DNA Aptamers Specific for Legionella Pneumophila: Systematic Evolution of Ligands by Exponential Enrichment in Whole Bacterial Cells

Legionella pneumophila is the major causative agent of Legionnaires’ disease and Pontiac fever, which pose major public health problems. Rapid detection of L. pneumophila is important for global control of these diseases. Aptamers, short oligonucleotides that bind to targets with high affinity and specificity, have great potential for use in pathogenic bacterium detection, diagnostics, and therapy. Here, we used a whole-cell SELEX (systematic evolution of ligands by exponential enrichment) method to isolate and characterize single-stranded DNA (ssDNA) aptamers against L. pneumophila. A total of 60 ssDNA sequences were identified after 17 rounds of selection. Other bacterial species (Escherichia coli, Bacillus subtilis, Pseudomonas syringae, Staphylococcus aureus, Legionella quateirensis, and Legionella adelaidensis) were used for counterselection to enhance the specificity of ssDNA aptamers against L. pneumophila. Four ssDNA aptamers showed strong affinity and high selectivity for L. pneumophila, with Kd values in the nanomolar range. Bioinformatic analysis of the most specific aptamers revealed predicted conserved secondary structures that might bind to L. pneumophila cell walls. In addition, the binding of these four fluorescently labeled aptamers to the surface of L. pneumophila was observed directly by fluorescence microscopy. This is the first study to use SELEX to target L. pneumophila whole cells. The aptamers identified in this study could be used in the future to develop medical diagnostic tools and public environmental detection assays for L. pneumophila.

Profiling Cancer Cells by Cell-SELEX: Use of Aptamers for Discovery of Actionable Biomarkers and Therapeutic Applications Thereof

The identification of tumor cell-specific surface markers is a key step towards personalized cancer medicine, allowing early assessment and accurate diagnosis, and development of efficacious targeted therapies. Despite significant efforts, currently the spectrum of cell membrane targets associated with approved treatments is still limited, causing an inability to treat a large number of cancers. What mainly limits the number of ideal clinical biomarkers is the high complexity and heterogeneity of several human cancers and still-limited methods for molecular profiling of specific cancer types. Thanks to the simplicity, versatility and effectiveness of its application, cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology is a valid complement to the present strategies for biomarkers’ discovery. We and other researchers worldwide are attempting to apply cell-SELEX to the generation of oligonucleotide aptamers as tools for both identifying new cancer biomarkers and targeting them by innovative therapeutic strategies. In this review, we discuss the potential of cell-SELEX for increasing the currently limited repertoire of actionable cancer cell-surface biomarkers and focus on the use of the selected aptamers as components of innovative conjugates and nano-formulations for cancer therapy.

Aptamer-based Cell Recognition and Detection

: Cells, regarded as the structural and functional units of organisms, have become one of the most important objects in many research areas. Specific recognition and detection of malignant cells are critical for disease diagnosis, therapy and prognosis. Aptamers are short; single-stranded oligonucleotides screened from a random library by an in vitro technology termed “Systematic Evolution of Ligands by Exponential Enrichment” (SELEX) on the basis of their specific binding to target cargos. With the advantages of small size, easy synthesis, convenient modification, high chemical stability and low immunogenicity, aptamers have attracted broad attention in bioanalysis. Using intact living cells as the selection target, the cell-SELEX technology enables the generation of many aptamers that can specifically recognize molecular signatures of target cells. These aptamers have been extensively utilized in various cell-based research. In this mini-review, we focus on recent advances in aptamer-based recognition and detection of cells, particularly circulating tumor cells (CTCs).

AutoCell Systematic Evolution of Ligands by Exponential Enrichment: The Software Designed and Developed for the Automated Screening System of Nucleic Acid Aptamers

Aptamers are a new kind of nano-probes for bioassays and drug delivery, etc. In this paper, software has been developed as an automatic control center for the automated aptamer selecting system which realized the high integration of aptamer selection, data acquisition and processing. This software, applied in windows system, is developed by C# with the Microsoft Visual Studio 2015 integrated developing environment and the database used in this software is implemented using open source relational database MYSQL. According to the requirement analysis, this software realized various important necessary functions including feasible experiment design, auto-control of the hardware, real time process monitoring and efficient data management which perfectly satisfies the users’ demands. During the actual experiment operation, this software worked smoothly and assumed stable serial port communication between it and the hardware, meanwhile, the interaction between the software and MYSQL remained good stability. As a consequence, it is practical and reasonable to apply this software to the automated aptamer selecting system for research.

The Application of Microfluidic Technologies in Aptamer Selection

Aptamers are sequences of single-strand oligonucleotides (DNA or RNA) with potential binding capability to specific target molecules, which are increasingly used as agents for analysis, diagnosis, and medical treatment. Aptamers are generated by a selection method named systematic evolution of ligands by exponential enrichment (SELEX). Numerous SELEX methods have been developed for aptamer selections. However, the conventional SELEX methods still suffer from high labor intensity, low operation efficiency, and low success rate. Thus, the applications of aptamer with desired properties are limited. With their advantages of low cost, high speed, and upgraded extent of automation, microfluidic technologies have become promising tools for rapid and high throughput aptamer selection. This paper reviews current progresses of such microfluidic systems for aptamer selection. Comparisons of selection performances with discussions on principles, structure, operations, as well as advantages and limitations of various microfluidic-based aptamer selection methods are provided.

Recent Advances in Micro/Nanomaterial-Based Aptamer Selection Strategies

Aptamers are artificial nucleic acid ligands that have been employed in various fundamental studies and applications, such as biological analyses, disease diagnostics, targeted therapeutics, and environmental pollutant detection. This review focuses on the recent advances in aptamer discovery strategies that have been used to detect various chemicals and biomolecules. Recent examples of the strategies discussed here are based on the classification of these micro/nanomaterial-mediated systematic evolution of ligands by exponential enrichment (SELEX) platforms into three categories: bead-mediated, carbon-based nanomaterial-mediated, and other nanoparticle-mediated strategies. In addition to describing the advantages and limitations of the aforementioned strategies, this review discusses potential strategies to develop high-performance aptamers.

Aptamer Selection Against Quinolones Antibiotics Using Immobilizing Free SELEX, and Studying Aptamer-Antibiotic Interaction By Spectroscopy and High-Resolution Mass Spectrometry

Over-consumption of antibiotics has led to antimicrobial resistance creating urgency to find fast and efficient techniques to monitor the level of antibiotics in the foods. The advantages of using aptamer for a variety of scientific challenges are becoming more abundant in different fields, making its applications more effective and cost-efficient. In this study, two aptamers against two quinolones antibiotics (Enrofloxacin and Ofloxacin) were isolated via GO-SELEX (graphene oxide assisted Systematic Evolution of Ligands by Exponential Enrichment). The interaction between the aptamer and its cognate antibiotic was investigated using spectroscopy and mass spectrometry. The obtained evidence from spectroscopy showed that the interaction of antibiotics with their aptamers resulted in a 41.98% and 22.45 % reduction in the extinction coefficient (ɛ) value of enrofloxacin and ofloxacin, respectively. This phenomenon could arise from the stacking interaction between the aromatic region of antibiotic and aptamer’s bases. The formation of the aptamer-antibiotic complex was studied by the high-resolution ESI-Q-TOF. In the obtained mass spectra, several peaks corresponding to the aptamer-antibiotic complexes with a wide range of charge distribution were identified, which could be explained by the fact that both aptamer-ENR and aptamer-OFLO complexes were stable in the gas phase and the intact complexes survived in the vacuum. This stability could be explained by the hydrogen bonding and electrostatic interactions between the aptamers and antibiotics. As both aptamers can distinguish quinolone from other antibiotic families, these aptamers may become great recognizing elements for apta-sensors.

Systematic Evolution of Ligands by Exponential Enrichment Technologies and Aptamer-Based Applications: Recent Progress and Challenges in Precision Medicine of Infectious Diseases

Infectious diseases are considered as a pressing challenge to global public health. Accurate and rapid diagnostics tools for early recognition of the pathogen, as well as individualized precision therapy are essential for controlling the spread of infectious diseases. Aptamers, which were screened by systematic evolution of ligands by exponential enrichment (SELEX), can bind to targets with high affinity and specificity so that have exciting potential in both diagnosis and treatment of infectious diseases. In this review, we provide a comprehensive overview of the latest development of SELEX technology and focus on the applications of aptamer-based technologies in infectious diseases, such as targeted drug-delivery, treatments and biosensors for diagnosing. The challenges and the future development in this field of clinical application will also be discussed.

Identification, systematic evolution and expression analyses of the AAAP gene family in Capsicum annuum

Background The amino acid/auxin permease (AAAP) family represents a class of proteins that transport amino acids across cell membranes. Members of this family are widely distributed in different organisms and participate in processes such as growth and development and the stress response in plants. However, a systematic comprehensive analysis of AAAP genes of the pepper (Capsicum annuum) genome has not been reported. Results In this study, we performed systematic bioinformatics analyses to identify AAAP family genes in the C. annuum ‘Zunla-1’ genome to determine gene number, distribution, structure, duplications and expression patterns in different tissues and stress. A total of 53 CaAAAP genes were identified in the ‘Zunla-1’ pepper genome and could be divided into eight subgroups. Significant differences in gene structure and protein conserved domains were observed among the subgroups. In addition to CaGAT1, CaATL4, and CaVAAT1, the remaining CaAAAP genes were unevenly distributed on 11 of 12 chromosomes. In total, 33.96% (18/53) of the CaAAAP genes were a result of duplication events, including three pairs of genes due to segmental duplication and 12 tandem duplication events. Analyses of evolutionary patterns showed that segmental duplication of AAAPs in pepper occurred before tandem duplication. The expression profiling of the CaAAAP by transcriptomic data analysis showed distinct expression patterns in various tissues and response to different stress treatment, which further suggest that the function of CaAAAP genes has been differentiated. Conclusions This study of CaAAAP genes provides a theoretical basis for exploring the roles of AAAP family members in C. annuum.