In silico SELEX screening and statistical analysis of newly designed 5mer peptide-aptamers as Bcl-xl inhibitors using the Taguchi method.

Drug development for cancer treatment is a complex process that requires special efforts. Targeting crucial proteins is the most essential purpose of drug design in cancers. Bcl-xl is an anti-apoptotic protein that binds to pro-apoptotic proteins and interrupts their signals. Pro-apoptotic Bcl-xl effectors are short BH3 sequences that form an alpha helix and bind to anti-apoptotic proteins to inhibit their activity. Computational systematic evolution of ligands by exponential enrichment (SELEX) is an exclusive approach for developing peptide aptamers as potential effectors. Here, the amino acids with a high tendency for constructing an alpha-helical structure were selected. Due to the enormous number of pentapeptides, Taguchi method was used to study a selected number of peptides. The binding affinity of the peptides to Bcl-xl was assessed using molecular docking, and after analysis of the obtained results, a final set of optimized peptides was arranged and constructed. For a better comparison, three chemical compounds with approved anti-Bcl-xl activity were selected for comparison with the top-ranked 5mer peptides. The optimized peptides showed considerable binding affinity to Bcl-xl. The molecular dynamics (MD) simulation indicated that the designed peptide (PO5) could create considerable interactions with the BH3 domain of Bcl-xl. The MM/GBSA calculations revealed that these interactions were even stronger than those created by chemical compounds. In silico SELEX is a novel approach to design and evaluate peptide-aptamers. The experimental design improves the SELEX process considerably. Finally, PO5 could be considered a potential inhibitor of Bcl-xl and a potential candidate for cancer treatment.

Designing of peptide aptamer targeting the receptor-binding domain of spike protein of SARS-CoV-2: an in silico study.

Short synthetic peptide molecules which bind to a specific target protein with a high affinity to exert its function are known as peptide aptamers. The high specificity of aptamers with small-molecule targets (metal ions, dyes and theophylline; ATP) is within 1 pM and 1 µM range, whereas with the proteins (thrombin, CD4 and antibodies) it is in the nanomolar range (which is equivalent to monoclonal antibodies). The recently identified coronavirus (SARS-CoV-2) genome encodes for various proteins, such as envelope, membrane, nucleocapsid, and spike protein. Among these, the protein necessary for the virus to enter inside the host cell is spike protein. The work focuses on designing peptide aptamer targeting the spike receptor-binding domain of SARS-CoV-2. The peptide aptamer has been designed by using bacterial Thioredoxin A as the scaffold protein and an 18-residue-long peptide. The tertiary structure of the peptide aptamer is modeled and docked to spike receptor-binding domain of SARS CoV2. Molecular dynamic simulation has been done to check the stability of the aptamer and receptor-binding domain complex. It was observed that the aptamer binds to spike receptor-binding domain of SARS-CoV-2 in a similar pattern as that of ACE2. The aptamer-receptor-binding domain complex was found to be stable in a 100 ns molecular dynamic simulation. The aptamer is also predicted to be non-antigenic, non-allergenic, non-hemolytic, non-inflammatory, water-soluble with high affinity toward ACE2 than serum albumin. Thus, peptide aptamer can be a novel approach for the therapeutic treatment for SARS-CoV-2.

Self-assembled RNA nanocarrier-mediated chemotherapy combined with molecular targeting in the treatment of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal cancer is the fifth most common cancer affecting men in China. The primary treatment options are surgery and traditional radio-chemotherapy; no effective targeted therapy exists yet. Self-assembled RNA nanocarriers are highly stable, easily functionally modified, and have weak off-tumor targeting effects. Thus, they are among the most preferred carriers for mediating the targeted delivery of anti-tumor drugs. miR-375 was found to be significantly down-regulated in esophageal squamous cell carcinoma (ESCC) tissues and its overexpression effectively inhibits the proliferation, migration, and invasion of ESCC cells. Moreover, epidermal growth factor receptor (EGFR) was overexpressed in ESCC cells, and accumulation of RNA nanoparticles in ESCC tumors was enhanced by EGFR-specific aptamer (EGFRapt) modification. RESULTS: Herein, a novel four-way junction RNA nanocarrier, 4WJ-EGFRapt-miR-375-PTX simultaneously loaded with miR-375, PTX and decorated with EGFRapt, was developed. In vitro analysis demonstrated that 4WJ-EGFRapt-miR-375-PTX possesses strong thermal and pH stabilities. EGFRapt decoration facilitated tumor cell endocytosis and promoted deep penetration into 3D-ESCC spheroids. Xenograft mouse model for ESCC confirmed that 4WJ-EGFRapt-miR-375-PTX was selectively distributed in tumor sites via EGFRapt-mediating active targeting and targeted co-delivery of miR-375 and PTX exhibited more effective therapeutic efficacy with low systemic toxicity. CONCLUSION: This strategy may provide a practical approach for targeted therapy of ESCC.

Evolution of KIPPIS as a versatile platform for evaluating intracellularly functional peptide aptamers.

Chimeric proteins have been widely used to evaluate intracellular protein-protein interactions (PPIs) in living cells with various readouts. By combining an interleukin-3-dependent murine cells and chimeric proteins containing a receptor tyrosine kinase c-kit, we previously established a c-kit-based PPI screening (KIPPIS) system to evaluate and select protein binders. In the KIPPIS components, proteins of interest are connected with a chemically inducible helper module and the intracellular domain of the growth-signaling receptor c-kit, which detects PPIs based on cell proliferation as a readout. In this system, proteins of interest can be incorporated into chimeric proteins without any scaffold proteins, which would be advantageous for evaluating interaction between small peptides/domains. To prove this superiority, we apply KIPPIS to 6 peptide aptamer-polypeptide pairs, which are derived from endogenous, synthetic, and viral proteins. Consequently, all of the 6 peptide aptamer-polypeptide interactions are successfully detected by cell proliferation. The detection sensitivity can be modulated in a helper ligand-dependent manner. The assay results of KIPPIS correlate with the activation levels of Src, which is located downstream of c-kit-mediated signal transduction. Control experiments reveal that KIPPIS clearly discriminates interacting aptamers from non-interacting ones. Thus, KIPPIS proves to be a versatile platform for evaluating the binding properties of peptide aptamers.

Predicting disease course in ulcerative colitis using stool proteins identified through an aptamer-based screen.

In the search for improved stool biomarkers for inflammatory bowel disease (IBD), an aptamer-based screen of 1129 stool proteins was conducted using stool samples from an IBD cohort. Here we report that of the 20 proteins subsequently validated by ELISA, stool Ferritin, Fibrinogen, Haptoglobin, Hemoglobin, Lipocalin-2, MMP-12, MMP-9, Myeloperoxidase, PGRP-S, Properdin, Resistin, Serpin A4, and TIMP-1 are significantly elevated in both ulcerative colitis (UC) and Crohn's disease (CD) compared to controls. When tested in a longitudinal cohort of 50 UC patients at 4 time-points, fecal Fibrinogen, MMP-8, PGRP-S, and TIMP-2 show the strongest positive correlation with concurrent PUCAI and PGA scores and are superior to fecal calprotectin. Unlike fecal calprotectin, baseline stool Fibrinogen, MMP-12, PGRP-S, TIMP-1, and TIMP-2 can predict clinical remission at Week-4. Here we show that stool proteins identified using the comprehensive aptamer-based screen are superior to fecal calprotectin alone in disease monitoring and prediction in IBD.

Binding Interactions of Peptide Aptamers.

Peptide aptamers are short amino acid chains that are capable of binding specifically to ligands in the same way as their much larger counterparts, antibodies. Ligands of therapeutic interest that can be targeted are other peptide chains or loops located on the surface of protein receptors (e.g., GCPR), which take part in cell-to-cell communications either directly or via the intermediary of hormones or signalling molecules. To confer on aptamers the same sort of conformational rigidity that characterises an antibody binding site, aptamers are often constructed in the form of cyclic peptides, on the assumption that this will encourage stronger binding interactions than would occur if the aptamers were simply linear chains. However, no formal studies have been conducted to confirm the hypothesis that linear peptides will engage in stronger binding interactions with cyclic peptides than with other linear peptides. In this study, the interaction of a model cyclic decamer with a series of linear peptide constructs was compared with that of a linear peptide with the same sequence, showing that the cyclic configuration does confer benefits by increasing the strength of binding.

Simultaneous Detection of VEGF and CEA by Time-Resolved Chemiluminescence Enzyme-Linked Aptamer Assay.

BACKGROUND: As two important tumor markers, vascular endothelial growth factor (VEGF) and carcinoembryonic antigen (CEA) have a great value for clinical application in the early diagnosis of cancer. Due to the complex composition of biological samples, the results from combined detection of CEA and VEGF are often taken as a comprehensive indicator in order to make an accurate judgment on a disease. However, most of the current methods can only be used to detect the content of one biomarker. Therefore, it is necessary to explore a simple, rapid, low-cost, and highly sensitive method for the simultaneous detection of CEA and VEGF. METHODS: Based on specific aptamers and magnetic separation, a time-resolved chemiluminescence enzyme-linked aptamer assay was developed for the simultaneous detections of CEA and VEGF in serum samples. RESULTS: Under the optimal conditions, the linear range of the calibration curve for VEGF was from 0.5 to 80 ng mL-1, and the limit of detection was 0.1 ng mL-1. The linear range of the calibration curve for CEA was 0.5 to 160 ng mL-1, and the limit of detection was 0.1 ng mL-1. The established method was applied to detect VEGF and CEA in serum samples. The results were consistent with those of commercial kits. CONCLUSION: The method has high sensitivity and can quickly obtain accurate results, which could greatly improve the measurement efficiency, reduce the cost, and also reduce the volume of sample consumed. It can be seen that the method established in this study has important application value and broad application prospect in clinical diagnosis.

Targeted doxorubicin-loaded mesenchymal stem cells-derived exosomes as a versatile platform for fighting against colorectal cancer.

Exosomes hold great potential for cancer treatment to deliver therapeutics due to its inherent low immunogenicity. Exosomes are biocompatible cell-exocytosed secreted vesicles by most cell types, which can be used to construct novel biomanufacturing platform for drug delivery and cancer therapy. In this study, we implemented nano-sized vesicles which were secreted by mesenchymal stem cell (MSC), to encapsulate doxorubicin (DOX) through electroporation method (DOX@exosome). DOX was loaded into exosomes, with an encapsulation efficiency of up to 35% and separated by ultracentrifugation. Subsequently, carboxylic acid-end MUC1 aptamer was used to covalently decorate the surface amine groups of the exosomes via amide bond formation to provide selective guided drug delivery (DOX@exosome-apt). The data showed that the DOX@exosome-apt provided highly efficient DOX transportation to MUC1-positive cancer cells in vitro as confirmed by MTT and flow cytometry experiments. Moreover, in vivo study on ectopic model of C26 (mouse colon adenocarcinoma) in BALB/c mice indicated that the single dose intravenous injection of DOX@exosome-apt significantly suppress tumor growth in comparison with free DOX. Ex vivo fluorescent imaging also verified the desirable biodistribution of DOX@exosome-apt by exhibiting higher tumor accumulation and faster liver clearance in comparison with DOX@exosome and free DOX. It could be concluded that MUC1 aptamer-decorated exosomes can be implemented therapeutically for the safe and versatile delivery of DOX to colon adenocarcinoma, thus offering valuable platform for clinical applications.

Harnessing Selectivity and Sensitivity in Electronic Biosensing: A Novel Lab-on-Chip Multigate Organic Transistor.

Electrolyte gated organic transistors can operate as powerful ultrasensitive biosensors, and efforts are currently devoted to devising strategies for reducing the contribution of hardly avoidable, nonspecific interactions to their response, to ultimately harness selectivity in the detection process. We report a novel lab-on-a-chip device integrating a multigate electrolyte gated organic field-effect transistor (EGOFET) with a 6.5 µL microfluidics set up capable to provide an assessment of both the response reproducibility, by enabling measurement in triplicate, and of the device selectivity through the presence of an internal reference electrode. As proof-of-concept, we demonstrate the efficient operation of our pentacene based EGOFET sensing platform through the quantification of tumor necrosis factor alpha with a detection limit as low as 3 pM. Sensing of inflammatory cytokines, which also include TNFα, is of the outmost importance for monitoring a large number of diseases. The multiplexable organic electronic lab-on-chip provides a statistically solid, reliable, and selective response on microliters sample volumes on the minutes time scale, thus matching the relevant key-performance indicators required in point-of-care diagnostics.

Aptamer supported in vitro endothelialization of poly(ether imide) films.

Implantation of synthetic small-diameter vascular bypass grafts is often associated with an increased risk of failure, due to thrombotic events or late intimal hyperplasia. As one of the causes an insufficient hemocompatibility of the artificial surface is discussed. Endothelialization of synthetic grafts is reported to be a promising strategy for creating a self-renewing and regulative anti-thrombotic graft surface. However, the establishment of a shear resistant cell monolayer is still challenging. In our study, cyto- and immuno-compatible poly(ether imide) (PEI) films were explored as potential biomaterial for cardiovascular applications. Recently, we reported that the initial adherence of primary human umbilical vein endothelial cells (HUVEC) was delayed on PEI-films and about 9 days were needed to establish a confluent and almost shear resistant HUVEC monolayer. To accelerate the initial adherence of HUVEC, the PEI-film surface was functionalized with an aptamer-cRGD peptide based endothelialization supporting system. With this functionalization the initial adherence as well as the shear resistance of HUVEC on PEI-films was considerable improved compared to the unmodified polymer surface. The in vitro results confirm the general applicability of aptamers for an efficient functionalization of substrate surfaces.

Targeting SOX2 Protein with Peptide Aptamers for Therapeutic Gains against Esophageal Squamous Cell Carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a predominant cancer type in developing countries such as China, where ESCC accounts for approximately 90% of esophageal malignancies. Lacking effective and targeted therapy contributes to the poor 5-year survival rate. Recent studies showed that about 30% of ESCC cases have high levels of SOX2. Herein, we aim to target this transcription factor with aptamer. We established a peptide aptamer library and then performed an unbiased screening to identify several peptide aptamers including P42 that can bind and inhibit SOX2 downstream target genes. We further found that P42 overexpression or incubation with a synthetic peptide 42 inhibited the proliferation, migration, and invasion of ESCC cells. Moreover, peptide 42 treatment inhibited the growth and metastasis of ESCC xenografts in mouse and zebrafish. Further analysis revealed that P42 overexpression led to alternations in the levels of proteins that are important for the proliferation and migration of ESCC cells. Taken together, our study identified the peptide 42 as a key inhibitor of SOX2 function, reducing the proliferation and migration of ESCC cells in vitro and in vivo, and thereby offering a potential therapy against ESCC.

Dual function of interleukin-23 Aptamer to suppress brain inflammation via attachment to macrophage stimulating 1 kinase and interleukin-23.

In the present study, the dual function of interleukin-23 (IL-23) Aptamer to suppress brain inflammation via attachment to macrophage stimulating 1 (MST1) kinase and IL-23, was introduced. Also, the anti-inflammatory property of IL-23 Aptamer has been investigated. This study showed that IL-23 Aptamer could reduce the clinical development of brain inflammation induced by Parathion, as an important organophosphate toxin. Both immunostaining and H&E staining indicated that the total inflammatory infiltration foci were remarkably decreased in IL-23 Aptamer-treated mice. Moreover, this study showed that IL-23 Aptamer reduced both absolute and relative numbers of MST1+CD4 + Th1 cells and IL-23-producing cells. Analysis of the Hippo signaling genes showed a sharp decrease of MST1 kinase compared with other genes (P < 0.001). Moreover, computer-assisted molecular docking demonstrated that both MST1 kinase and IL-23 could tightly attach to IL-23 Aptamer, and maybe block it. Taken together, IL-23 Aptamer coud decrease brain inflammation via suppressing MST1 kinase and IL-23.

A CMOS VEGF Sensor for Cancer Diagnosis Using a Peptide Aptamer-Based Functionalized Microneedle.

This paper presents the first CMOS Vascular Endothelial Growth Factor (VEGF) sensor for cancer diagnosis directly from human blood. The sensor incorporates a peptide aptamer-based microneedle that allows the detection of electrochemical reactions with VEGF. This results in a capacitance change between the microneedles and then reads out by a two-step capacitance-to-digital converter (CDC). The proposed two-step CDC consists of a coarse 5b slope ADC and a fine 14b continuous-time delta-sigma modulator (CTDSM). During slow peptide-binding, the slope ADC performs a coarse conversion and the results are used to adjust the current level of the stimulator. After settling of the peptide-binding, based on an adjusted stimulation current, the CTDSM measures the small capacitance changes of the sensor. The prototype chip is fabricated in a 65-nm CMOS process, occupying a 0.87 mm 2 active area. The power consumption is 270 muW. Thanks to the two-step approach, this work achieves a wide dynamic range of 18.3b, covering a large sensor-to-sensor variation. It also achieves a peak resolution of 13.7b, while maintaining errors in 1 to 100 nF baseline capacitance. The overall sensor system successfully detects the VEGF in both phosphate-buffered saline (PBS) and human blood serum. Without the use of precision instruments, this work achieves a resolution of 15 fM [Formula: see text] in range of 0.1 to 1000 pM and denotes the clear VEGF selectivity at 40× in PBS and 5× in the blood serum compared to other proteins (IgG, Con A, and cholera toxin).

A new small cell lung cancer biomarker identified by Cell-SELEX generated aptamers.

Small cell lung cancer (SCLC) has been a recalcitrant cancer without significant breakthroughs in clinical treatment during the past three decades. As there is a lack of effective protein inhibitor for SCLC targeted therapy, the discovery of new druggable SCLC biomarkers is a pressing work. Here we identified a new protein biomarker of SCLC, which is high density lipoprotein binding protein (HDLBP), through the aptamer generated by cell-SELEX against SCLC cells. Immunohistochemistry results showed an elevated HDLBP level in SCLC tissues from clinical samples. Attenuating HDLBP expression with siRNA inhibited proliferation and metastasis of SCLC cells in vitro and tumor formation in vivo. Mechanism study revealed the new function of HDLBP in promoting G1/S cell cycle transition for tumor progression. While the inhibitor of HDLBP has been reported, our work suggested a promising potential of targeting HDLBP to improve the treatment of fatal SCLC and a powerful tool of using cell-SELEX in cancer medicine.

AAV-encoded CaV2.2 peptide aptamer CBD3A6K for primary sensory neuron-targeted treatment of established neuropathic pain.

Transmission of pain signals from primary sensory neurons to secondary neurons of the central nervous system is critically dependent on presynaptic voltage-gated calcium channels. Calcium channel-binding domain 3 (CBD3), derived from the collapsin response mediator protein 2 (CRMP2), is a peptide aptamer that is effective in blocking N-type voltage-gated calcium channel (CaV2.2) activity. We previously reported that recombinant adeno-associated virus (AAV)-mediated restricted expression of CBD3 affixed to enhanced green fluorescent protein (EGFP) in primary sensory neurons prevents the development of cutaneous mechanical hypersensitivity in a rat neuropathic pain model. In this study, we tested whether this strategy is effective in treating established pain. We constructed AAV6-EGFP-CBD3A6K (AAV6-CBD3A6K) expressing a fluorescent CBD3A6K (replacing A to K at position 6 of CBD3 peptide), which is an optimized variant of the parental CBD3 peptide that is a more potent blocker of CaV2.2. Delivery of AAV6-CBD3A6K into lumbar (L) 4 and 5 dorsal root ganglia (DRG) of rats 2 weeks following tibial nerve injury (TNI) induced transgene expression in neurons of these DRG and their axonal projections, accompanied by attenuation of pain behavior. We additionally observed that the increased CaV2.2α1b immunoreactivity in the ipsilateral spinal cord dorsal horn and DRG following TNI was significantly normalized by AAV6-CBD3A6K treatment. Finally, the increased neuronal activity in the ipsilateral dorsal horn that developed after TNI was reduced by AAV6-CBD3A6K treatment. Collectively, these results indicate that DRG-restricted AAV6 delivery of CBD3A6K is an effective analgesic molecular strategy for the treatment of established neuropathic pain.

The protean world of non-coding RNAs in glioblastoma.

Non-coding ribonucleic acids (ncRNAs) are a diverse group of RNA molecules that are mostly not translated into proteins following transcription. We review the role of ncRNAs in the pathobiology of glioblastoma (GBM), and their potential applications for GBM therapy. Significant advances in our understanding of the protean manifestations of ncRNAs have been made, allowing us to better decipher the molecular complexity of GBM. A large number of regulatory ncRNAs appear to have a greater influence on the molecular pathology of GBM than thought previously. Importantly, also, a range of therapeutic approaches are emerging whereby ncRNA-based systems may be used to molecularly target GBM. The most successful of these is RNA interference, and some of these strategies are being evaluated in ongoing clinical trials. However, a number of limitations exist in the clinical translation of ncRNA-based therapeutic systems, such as delivery mechanisms and cytotoxicity; concerted research endeavors are currently underway in an attempt to overcome these. Ongoing and future studies will determine the potential practical role for ncRNA-based therapeutic systems in the clinical management of GBM. These applications may be especially promising, given that current treatment options are limited and prognosis remains poor for this challenging malignancy.

Protein-Catalyzed Capture Agents.

Protein-catalyzed capture agents (PCCs) are synthetic and modular peptide-based affinity agents that are developed through the use of single-generation in situ click chemistry screens against large peptide libraries. In such screens, the target protein, or a synthetic epitope fragment of that protein, provides a template for selectively promoting the noncopper catalyzed azide-alkyne dipolar cycloaddition click reaction between either a library peptide and a known ligand or a library peptide and the synthetic epitope. The development of epitope-targeted PCCs was motivated by the desire to fully generalize pioneering work from the Sharpless and Finn groups in which in situ click screens were used to develop potent, divalent enzymatic inhibitors. In fact, a large degree of generality has now been achieved. Various PCCs have demonstrated utility for selective protein detection, as allosteric or direct inhibitors, as modulators of protein folding, and as tools for in vivo tumor imaging. We provide a historical context for PCCs and place them within the broader scope of biological and synthetic aptamers. The development of PCCs is presented as (i) Generation I PCCs, which are branched ligands engineered through an iterative, nonepitope-targeted process, and (ii) Generation II PCCs, which are typically developed from macrocyclic peptide libraries and are precisely epitope-targeted. We provide statistical comparisons of Generation II PCCs relative to monoclonal antibodies in which the protein target is the same. Finally, we discuss current challenges and future opportunities of PCCs.

Advances in the Study of Aptamer-Protein Target Identification Using the Chromatographic Approach.

Ever since the development of the process known as the systematic evolution of ligands by exponential enrichment (SELEX), aptamers have been widely used in a variety of studies, including the exploration of new diagnostic tools and the discovery of new treatment methods. Aptamers' ability to bind to proteins with high affinity and specificity, often compared to that of antibodies, enables the search for potential cancer biomarkers and helps us understand the mechanisms of carcinogenesis. The blind spot of those investigations is usually the difficulty in the selective extraction of targets attached to the aptamer. There are many studies describing the cell SELEX for the prime choice of aptamers toward living cancer cells or even whole tumors in the animal models. However, a dilemma arises when a large number of proteins are being identified as potential targets, which is often the case. In this article, we present a new analytical approach designed to selectively target proteins bound to aptamers. During studies, we have focused on the unambiguous identification of the molecular targets of aptamers characterized by high specificity to the prostate cancer cells. We have compared four assay approaches using electrophoretic and chromatographic methods for "fishing out" aptamer protein targets followed by mass spectrometry identification. We have established a new methodology, based on the fluorescent-tagged oligonucleotides commonly used for flow-cytometry experiments or as optic aptasensors, that allowed the detection of specific aptamer-protein interactions by mass spectrometry. The use of atto488-labeled aptamers for the tracking of the formation of specific aptamer-target complexes provides the possibility of studying putative protein counterparts without needing to apply enrichment techniques. Significantly, changes in the hydrophobic properties of atto488-labeled aptamer-protein complexes facilitate their separation by reverse-phase chromatography combined with fluorescence detection followed by mass-spectrometry-based protein identification. These comparative results of several methodological approaches confirmed the universal applicability of this method to studying aptamer-protein interactions with high sensitivity, showing superior properties compared with pull-down techniques.

Dual-target recognition sandwich assay based on core-shell magnetic mesoporous silica nanoparticles for sensitive detection of breast cancer cells.

A novel dual-target recognition sandwich strategy for selective capture and detection of MCF-7 breast cancer cells based on core-shell magnetic mesoporous silica (Fe3O4@nSiO2@mSiO2@apt) nanoparticles was developed. Fe3O4@nSiO2@mSiO2@apt nanoparticles, which were prepared by a layer-by-layer method and were used for the first time to capture cancer cells, have large surface areas, particularly accessible mesochannels, and good biocompatibility, enabling aptamers to be compactly anchored onto the surface of the core-shell magnetic nanoparticles. A mucin 1 protein (MUC1)-targeted Fe3O4@nSiO2@mSiO2@apt nanoparticle was used as an affinity magnetic isolate material to capture target MCF-7 cells selectively and to reduce interference through affinity interaction between the anti-MUC1 aptamer and the MUC1 protein over-expressed on the surface of the MCF-7 cells. Meanwhile, a folate receptor (FR)-targeted affinity fluorescent probe (FA-BSA-FITC) was developed by coupling folic acid and FITC to the surface of BSA, enabling high sensitivity, selective fluorescent labeling of FR over-expressed MCF-7 cells. A dual-target recognition sandwich assay was developed based on the MUC1-targeted magnetic nanoparticles and the FR-targeted fluorescent probes. Under optimum conditions, a quantitative assay of MCF-7 cells was achieved with a dynamic range of 102-105 cells/mL (R2 = 0.9991). This assay showed high specificity and sensitivity to the target MCF-7 cells. Finally, the proposed strategy could be extended to detect MCF-7 cells in human plasma and whole blood with a recovery range of 86.1-104.0% and a RSD range of 1.2-8.4%, respectively. This indicates that the dual-target recognition method developed in this research exhibits good selectivity, anti-interference capability, and reliability even in plasma and whole blood samples and is more suitable for complex samples than previous targeted assays. Therefore, the approach proposed here may have great potential for early breast cancer diagnosis.

Interaction of Peptide Aptamers with Prion Protein Central Domain Promotes α-Cleavage of PrPC.

Prion diseases are infectious and fatal neurodegenerative diseases affecting humans and animals. Transmission is possible within and between species with zoonotic potential. Currently, no prophylaxis or treatment exists. Prions are composed of the misfolded isoform PrPSc of the cellular prion protein PrPC. Expression of PrPC is a prerequisite for prion infection, and conformational conversion of PrPC is induced upon its direct interaction with PrPSc. Inhibition of this interaction can abrogate prion propagation, and we have previously established peptide aptamers (PAs) binding to PrPC as new anti-prion compounds. Here, we mapped the interaction site of PA8 in PrP and modeled the complex in silico to design targeted mutations in PA8 which presumably enhance binding properties. Using these PA8 variants, we could improve PA-mediated inhibition of PrPSc replication and de novo infection of neuronal cells. Furthermore, we demonstrate that binding of PA8 and its variants increases PrPC α-cleavage and interferes with its internalization. This gives rise to high levels of the membrane-anchored PrP-C1 fragment, a transdominant negative inhibitor of prion replication. PA8 and its variants interact with PrPC at its central and most highly conserved domain, a region which is crucial for prion conversion and facilitates toxic signaling of Aß oligomers characteristic for Alzheimer's disease. Our strategy allows for the first time to induce α-cleavage, which occurs within this central domain, independent of targeting the responsible protease. Therefore, interaction of PAs with PrPC and enhancement of α-cleavage represent mechanisms that can be beneficial for the treatment of prion and other neurodegenerative diseases.