aptamer to be useful for reporting this chemical transformation in real time it must exhibit a different level of fluorescence in the presence of A than with B. If the signaling aptamer has a higher level of fluorescence in the presence of B, the A-to-B transformation can be monitored conveniently by following the increase in the fluorescence intensity of the signaling aptamer. If an enzyme mediates the chemical reaction, the presence of the fluorescent aptamer reporter should permit real-time monitoring of the activity of the enzyme, as well as screening for small-molecule inhibitors. We found previously that the structure-switching ATP reporter shown in Figure 1A is able to generate signals with different fluorescence intensities depending on whether adenosine or one of its 5’-phosphorylated analogues is used as the target.