Monoclonal antibodies and aptamers: The future therapeutics for Alzheimer's disease.

Alzheimer's disease (AD) is considered the most common and prevalent form of dementia of adult-onset with characteristic progressive impairment in cognition and memory. The cure for AD has not been found yet and the treatments available until recently were only symptomatic. Regardless of multidisciplinary approaches and efforts made by pharmaceutical companies, it was only in the past two years that new drugs were approved for the treatment of the disease. Amyloid beta (Aß) immunotherapy is at the core of this therapy, which is one of the most innovative approaches looking to change the course of AD. This technology is based on synthetic peptides or monoclonal antibodies (mAb) to reduce Aß levels in the brain and slow down the advance of neurodegeneration. Hence, this article reviews the state of the art about AD neuropathogenesis, the traditional pharmacologic treatment, as well as the modern active and passive immunization describing approved drugs, and drug prototypes currently under investigation in different clinical trials. In addition, future perspectives on immunotherapeutic strategies for AD and the rise of the aptamer technology as a non-immunogenic alternative to curb the disease progression are discussed.

In Vitro Characterization of Protein:Nucleic Acid Liquid-Liquid Phase Separation by Microscopy Methods and Nanoparticle Tracking Analysis.

Uncontrolled assembly/disassembly of physiologically formed liquid condensates is linked to irreversible aggregation. Hence, the quest for understanding protein-misfolding disease mechanism might lie in the studies of protein:nucleic acid coacervation. Several proteins with intrinsically disordered regions as well as nucleic acids undergo phase separation in the cellular context, and this process is key to physiological signaling and is related to pathologies. Phase separation is reproducible in vitro by mixing the target recombinant protein with specific nucleic acids at various stoichiometric ratios and then examined by microscopy and nanotracking methods presented herein. We describe protocols to qualitatively assess hallmarks of protein-rich condensates, characterize their structure using intrinsic and extrinsic dyes, quantify them, and analyze their morphology over time. Analysis by nanoparticle tracking provides information on the concentration and diameter of high-order protein oligomers formed in the presence of nucleic acid. Using the model protein (globular domain of recombinant murine PrP) and DNA aptamers (high-affinity oligonucleotides with 25 nucleotides in length), we provide examples of a systematic screening of liquid-liquid phase separation in vitro.

Colorimetric detection of Pseudomonas aeruginosa by aptamer-functionalized gold nanoparticles.

Novel rapid methodologies for the detection of bacteria have been recently investigated and applied. In hospital environments, infections by pathogens are very common and can cause serious health problems. Pseudomonas aeruginosa is one of the most common bacteria, which can grow in hospital equipment such as catheters and respirators. Even at low concentrations, it can cause severe infections as it is resistant to antibiotics and other treatments. Based on this subject's relevance, this work aimed to develop a colorimetric biosensor using aptamer-functionalized gold nanoparticles for identifying P. aeruginosa. The detection mechanism is based on the color change of gold nanoparticles (AuNPs) from red to blue-purple through NaCl induction after bacteria incubation and aptamer-target binding. First, AuNPs were synthesized and characterized. The influence of aptamer and sodium chloride concentration on the agglomeration of AuNPs was investigated. Optimization of aptamer concentration and salt addition were performed. The best condition for detection was 5 µM aptamers and 200 mM of NaCl. In this case, P. aeruginosa was detected after 5 h for concentrations from 108 to 105 CFU mL-1, being 105 and 104 CFU mL-1 the detection limit for color change by the naked eye and UV-Vis spectrometry, respectively. In addition, other bacteria such as E. coli, S. typhimurium, and Enterobacteriaceae bacterium were also detected with color changing from red to gray. Finally, it was confirmed that the salt incubation time can be 2 h, and that the ideal aptamer concentration is 5 µM. Thus, the colorimetric analysis can be a simple and fast detection method for P. aeruginosa in the range of 108 to 105 CFU mL-1 to the naked eye. KEY POINTS: • A new method for rapid detection of Pseudomonas aeruginosa • Aptamers conjugated with gold nanoparticles allow pathogen detection by colorimetry • No need for previous surface modification of nanoparticles.

Selection and Characterization of Single-Stranded DNA Aptamers of Diagnostic Potential against the Whole Zika Virus.

Zika virus became a major public health problem in early 2015, when cases of Guillain-Barré syndrome and microcephaly were associated with viral infection. Currently, ZIKV is endemic in all tropical areas of the world, and the chance for future Zika epidemics remains very real and accurate diagnosis is crucial. The aim of this work was to select specific ssDNA aptamers that bind to the entire Zika virus and can be used to compose specific diagnostics, without cross-reactivity with other flaviviruses. Zika virus was cultivated in Vero cells and used as a target for aptamer selection. Aptamers specific for the ZIKV were selected using whole-virus SELEX, with counterselection for other flavivirus. Secondary and tertiary structures were evaluated and the molecular anchoring between the aptamers and target were simulated by the HDOCK server. Aptamer interaction was evaluated by ELISA/ELASA and the dissociation constant (Kd) was calculated by thermophoresis. Four ZIKV-specific aptamers were selected. The best two were further characterized and proved to be specific for ZIKV. Aptamers are capable of binding specifically to the ZIKV and differentiate from Dengue virus. The aptamers selected in this work can be used as capture agents in the composition of diagnostic tests to specifically detect ZIKV infection.

Selection of DNA Aptamers for Differentiation of Human Adipose-Derived Mesenchymal Stem Cells from Fibroblasts.

In recent years, stem cell therapy has shown promise in regenerative medicine. The lack of standardized protocols for cell isolation and differentiation generates conflicting results in this field. Mesenchymal stem cells derived from adipose tissue (ASC) and fibroblasts (FIB) share very similar cell membrane markers. In this context, the distinction of mesenchymal stem cells from fibroblasts has been crucial for safe clinical application of these cells. In the present study, we developed aptamers capable of specifically recognize ASC using the Cell-SELEX technique. We tested the affinity of ASC aptamers compared to dermal FIB. Quantitative PCR was advantageous for the in vitro validation of four candidate aptamers. The binding capabilities of Apta 2 and Apta 42 could not distinguish both cell types. At the same time, Apta 21 and Apta 99 showed a better binding capacity to ASC with dissociation constants (Kd) of 50.46 ± 2.28 nM and 72.71 ± 10.3 nM, respectively. However, Apta 21 showed a Kd of 86.78 ± 9.14 nM when incubated with FIB. Therefore, only Apta 99 showed specificity to detect ASC by total internal reflection microscopy (TIRF). This aptamer is a promising tool for the in vitro identification of ASC. These results will help understand the differences between these two cell types for more specific and precise cell therapies.

Selection of DNA aptamers for extra cellular domain of human epidermal growth factor receptor 2 to detect HER2 positive carcinomas.

BACKGROUND: Human epidermal growth factor receptor 2 (Her2, an orphan receptor of ErbB family) is considered as an important biomarker as it plays a key role in the development and progression of aggressive types of breast, ovarian, stomach and gastric cancer. In the present study, we developed novel DNA aptamers against the extra-cellular domain (ECD) of Her2 protein for detection of Her2-positive carcinomas. METHODS: We cloned and expressed Her2-ECD protein in E. coli system. After purification, the protein was used as a bait for screening of specific DNA aptamer candidate from a pool of 1014-15 random oligonucleotides through in vitro Systematic Evaluation of Ligands by Exponential Enrichment (SELEX) process. The aptamer-protein binding kinetics was elucidated by isothermal calorimetry. The specificity of FAM-labelled ECD_Apt1 towards Her2-positive cell lines was estimated by FACS and immunofluorescence assay. The specificity of the candidate was also verified with the tissue samples of breast cancer patients by immunohistochemistry process. RESULTS: Among four selected candidates, ECD_Apt1 (having minimum ∆G = -3.24) showed the highest binding affinity (K d = 6.33 ± 0.86 nM) to Her2-ECD protein. The aptamer-protein sandwich assay showed a linear rise in chemiluminescence (at 490 nm wavelength) in the dynamic range of 100-700 nM ECD_Apt1 with a detection limit of 12.5 ± 2.5 ng/mL. Biotinylated ECD_Apt1 showed stronger cytoplasmic staining in Her2-positive breast cancer cell lines (SKBR3) compared to Her2-negative cells (MDA MB 231, MCF7). In paraffin-embedded breast cancer tissue sections, it showed specific and selective localization in the cytoplasmic niche of malignant duct cancer cells without any cross-reactivity to fibroblasts, inflammatory cells and adipocytes. CONCLUSIONS: Binding assays, cytochemical and histochemical studies support ECD_Apt1 as a potential theranostic agent for Her2-positive carcinomas. ECD_Apt1 could be an effective low-cost alternative to conventional anti-Her2 antibody in solid phase immunoassays for cancer diagnosis and related applications.