Detection And Aggregation Regulation Of β Amyloid By Using Aptamers Based On Atomic Force Microscopy

Alzheimer’s disease（AD）is a progressive and irreversible neurodegenerative disease.Various neurotoxic aggregates formed during the aggregation ofβ-amyloid peptide（Aβ）and amyloid plaques formed by fibril deposition are considered as important factors for predisposing AD.Aβmonomer and aggregates have been broadly considered as an important biomarker and therapeutic target for AD.Therefore,the accurate and sensitive quantification of Aβmonomer and aggregates or the regulation of Aβaggregation process is contribute to the early diagnosis and treatment of AD.Thanks to the development of aptamers against Aβ,its application in neurodegenerative diseases has attracted increasing attention.On the one hand,aptamers could be applied to the detection of Aβaggregates,which provides the possibility of early diagnosis of AD.However,the achievement of accurate and highly sensitive quantification of Aβaggregates in complex systems based on aptamers still faces challenges.On the other hand,it has been reported that RNA aptamers could be used for regulating the Aβaggregation process,which not only provides a potential tool for regulating Aβaggregation,but also provides a new strategy for AD treatment.However,RNA aptamers are easily contaminated and degraded by enzymes,resulting in low inhibition efficiency.Hence,developing new aptamers for inhibiting Aβaggregation with high efficiency is the persevered pursuit.Given that atomic force microscopy（AFM）technology possesses the characteristics of nanometer-level temporal and spatial resolution,pico-Newton force measurement sensitivity,free label,and flexible operating environment,herein,aptamers were used for detecting and regulating aggregation of Aβbased on AFM.The details are as follows:1.Investigation of the interactions between the aptamer and amyloid using single-molecule force spectroscopyUnderstanding the interaction of misfolded amyloid and the aptamer is crucial for the development of aggregation inhibitors and the diagnosis of amyloid diseases.In this work,the lysozyme as a model,the single-molecule force spectroscopy（SMFS）technology based on AFM was use for exploring the interaction of aptamer with lysozyme monomer,oligomer and amyloid fibril at different loading rates.It was found that the aptamer against lysozyme monomer can also recognize oligomer and amyloid fibril.In addition,although the lysozyme monomer-aptamer,oligomer-aptamer and amyloid fibril-aptamer complexes went through the similar dissociation pathway during the dissociation process,the oligomer-aptamer and amyloid fibril-aptamer complexes were more stable than lysozyme monomer-aptamer complexes.This work showed that SMFS could investigate the interaction between amyloid and aptamer at the single-molecule level,so as to provided valuable information for the application of aptamers in amyloid-related research.2.Aptamers as recognition probes for the detection ofβamyloid aggregatesThe interaction between Aβ₄₀ and two Aβ₄₀ aptamers（Apt1 and Apt2）was investigated using SMFS.It was found that the interaction force histograms of Apt1-Aβ₄₀ oligomer,Apt2-Aβ₄₀ oligomer and Apt2-Aβ₄₀ fibril showed a unimodal distribution,while the interaction force histogram of Apt1-Aβ₄₀ fibril showed an obvious bimodal distribution.On this basis,the result from SMFS measurement indicated that the two aptamers could simultaneously recognize Aβ₄₀ aggregates(including Aβ₄₀ oligomer and fibril).Accordingly,surface plasmon resonance sensor based on dual aptamers was constructed for the detection of Aβ₄₀ aggregates.The detection limit of Aβ₄₀ oligomer was as low as 0.2 p M,and the detection limit of Aβ₄₀ fibiril was as low as 0.05 p M.The sensor could be applied to sensitive detection of Aβ₄₀ aggregates in complex systems（artificial cerebrospinal fluid and undiluted real cerebrospinal fluid）.This work provided a strategy for monitoring simultaneous recognition events,which provided valuable information for the design of biosensing elements,and broadened the application of aptamers in the diagnosis of neurodegenerative diseases.3.Aptamer as a tool for investigating the effects of electric field on Aβ40monomer and aggregatesAlthough the research with regard to the effect of electric field on the aggregation of Aβhas attracted more attention,related reports have been still controversial.In this work,aptamer was used as a tool for investigating the effects of electric field on Aβ₄₀ monomer and aggregates by SMFS.It was found that after Aβ₄₀monomer and fibril were treated with electric field,the interaction of aptamer and Aβ₄₀ monomer changed from non-specific interaction to specific interaction,and the interaction force distribution of aptamer and Aβ₄₀ fibril changed from the bimodal peak to unimodal peak.However,after Aβ₄₀ oligomer was treated with electric field,the specific interaction force and binding probability of aptamer-Aβ₄₀ oligomer increased slightly.It was speculated that the phenomenon may be due to the changes in the conformation of Aβ₄₀ monomer and Aβ₄₀ aggregates treated with electric field.The results from AFM imaging also confirmed this speculation.This work was not only helpful for analyzing the effect of electric field on Aβmonomer and aggregates,but also expanded the research on the influence of other external factors（such as medicine）on Aβmonomer and aggregates.4.Aptamer as a regulator for the study ofβamyloid aggregation regulationDeveloping a new strategy with low-cost,high-efficiency and biosafety for regulating the Aβaggregation process is of great significance for the treatment of AD.Aptamers have attracted much attention as one of the promising inhibitors of amyloid aggregation.The effect of Aβ₄₂-specific aptamer Apt₄₂ screened by our research group on the Aβaggregation process was explored.It was found that the aptamer could completely inhibit the Aβ₄₂ fibrillation process and delay the Aβ₄₀ aggregation process and decrease the number of Aβ₄₀ fibril.In addition,it was also found that the inhibition effect of aptamer in the process of Aβoligomer aggregation was significantly better than in the fibrillation process of Aβmonomer.This work is expected to provide an effectiveβamyloid aggregation inhibitor,which is helpful for the treatment of AD.5.Constructed nanostructure based on the aptamer for study ofβamyloid aggregation regulationIn view of the stable structure,good biocompatibility,easy synthesis and design of DNA nanostructure,it was combined with the nucleic acid aptamer Apt₄₂ to construct the nanostructure HCR-Apt.Further,its effect on regulation of the Aβ₄₀aggregation process was investigated.The results showed that the nanostructure can regulate Aβ₄₀ aggregation process at lower concentration(Aβ₄₀:HCR-Apt=8:1).Compared to the study in the previous work,the constructed nanostructure based on aptamer Apt₄₂ could effectively inhibit the aggregation of Aβ₄₀ aggregation process.Meanwhile,due to the introduction of nanostructure,it will be easier to cross the blood-brain barrier,which is expected to be applied in practice.