New methods for developing nucleic acid aptamers could support their wider use in molecular diagnostics, according to a review published in Signal Transduction and Targeted Therapy.
Aptamers are short, single-stranded DNA or RNA molecules that fold into shapes capable of binding specific targets. Often described as “chemical antibodies,” they can recognize proteins, small molecules, ions, viruses, bacteria, and whole cells.
Aptamers are commonly developed using systematic evolution of ligands by exponential enrichment (SELEX). Researchers begin with a large library of nucleic acid sequences and isolate those that bind the intended target. These sequences are copied and tested repeatedly until the strongest candidates remain.
Traditional SELEX can require many labor-intensive selection cycles. It may also identify aptamers that bind purified targets but perform less reliably in blood, tissue, or other complex clinical samples.
Newer approaches aim to accelerate selection and improve performance. Microfluidic platforms can sort candidate sequences more rapidly, while nuclease-assisted methods remove unstable or weakly binding candidates. Hydrogel-based systems may reduce nonspecific binding.
Artificial intelligence and machine learning are also being used to predict aptamer structures, generate new sequences, and analyze large datasets. Some platforms combine aptamer screening with CRISPR gene editing and single-cell analysis to identify the target and determine how binding affects cell function.
The authors emphasized that identifying a candidate is only the first step. Before an aptamer can be developed into a diagnostic test, laboratories must confirm its structure, binding strength, specificity, and performance in clinically relevant samples.
Nuclear magnetic resonance spectroscopy and cryo-electron microscopy can help determine aptamer structure. Computational tools can provide faster predictions, but these results still require experimental confirmation. Other laboratory methods, including surface plasmon resonance, biolayer interferometry, and flow cytometry, can measure binding strength, kinetics, and specificity.
An aptamer-based diagnostic platform usually includes three elements: an aptamer that recognizes the target, a reporting system that produces a measurable signal, and, when needed, a method to amplify that signal.
Target binding can be detected through fluorescence, electrochemical measurements, or surface-enhanced Raman scattering. Amplification methods include rolling circle amplification, hybridization chain reaction, and CRISPR-based systems.
Potential applications include liquid biopsy, molecular imaging, biomarker testing, point-of-care testing, and wearable monitoring. Multiplex platforms could measure several targets at once, supporting disease detection, classification, and treatment monitoring. Research is underway in cancer, infectious disease, cardiovascular disease, neurologic disorders, and metabolic conditions.
Aptamers offer several practical advantages over antibodies. They can be chemically synthesized, modified at specific sites, and produced with limited batch-to-batch variation. Their small size and thermal stability may also simplify assay development, storage, and transportation.
However, clinical translation remains limited. Aptamers may change shape or bind nonspecifically in complex samples. Their performance can be affected by temperature, pH, ionic conditions, specimen type, and interfering substances. Some are also vulnerable to degradation by nucleases.
For clinical laboratories, aptamer-based tests will require the same evidence expected for other diagnostic technologies. This includes analytical sensitivity and specificity, reproducibility, interference studies, standardized quality control, and validation in representative patient populations. New tests must also demonstrate that they provide useful information beyond existing methods.
Although thousands of aptamers have been developed, relatively few have reached routine clinical use. The authors concluded that improved selection methods, stronger target validation and standardized clinical testing will be needed before these probes can be widely adopted.
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