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Non-structural 1 (NS1) is a protein biomarker that can be found in blood in the early stages of dengue and related infections (Zika and Chikungunya). This study aims to develop a biosensor to selectively quantify NS1 using DNA aptamer co-immobilized on gold electrodes with 6-(ferrocenyl)hexanethiol (FCH) using electrochemical capacitive spectroscopy. This technique uses a redox probe (FCH) immobilized on the self-assembled monolayer to convert impedance into capacitance information. The developed platform was blocked with bovine serum albumin before NS1 exposure and the ratio between aptamers and FCH was optimized. The aptasensor was tested using commercial NS1 serotype 4 in phosphate-buffered saline and commercial undiluted human serum. Using the optimum applied potential provides high sensitivity (3 and 4 nF per decade) and low limit of detection (30.9 and 41.8 fg/mL) with a large linear range (10 pg to 1 µg/mL and 10 pg to 100 ng/mL, respectively). Both results exhibit a residual standard deviation value < 1%. The results suggested that this aptasensor was capable of detecting NS1 in the clinical range and can be applied to any other specific aptamer with FCH, opening the path for label-free miniaturized point-of-care devices with high sensitivity and specificity.

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BACKGROUND: Scaffold (SCA) functionalization with aptamers (APT) provides adsorption of specific bioactive molecules on biomaterial surfaces. The aim of this study was to observe if SCA enriched with anti-fibronectin APT can favor coagulum (PhC) and osteoblasts (OSB) differentiation. METHODS: 20 µg of APT was functionalized on SCA by simple adsorption. For PhC formation, SCAs were inserted into rat calvaria defects for 17 h. Following proper transportation (buffer solution PB), OSBs (UMR-106 lineage) were seeded over PhC + SCAs with and without APT. Cells and PhC morphology, PhC cell population, protein labeling and gene expression were observed in different time points. RESULTS: The APT induced higher alkaline phosphatase and bone sialoprotein immunolabeling in OSB. Mesenchymal stem cells, leukocytes and lymphocytes cells were detected more in the APT group than when scaffolds were not functionalized. Additionally, an enriched and dense fibrin network and different cell types were observed, with more OSB and white blood cells in PhC formed on SCA with APT. The gene expression showed higher transforming growth factor beta 1 (TGF-b1) detection in SCA with APT. CONCLUSIONS: The SCA functionalization with fibronectin aptamers may alter key morphological and functional features of blood clot formation, and provides a selective expression of proteins related to osteo differentiation. Additionally, aptamers increase TGF-b1 gene expression, which is highly associated with improvements in regenerative therapies.

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The methylotrophic yeast Komagataella phaffii is one of the most important microbial platforms to produce recombinant proteins. Despite its importance in the context of industrial biotechnology, the use of synthetic biology approaches in K. phaffii is hampered by the fact that few genetic tools are available for precise control of gene expression in this system. In this work, we used an RNA aptamer activated by tetracycline to modulate protein production at the translational level. Using lacZ as gene reporter, we have demonstrated significant reduction of the heterologous protein upon addition of tetracycline. Furthermore, this genetic control device was applied for the control of Ku70p. This protein is involved in non-homologous recombination and the control of its production paves the way for the development of strains exhibiting higher rates of homologous recombination.

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Despite improvements in B cell acute lymphoblastic leukemia (B-ALL) treatment, a significant number of patients experience relapse of the disease, resulting in poor prognosis and high mortality. One of the drawbacks of current B-ALL treatments is the high toxicity associated with the non-specificity of chemotherapeutic drugs. Targeted therapy is an appealing strategy to treat B-ALL to mitigate these toxic off-target effects. One such target is the B cell surface protein CD22. The restricted expression of CD22 on the B-cell lineage and its ligand-induced internalizing properties make it an attractive target in cases of B cell malignancies. To target B-ALL and the CD22 protein, we performed cell internalization SELEX (Systematic Evolution of Ligands by EXponential enrichment) followed by molecular docking to identify internalizing aptamers specific for B-ALL cells that bind the CD22 cell-surface receptor. We identified two RNA aptamers, B-ALL1 and B-ALL2, that target human malignant B cells, with B-ALL1 the first documented RNA aptamer interacting with the CD22 antigen. These B-ALL-specific aptamers represent an important first step toward developing novel targeted therapies for B cell malignancy treatments.

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Rapidly reversible anticoagulant agents have great clinical potential. Oligonucleotide-based anticoagulant agents are uniquely positioned to fill this clinical niche, as they are able to be deactivated through the introduction of the reverse complement oligo. Once the therapeutic and the antidote oligos meet in solution, they are able to undergo isothermal reassociation to form short, inactive, duplexes that are rapidly secreted via filtration by the kidneys. The formation of the duplexes interrupts the structure of the anticoagulant oligo, allowing normal coagulation to be restored. To effectively assess these new anticoagulants, a variety of methods may be employed. The measurement of thrombin generation (TG) reflects the overall capacity of plasma to produce active thrombin and provides a strong contribution to identifying new anticoagulant drugs, including DNA/RNA thrombin binding aptamer carrying fibers which are used through this chapter as an example. Here we describe the TG assessed by Calibrated Automated Thrombogram (CAT) assay in a fully automated system. This method is based on the detection of TG in plasma samples by measuring fluorescent signals released from a quenched fluorogenic thrombin substrate and the subsequent conversion of these signals in TG curves.

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Staphylococcal enterotoxin B (SEB) is a protein produced by Staphylococcus aureus, which is toxic to humans. It is well known for its ability to stimulate the exacerbated activation of proinflammatory CD4+ T cells (Th1 profile), and in vitro studies have been conducted to understand its mechanism of action and its potential use as an immune therapy. However, the efficiency of the SEB1741 aptamer in blocking SEB has not been experimentally demonstrated. METHODS: Enrichment CD4+ T cells were stimulated with SEB, and as a blocker, we used the SEB1741 aptamer, which was previously synthesised by an "in silico" analysis, showing high affinity and specificity to SEB. The efficiency of the SEB1741 aptamer in blocking CD4+ T cell activation was compared with that of an anti-SEB monoclonal antibody. Flow cytometry and Bio-Plex were used to evaluate the T-cell function. RESULTS: In vitro, SEB induced the activation of CD4+ T cells and favoured a Th1 profile; however, the SEB1741 aptamer was highly efficient in decreasing the frequency of CD4+ T cells positive to ki-67 and CD69 cells, this means that proliferation and activation of CD4+ T cells was decreased. Moreover, the production of interleukin 2 (IL-2) and interferon-gamma (IFN-γ) was affected, suggesting that the Th1 profile is not present when the SEB1441 aptamer is used. Thus, the SEB1741 function was similar to that of anti-SEB. CONCLUSIONS: The SEB1741 aptamer is a valuable tool for blocking CD4+ T cell activation and the subsequent release of proinflammatory cytokines by SEB stimulation.

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Aptamers are emerging as a promising new class of functional nucleic acids because they can specifically bind to any target with high affinity and be easily modified chemically with different pharmacophoric subunits for therapy. The truncated aptamer, Sgc8-c, binds to tyrosine-protein kinase-like 7 receptor, a promising cancer therapeutic target, allowing the recognition of haemato-oncological malignancies, among others. We have previously developed aptamer-drug conjugates by chemical synthesis, hybridizing Sgc8-c and dasatinib, a drug proposed for lymphoma chemotherapy. One of the best-characterised Sgc8-c-dasatinib hybrids, namely Sgc8-c-carb-da, was capable of releasing dasatinib at an endosomal-pH. Herein, we probed the therapeutic potential of this aptamer-drug conjugate. Sgc8-c-carb-da specifically inhibited murine A20 B lymphocyte growth and produced cell death, mainly by late apoptosis and necrosis. In addition, Sgc8-c-carb-da generated an arrest in cell proliferation, with a cell cycle arrest in the Sub-G1-peak. The mitochondrial potential was altered accordingly to these pathways. Moreover, using an in vitro cell-targeting assay that mimics in vivo conditions, we showed that Sgc8-c-carb-da displayed higher (2.5-fold) cytotoxic effects than dasatinib. These findings provide proof-of-concept of the therapeutic value of Sgc8-c-carb-da for lymphoma, creating new opportunities for the chemical synthesis of targeted biotherapeutics.

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The escape from immune surveillance is a hallmark of cancer progression. The classic immune checkpoint molecules PD-1, PD-L1, CTLA-4, LAG-3, TIM-3 novel ones are part of a sophisticated system of up- and downmodulation of the immune system, which is unregulated in cancer. In recent years, there have been remarkable advances in the development of targeting strategies, focused principally on immunotherapies aiming at blocking those molecules involved in the evasion of the immune system. However, there are still challenges to predicting their efficacy due to the wide heterogeneity of clinical responses. Thus, there is a need to develop new strategies, and theranostics has much to contribute in this field. Besides that, aptamers have emerged as promising molecules with the potential to generate a huge impact in the immunotheranostic field. They are single-stranded oligonucleotides with a unique self-folding tridimensional structure, with high affinity and specificity for the target. In particular, their small size and physicochemical characteristics make them a versatile tool for designing theranostic strategies. Here, we review the progress in theranostic strategies based on aptamers against immune checkpoints, and highlight the potential of those approaches.

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Foot-and-mouth disease (FMD) is a devastating livestock disease caused by foot-and-mouth disease virus (FMDV), a member of the Picornaviridae family. The 3ABC is a non-structural protein of FMDV, produced during viral replication and absent from inactivated FMD vaccines. Nucleic acid aptamers are DNA or RNA oligonucleotides capable of binding with high specificity and affinity to a molecular target. The aim of this study was to obtain DNA aptamers specific for 3ABC protein with a view of their application in the FMD diagnosis. Aptamers are usually obtained through SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. In this study, an aptamer (termed FMDV1) was selected by a variation of this technique called Capillary Electrophoresis SELEX (CE-SELEX). The FMDV1 aptamer showed high binding affinity to the 3ABC protein with Kd value in the nano molar range: 22.69 ± 1.79 nM. The FMDV1 aptamer binding to 3ABC was significantly higher when compared with the BSA protein, used as control, demonstrating its specificity.

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Abnormal phase transitions have been implicated in the occurrence of proteinopathies. Disordered proteins with nucleic acidbinding ability drive the formation of reversible micron-sized condensates capable of controlling nucleic acid processing/transport. This mechanism, achieved via liquid-liquid phase separation (LLPS), underlies the formation of long-studied membraneless organelles (e.g., nucleolus) and various transient condensates formed by driver proteins. The prion protein (PrP) is not a classical nucleic acid-binding protein. However, it binds nucleic acids with high affinity, undergoes nucleocytoplasmic shuttling, contains a long intrinsically disordered region rich in glycines and evenly spaced aromatic residues, among other biochemical/biophysical properties of bona fide drivers of phase transitions. Because of this, our group and others have characterized LLPS of recombinant PrP. In vitro phase separation of PrP is modulated by nucleic acid aptamers, and depending on the aptamer conformation, the liquid droplets evolve to solid-like species. Herein, we discuss recent studies and previous evidence supporting PrP phase transitions. We focus on the central role of LLPS related to PrP physiology and pathology, with a special emphasis on the interaction of PrP with different ligands, such as proteins and nucleic acids, which can play a role in prion disease pathogenesis. Finally, we comment on therapeutic strategies directed at the non-functional phase separation that could potentially tackle prion diseases or other protein misfolding disorders.

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Introduction: Chagas disease (CD) is caused by the protozoan parasite Trypanosoma cruzi. Although endemic mainly in Latin America, CD has become a global public health problem due to migration of infected individuals to non-endemic regions. Despite progress made in drug development, preclinical assays for drug discovery are required to accelerate the development of new drugs with reduced side effects, which are much needed for human treatment. Methods: We used a cure model of infected mice treated with Fexinidazole (FZ) to further validate a novel Enzyme Linked Aptamer (ELA) assay that detects parasite biomarkers circulating in the blood of infected animals. Results: The ELA assay showed cure by FZ in ~71% and ~77% of mice infected with the VL-10 and Colombiana strains of T. cruzi, respectively. The ELA assay also revealed superior treatment efficacy of FZ compared to Benznidazole prior to immunosuppression treatment. Discussion: Our study supports the use of ELA assay as an alternative to traditional serology or blood PCR to assess the efficacy of antichagasic drugs during their preclinical phase of development. Further, the combination of high sensitivity and ease of use make this parasite antigen detection assay an attractive new tool to facilitate the development of much needed new therapies for CD.

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Aflatoxin contamination of cattle feed is responsible for serious adverse effects on animal and human health. A number of approaches have been reported to determine aflatoxin B1 (AFB1) in a variety of feed samples using aptasensors. However, rapid analysis of AFB1 in these matrices remains to be addressed in light of the complexity of the preanalytical process. Herein we describe an optimization on the preanalytical stage to minimize the sample processing steps required to perform semi-quantitative colorimetric detection of AFB1 in cattle feed using a gold nanoparticle-based aptasensor (nano-aptasensor). The optical behavior of the nano-aptasensor was characterized in different organics solvents, with acetonitrile showing the least interference on the activity of the nan-aptasensor. This solvent was selected as the extractant agent for AFB1-containing feed, allowing for the first time, direct colorimetric detection from the crude extract (detection limit of 5 µg/kg). Overall, these results lend support to the application of this technology for the on-site detection of AFB1 in the dairy sector.

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The COVID-19 pandemic has been a main concern over the last two years and has become one of the most important crises in the history of human health. Today, there is still a need for affordable and reliable diagnostic tests for massive disease monitoring. Previously, a set of highly specific DNA-aptamers (C7/C9) binding to the SARS-CoV-2 Spike (S) protein were isolated but its performance in clinical samples remained to be tested. Here, 242 samples were collected through three different methods and subjected to florescence-linked aptamer assays (FLAA) based on C7/C9 aptamers through two readout protocols. Then, a step-by-step statistical approach which included agreement tests, proportion comparisons and binomial and multinomial logistic regressions was used to predict optimal conditions for the novel C7/C9 FLAA test. RTqPCR threshold cycles, symptoms onset and processing time were influential factors on FLAA test results. Naturally occurring mutations on S were also detected and analyzed. Aminoacidic substitutions D614G and T732A appeared relevant for aptamer recognition although further studies are necessary. The methodology presented here is the first step to determine the performance and diagnosis across a range of clinical contexts and it might serve as a base for a complete analysis applicable to other designs of new diagnostic tests.

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Zika virus (ZIKV) is a mosquito-borne virus that is phylogenetically close to other medically important flaviviruses with high global public health significance, such as dengue (DENV) and yellow fever (YFV) viruses. Correct diagnosis of a flavivirus infection can be challenging, particularly in world regions where more than one flavivirus co-circulates and YFV vaccination is mandatory. Acid nucleic aptamers are oligonucleotides that bind to a specific target molecule with high affinity and specificity. Because of their unique characteristics, aptamers are promising tools for biosensor development. Aptamers are usually obtained through a procedure called "systematic evolution of ligands by exponential enrichment" (SELEX). In this study, we select an aptamer (termed ZIKV60) by capillary electrophoresis SELEX (CE-SELEX) to the Zika virus non-structural protein 1 (NS1) and counterselection against the NS1 proteins of DENV (serotypes 1, 2, 3, and 4) and YFV. The ZIKV60 dissociation constant (K d) is determined by enzyme-linked oligonucleotide assay (ELONA) and the aptamer specificity is evaluated by quantitative real-time polymerase chain reaction. ZIKV60 shows a high binding affinity to the ZIKV NS1 protein with a K d value of 2.28 ± 0.28 nM. The aptamer presents high specificity for ZIKV NS1 compared to NS1 of DENV and YFV. Furthermore, graphene field-effect transistor devices functionalized with ZIKV60 exhibit an evident identification of NS1 protein diluted in human serum. These results point to the applicability of biosensors based on the ZIKV60 aptamer for the differential diagnosis of the Zika virus.

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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.

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Infections caused by multidrug-resistant A. baumannii are a worldwide health concern with high mortality rates. Rapid identification of this infectious agent is critical as it can easily spread with difficult or no options for treatment. In this context, the development of reliable and economically viable detection and therapeutic methodologies are still challenging. One of the promising solutions is the development of nucleic acid aptamers capable of interacting with bacteria. These aptamers can be used for specific recognition of infectious agents as well as for blocking their functions. Cell-SELEX technology currently allows the selection and identification of aptamers and is flexible enough to target molecules present in an entire bacterial cell without their prior knowledge. However, the aptamer technology is still facing many challenges, such as the complexity of the screening process. Here, we describe the selection and identification of a new aptamer A01, using an in-house whole-cell SELEX-based methodology, against multi-resistant Acinetobacter baumannii, with rapid execution and low cost. In addition, this protocol allowed the identification of the aptamer A01 with the whole A. baumannii cell as a target. The aptamer A01 demonstrated a binding preference to A. baumannii when compared to K. pneumoniae, C. albicans, and S. aureus in fluorescence assays. Although the time-kill assay did not show an effect on bacterial growth, the potential bactericidal or bacteriostatic cannot be totally discarded. The new categorized aptamer (A01) displayed a significant binding affinity to MDR A. baumannii.

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BACKGROUND: Prostate cancer cells have very high PCA3 messenger RNA levels, which turns them into one of the new biomarkers for prostate cancer prognosis and diagnosis. OBJECTIVE: Our goal here is to develop a new aptasensor to detect PCA3 release by the cancer cell. METHODS: DNA hairpin containing PCA3 aptamer was thiolated, conjugated to methylene blue (MB) redox probe, and immobilized on gold electrode through self-assembly to detect label-free cancer cells. RESULTS: Our data have evidenced stable and sensitive sensors presenting a wide linear detection range (0-150ng/mL). In addition, monitoring PCA3 released by different types of prostate cells can provide in-depth knowledge about prostate cancer dynamics; therefore, it is a powerful platform for earlier clinical diagnostic. The released PCA3 can vary depending on the type of adopted prostate cells. CONCLUSION: PCA3 release was monitored in a group of cells for 2 h; it showed significantly higher expression in both LNCaP and PC-3 cells. This strategy provides a unique and simple methodology to achieve more sensitive and specific PCA3 detection; thus, it emerged as a promising tool for early cost-effective diagnosis.

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Aptamers are affinity-based oligonucleotide ligands raised against a target molecule, which might be of proteic or other nature. Aptamers are developed by using a reiterative in vitro selection procedure, named SELEX, in which the target is exposed to a combinatorial oligonucleotide combinatorial library. Target bound oligonucleotides are eluted, and PCR amplified followed by the next SELEX round. The process is repeated until no further increase in target binding affinity and specificity is achieved. Selected aptamers are identified and immobilized for protein purification. In view of their stability against denaturation and capability of renaturation, low costs of production, easiness of modification and stabilization, oligonucleotide aptamers are excellent tools as high-affinity ligands for applications of protein purification.

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Cancer is the second cause of mortality worldwide. Early diagnosis of this multifactorial disease is challenging, especially in populations with limited access to healthcare services. A vast repertoire of cancer biomarkers has been studied to facilitate early diagnosis; particularly, the use of antibodies against these biomarkers has been of interest to detect them through biorecognition. However, there are certain limitations to this approach. Emerging biorecognition engineering technologies are alternative methods to generate molecules and molecule-based scaffolds with similar properties to those presented by antibodies. Molecularly imprinted polymers, recombinant antibodies, and antibody mimetic molecules are three novel technologies commonly used in scientific studies. This review aimed to present the fundamentals of these technologies and address questions about how they are implemented for cancer detection in recent scientific studies. A systematic analysis of the scientific peer-reviewed literature regarding the use of these technologies on cancer detection was carried out starting from the year 2000 up to 2021 to answer these questions. In total, 131 scientific articles indexed in the Web of Science from the last three years were included in this analysis. The results showed that antibody mimetic molecules technology was the biorecognition technology with the highest number of reports. The most studied cancer types were: multiple, breast, leukemia, colorectal, and lung. Electrochemical and optical detection methods were the most frequently used. Finally, the most analyzed biomarkers and cancer entities in the studies were carcinoembryonic antigen, MCF-7 cells, and exosomes. These technologies are emerging tools with adequate performance for developing biosensors useful in cancer detection, which can be used to improve cancer diagnosis in developing countries.

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Photodynamic therapy (PDT) may be an excellent alternative in the treatment of breast cancer, mainly for the most aggressive type with limited targeted therapies such as triple-negative breast cancer (TNBC). We recently generated conjugated polymer nanoparticles (CPNs) as efficient photosensitizers for the photo-eradication of different cancer cells. With the aim of improving the selectivity of PDT with CPNs, the nanoparticle surface conjugation with unique 2'-Fluoropyrimidines-RNA-aptamers that act as effective recognition elements for functional surface signatures of TNBC cells was proposed and designed. A coupling reaction with carbodiimide was used to covalently bind NH2-modified aptamers with CPNs synthetized with two polystyrene-based polymer donors of COOH groups for the amide reaction. The selectivity of recognition for TNBC membrane receptors and PDT efficacy were assayed in TNBC cells and compared with non-TNBC cells by flow cytometry and cell viability assays. Furthermore, in vitro PDT efficacy was assayed in different TNBC cells with significant improvement results using CL4, sTN29 and sTN58 aptamers compared to unconjugated CPNs and SCR non-specific aptamer. In a chemoresistance TNBC cell model, sTN58 was the candidate for improving labelling and PDT efficacy with CPNs. We proposed sTN58, sTN29 and CL4 aptamers as valuable tools for selective TNBC targeting, cell internalization and therapeutic improvements for CPNs in PDT protocols.