Advances and Challenges in Small-Molecule DNA Aptamer Isolation, Characterization, and Sensor Development

Abstract

Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor-intensive to develop aptamer-based sensors for small-molecule detection. Here, we review the challenges and advances in the isolation and characterization of small-molecule-binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer-target binding and structure. Afterwards, we discuss various small-molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer-based small-molecule sensors for real-world applications.

Keywords: DNA; SELEX; aptamers; biosensors.

Figure 1.

The general process of SELEX entails multiple cycles of target incubation with a nucleic acid library, target-binding strand separation, PCR amplification, and single-stranded DNA generation to regenerate a new library for another iteration of this process.

Figure 2.

SELEX library designs including (A) unstructured linear libraries as well as functionalized (B) G-quadruplex, (C) stem-loop, (D) three-way junction, and (E) two-way junction structured libraries. Black- and red-colored regions indicate conserved and randomized regions, respectively.

Figure 3.

Common strategies to separate aptamers from non-binding strands in oligonucleotide libraries including (A) target-immobilized SELEX, (B) library-immobilized SELEX, and (C) homogeneous SELEX. The red sphere represents a solid substrate like a microbead.

Figure 4.

Common SELEX strategies for modulating aptamer specificity. Working principle of (A) negative/counter-SELEX, (B) Toggle-SELEX, and (C) parallel-serial SELEX.

Figure 5.

Different sensing strategies to develop aptamer-based sensors from a fully-folded aptamer through a variety of strategies, including (A) aptamer truncation, (B) splitting, (C) introducing a competitive strand, (D) use of a dye probe, and (E) exonuclease digestion.