Neurofilament light chain (NfL) is a structural protein component of large nerve fibers that has emerged as a biomarker of neuronal injury related to a broad range of diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS), among others. Now, researchers at the Tokyo University of Science report they have developed DNA aptamers that bind to NfL with high affinity and specificity, pointing to its potential for use in blood-based diagnostics for neurodegeneration. Their research is published in the journal Biochemical and Biophysical Research Communications.
“We have reported the world’s first DNA aptamer that binds to NfL, which is released into the blood in response to neurodegeneration in various neurodegenerative diseases. The developed aptamer has a binding affinity comparable to commercially available antibodies, and is expected to have a variety of applications in the future, such as diagnosing the progression of AD,” said senior author Kaori Tsukakoshi, PhD, an associate professor of chemistry at Tokyo University of Science.
Prior research has shown that when axons are injured, fragments of NfL enter cerebrospinal fluid and eventually circulate in blood. “Neurofilament light chain (NfL) is a 68-kDa cytoskeletal protein expressed in large, myelinated axons, where it contributes to axonal structure and mechanical stability. Axonal injury leads to the release of NfL into cerebrospinal fluid and blood, and circulating NfL has emerged as a robust and quantitative biomarker of neurodegeneration,” the researchers wrote.
Evidence from prior studies suggested the biomarker could be used for detecting a number of different neurological conditions, which has led researchers to look for methods to develop reliable methods to use it to identify the presence of disease. “Several landmark clinical studies have demonstrated that plasma NfL correlates with neuronal injury severity in Alzheimer’s disease (AD), Parkinson’s disease, multiple sclerosis, and frontotemporal dementia, and predicts cognitive decline and neurodegenerative progression,” the researchers wrote.
To identify molecules capable of binding NfL, the research team used Systematic Evolution of Ligands by Exponential Enrichment, or SELEX, a screening process that identifies and isolates DNA sequences that have strong binding properties. The team used large libraries of random DNA strands and tested them repeatedly to those sequences that attached to NfL. These sequences were then recovered and amplified while strands that bound to unrelated tags were removed. This identified 86 potential aptamer candidates. The researchers then removed sequences that were likely to form multimers and cause non-specific interactions, further narrowing the field to 30 candidates that recognized the full-length NfL protein.
“Here, we report the identification of novel single-stranded DNA aptamers that specifically recognize NfL with high affinity,” the researchers wrote. “Two lead aptamers, MN711 and MN734, displayed nanomolar dissociation constants of 11 nM and 8.1 nM, respectively, comparable to those of widely used anti-NfL antibodies.”
In lab experiments the researchers showed that both aptamers bound to a region of the NfL protein containing amino acid residues 281–338. This region is present in several fragments detected in blood plasma of patients with Alzheimer’s disease. Continuing on this path, the team then used human plasma mixed with this protein and found that it maintained its binding ability, further strengthening the case of it potential for use in a blood-based diagnostic.
Specificity testing showed that the aptamers had minimal binding to other proteins associated with Alzheimer’s disease, including amyloid-beta and phosphorylated tau, a potentially important measure as selectivity may be important for future diagnostic systems designed to measure NfL without interference from related biomarkers.
Currently, NfL detection relies on immunoassays that use monoclonal antibodies to find the protein in blood. While this method can achieve high sensitivity, the antibodies needed for this can be expensive and more difficult to adapt for integration with some sensor technologies.
DNA aptamers offer a different approach. “A key advantage of DNA aptamers is their compatibility with chemical modification,” the researchers noted. “Aptamers can be synthesized with terminal functional groups that enable straightforward immobilization onto metallic or carbon-based electrode surfaces commonly used in electrochemical biosensors.”
The researchers will continue their work to find ways to integrated these aptamers for different sensing platforms and conduct broader testing of their method on clinical samples from patient with different neurodegenerative diseases. In addition, the team will explore making structural modifications to one of the aptamers to improve its binding properties, potentially enhancing its potential as a diagnostic tool.
