Detection of Circulating Serum microRNA/Protein Complexes in ASD Using Functionalized Chips for an Atomic Force
Anna L Kaysheva1, Arina I Isaeva1, Tatyana O Pleshakova1
1Laboratory of Nanobiotechnology, Institute of Biomedical Chemistry, Pogodinskaya St. 10/8, 119121 Moscow, Russia.
Abstract:
MicroRNAs, which circulate in blood, are characterized by high diagnostic value; in biomedical research, they can be considered as candidate markers of various diseases. Mature microRNAs of glial cells and neurons can cross the blood-brain barrier and can be detected in the serum of patients with autism spectrum disorders (ASD) as components of macrovesicles, macromolecular protein and low-density lipoprotein particles. In our present study, we have proposed an approach, in which microRNAs in protein complexes can be concentrated on the surface of AFM chips with oligonucleotide molecular probes, specific against the target microRNAs. MicroRNAs, associated with the development of ASD in children, were selected as targets. The chips with immobilized molecular probes were incubated in serum samples of ASD patients and healthy volunteers. By atomic force microscopy (AFM), objects on the AFM chip surface have been revealed after incubation in the serum samples. The height of these objects amounted to 10 nm and 6 nm in the case of samples of ASD patients and healthy volunteers, respectively. MALDI-TOF-MS analysis of protein components on the chip surface allowed us to identify several cell proteins. These proteins are involved in the binding of nucleic acids (GBG10, RT24, RALYL), in the organization of proteasomes and nucleosomes (PSA4, NP1L4), and participate in the functioning of the channel of active potassium transport (KCNE5, KCNV2).
Insights
Researchers developed a novel method to detect autism spectrum disorder (ASD) biomarkers in blood. This technique uses atomic force microscopy (AFM) to identify specific microRNAs linked to ASD in children.
Area of Science:
- Biomedical research
- Molecular biology
- Neuroscience
Background:
- Circulating microRNAs in blood show significant diagnostic potential for various diseases.
- Mature microRNAs from glial cells and neurons can cross the blood-brain barrier and are detectable in serum of individuals with autism spectrum disorders (ASD).
- These microRNAs are found within macrovesicles and lipoprotein particles in serum.
Purpose of the Study:
- To propose and validate an approach for concentrating and detecting microRNAs associated with ASD development in children.
- To utilize atomic force microscopy (AFM) chips with specific oligonucleotide probes for targeted microRNA capture.
- To differentiate between serum samples from ASD patients and healthy volunteers based on microRNA detection.
Main Methods:
- Developed AFM chips functionalized with oligonucleotide molecular probes specific to target microRNAs.
- Incubated functionalized chips with serum samples from children with ASD and healthy controls.
- Analyzed chip surfaces using atomic force microscopy (AFM) to measure object heights.
- Performed MALDI-TOF-MS analysis to identify protein components associated with captured microRNAs.
Main Results:
- AFM revealed distinct object heights on the chip surfaces: 10 nm for ASD patient samples and 6 nm for healthy volunteers.
- MALDI-TOF-MS identified cell proteins involved in nucleic acid binding, proteasome and nucleosome organization, and potassium channel function.
- Specific microRNAs associated with ASD development were successfully concentrated and detected.
Conclusions:
- The proposed method effectively concentrates and detects microRNAs in protein complexes from serum using AFM chips.
- Distinct physical characteristics (height) of captured complexes can differentiate between ASD patients and healthy individuals.
- Identified proteins provide insights into the molecular mechanisms potentially underlying ASD.
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