Recent Advances in Aptamer Discovery and Applications

Abstract

Aptamers are short, single-stranded DNA, RNA, or synthetic XNA molecules that can be developed with high affinity and specificity to interact with any desired targets. They have been widely used in facilitating discoveries in basic research, ensuring food safety and monitoring the environment. Furthermore, aptamers play promising roles as clinical diagnostics and therapeutic agents. This review provides update on the recent advances in this rapidly progressing field of research with particular emphasis on generation of aptamers and their applications in biosensing, biotechnology and medicine. The limitations and future directions of aptamers in target specific delivery and real-time detection are also discussed.

Keywords: aptamer; biosensor; diagnostics; nanorocket; systematic evolution of ligands by exponential enrichment (SELEX); therapeutics.

Figure 1

Schematic representation of SELEX (Systematic Evolution of Ligands by EXponential Enrichment). The starting single-stranded DNA or RNA library (10¹⁴~10¹⁶ random oligonucleotides) is composed of sequences 20~100 nucleotides in length with a random region in the middle flanked by fixed primer sequences. After incubation with the target of interest, the bound oligonucleotides are partitioned from unbound sequences and amplified by PCR. The resulting enriched DNA pool is used for the next round of selection.

Figure 2

Flowchart of animal SELEX. Animal SELEX can be used to generate aptamers specific to target tissues. (A) Aptamer libraries are first injected into the target mice. (B) After inoculation, the organs of interest are harvested. (C) The selected aptamers are isolated and amplified by PCR. (D) After rounds of selection, counter selection can be performed by inoculating aptamer pool into the healthy mouse tissues. (E) The aptamer sequences with high affinity and specificity to the target tissues of interest are selected and identified by sequencing.

Figure 3

Aptamers used in biosensors. Aptamer-based biosensors are used to detect disease biomarkers, monitor environmental contaminants, or to ensure food safety. Aptamers can be further enhanced by different nanomaterials or biomaterials. The signal of detection in the most of the recently developed aptamer-based biosensors is based on electric or optic/fluorescent signal.

Figure 4

Aptamer-tethered multistage “nanorocket” for target-specific delivery. (A) An aptamer-tethered multistage “nanorocket” is a complex of two or more aptamers, each of which contains its own functional oligonucleotide. Serial stages of aptamers are mounted on top of another to build a carrier “nanorocket” for the target specific recognition and delivery. With appropriate linkages, each stage of aptamer “nanorocket” can freely rotate and perform its function. (B) The selected aptamers can be used in different stages of “nanorockets”, designed for tissue penetration, cell target recognition and cellular internalization. After reaching target, the recognition and delivering stages of the “nanorockets” can be cleaved and degraded by the cell leaving only the cargo oligos. Aptamers in multi-stage “nanorockets” can be used as tissue-, cell-type-, and cellular compartment-specific delivery systems.

Figure 5

Flowchart of nanopore-based aptamer assaying. Nanopore sensors can be used to detect aptamer binding and selection in a real-time. Aptamer and target interaction can be detected by measuring the current disruption caused by molecules electrophoretically driven through the pore. (A) An ionic current is passed through the nanopore. The current changes as molecular target (B), aptamer (C), or aptamer–target complex (D) passes through the nanopore.