Aptamers, synthetic oligonucleotide-based molecular recognition probes, have found use in a wide array of biosensing technologies based on their tight and highly selective binding to a variety of molecular targets. However, the inherent challenges associated with the selection and characterization of aptamers for small molecule targets have resulted in their underrepresentation, despite the need for small molecule detection in fields such as medicine, the environment, and agriculture. This protocol describes the steps in the selection, sequencing, affinity characterization, and truncation of DNA aptamers that are specific for small molecule targets. © 2017 by John Wiley & Sons, Inc.

The delivery of therapeutics across the blood-brain barrier remains a considerable challenge in investigating central nervous system related processes. In this work, a liposome vehicle was surface-modified with an aptamer that binds to the transferrin receptor and was loaded with two different dopamine-binding aptamer payloads. This system was effectively used to promote the delivery of the aptamer cargo from the peripheral injection site into the brain. The effect of these delivered aptamers on behavior was investigated in vivo in a locomotor task. The first dopamine binding aptamer assessed was a DNA aptamer, the binding of which had been previously validated through the aptamer-based biosensor development reported by several independent research groups. The second aptamer investigated was the result of a novel in vitro selection experiment described herein. Our data suggest that systemic administration of the modified liposomes led to delivery of the dopamine aptamers into the brain. Fluorescence microscopy revealed differential distribution of fluorescence based on the presence or absence of the transferrin receptor aptamer on the surface of fluorescently modified liposomes. In a behavioral experiment using cocaine administration to induce elevated concentrations of neural dopamine, systemic pretreatment with the dopamine aptamer-loaded liposomes reduced cocaine-induced hyperlocomotion. Multiple controls including a transferrin-negative liposome control and transferrin-positive liposomes loaded with either a nonbinding, base-substituted dopamine aptamer or a random oligonucleotide were investigated. None of these controls altered cocaine-induced hyperlocomotion. Chronic systemic administration of the modified liposomes produced no deleterious neurobehavioral or neural degenerative effects. Importantly, this work is one example of an application for this versatile multiaptamer payload/targeting system. Its general application is limited only by the availability of aptamers for specific neural targets.

Aptamers are functional nucleic acids that function in a wide variety of applications, from biosensing to catalysis to therapeutics. Considered functionally analogous to antibodies, aptamers are single-stranded oligonucleotides capable of binding to target molecules with great affinity and specificity. In the field of medicine, the potential biological application of aptamers as a therapeutic or diagnostic is ever growing. Aptamers designed to target α-synuclein, a protein implicated in the pathogenesis of Parkinson's disease due to its propensity to aggregate, were investigated to probe their affinity for the protein as well as their ability to hinder the aggregation of the protein in vitro. Aggregation assays have proven that the presence of the aptamer candidates targeting monomeric α-synuclein stunt the formation of protein fibrils. One promising aptamer from the research, a-syn-1, was successfully packaged into a liposome vesicle modified to cross the blood-brain barrier and used in in vivo applications. The a-syn-1 aptamer has been delivered to the brains of transgenic mice, expressing human α-synuclein, where it has been colocalized with α-synuclein, and ongoing pharmacokinetic studies are being pursued to investigate the lifetime and distribution of the aptamer in a mouse model through extraction and PCR amplification of the aptamer. Analysis of the liposomes used for the packaging and delivery of the aptamer has provided insight into the loading efficiency and production efficiency of liposome batches.