Small‐Molecule Screening Made Simple for a Difficult Target with a Signaling Nucleic Acid Aptamer that Reports on Deaminase Activity

Abstract

Conventional approaches to small-molecule screening are currently being challenged by the availability of vast amounts of genomic sequence information and a plethora of potential targets. This challenge has precipitated a need for new screening paradigms that emphasize simplicity, capacity, and parallelization. Herein we report the application of signaling DNA-aptamer technology for the development and execution of a high-throughput screen for an otherwise problematic target, adenosine deaminase (ADA). The approach employed a signaling DNA aptamer that reports on adenosine concentration over the course of the enzymatic reaction. The assay was extremely robust in a screen of more than 44 000 molecules and revealed a new competitive inhibitor of the deaminase. Nucleic acid aptamers have proven worthy as a routinely selectable species for a wide variety of small molecules and so this proof-of-principle work has broad

Applications of Aptamers as Sensors

“…The aptamer can be labeled with a fluorophore and the antisense oligonucleotide with a quencher, leading to a quenched fluorescent signal in the absence of target, similar to the unimolecular aptamer beacons described above. Nutiu & Li (37) have pioneered this approach for aptamer beacon design by making antisense aptamer beacons that can detect ATP (K d = 600 μM) and thrombin (K d = 400 nM), and such aptamer beacons have been further adapted to high-throughput drug screening (38). The antisense oligonucleotide can also contain fluorescent nucleobases, rather than dyes (39).…”

Functional Nucleic Acids in High Throughput Screening and Drug Discovery

“…Therefore, development of a homogeneous and sensitive assay amenable to HTS format will be of great significance. Elowe and coworkers developed a screening assay similar to the one discussed above, by using a signaling aptamer, which exhibited high affinity for adenosine and practically no affinity for inosine [36]. The aptamer was fluorescently modified at the 5' end with fluorescein and contained a complementary binding region for the quencherlabeled antisense strand (Fig.…”

Strategy for Use of Smart Routes to Prepare Label‐Free Aptasensors for Bioassay Using Different Techniques

“…The aptamer beacon approach contains two modes of signal-on (A1) (Yamamoto and Kumar, 2000;Hamaguchi et al, 2001) and signal-off/FRET (A2) (Li et al, 2002;Ueyama et al, 2002). The duplex-to-complex switching approach contains two signal-on modes: B1 (Rupcich et al, 2005;Elowe et al, 2006) and B2 .…”

“…One often places an F-labeled (Q-labeled) aptamer in a duplex structure with a (partial) complementary sequence labeled with a Q (F) (Figure 12.11B1) (Rupcich et al, 2005;Elowe et al, 2006). Addition of the target forces the departure of the partial complementary strand from the aptamer, accompanied by an increase in fluorescence.…”