Optimization of Surface-Enhanced Raman Spectroscopy Detection Conditions for Interaction between Gonyautoxin and Its Aptamer

This study aimed to optimize the detection conditions for surface-enhanced Raman spectroscopy (SERS) of single-stranded DNA (ssDNA) in four different buffers and explore the interaction between gonyautoxin (GTX1/4) and its aptamer, GO18. The influence of the silver colloid solution and MgSO4 concentration (0.01 M) added under four different buffered conditions on DNA SERS detection was studied to determine the optimum detection conditions. We explored the interaction between GTX1/4 and GO18 under the same conditions as those in the systematic evolution of ligands by exponential enrichment technique, using Tris-HCl as the buffer. The characteristic peaks of GO18 and its G-quadruplex were detected in four different buffer solutions. The change in peak intensity at 1656 cm−1 confirmed that the binding site between GTX1/4 and GO18 was in the G-quadruplex plane. The relative intensity of the peak at 1656 cm−1 was selected for the GTX1/4–GO18 complex (I1656/I1099) to plot the ratio of GTX1/4 in the Tris-HCl buffer condition (including 30 μL of silver colloid solution and 2 μL of MgSO4), and a linear relationship was obtained as follows: Y = 0.1867X + 1.2205 (R2 = 0.9239). This study provides a basis for subsequent application of SERS in the detection of ssDNA, as well as the binding of small toxins and aptamers.

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.

Pheno-SELEX: Engineering Anti-Metastatic Aptamers through Targeting the Invasive Phenotype Using Systemic Evolution of Ligands by Exponential Enrichment

Multiple methods (e.g., small molecules and antibodies) have been engineered to target specific proteins and signaling pathways in cancer. However, many mediators of the cancer phenotype are unknown and the ability to target these phenotypes would help mitigate cancer. Aptamers are small DNA or RNA molecules that are designed for therapeutic use. The design of aptamers to target cancers can be challenging. Accordingly, to engineer functionally anti-metastatic aptamers we used a modification of systemic evolution of ligands by exponential enrichment (SELEX) we call Pheno-SELEX to target a known phenotype of cancer metastasis, i.e., invasion. A highly invasive prostate cancer (PCa) cell line was established and used to identify aptamers that bound to it with high affinity as opposed to a less invasive variant to the cell line. The anti-invasive aptamer (AIA1) was found to inhibit in vitro invasion of the original highly invasive PCa cell line, as well as an additional PCa cell line and an osteosarcoma cell line. AIA1 also inhibited in vivo development of metastasis in both a PCa and osteosarcoma model of metastasis. These results indicate that Pheno-SELEX can be successfully used to identify aptamers without knowledge of underlying molecular targets. This study establishes a new paradigm for the identification of functional aptamers.

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).

Efficient Screening of Pesticide Diazinon-Binding Aptamers Using The Sol-Gel Coated Nanoporous Membrane-Assisted SELEX Process and Next-Generation Sequencing

BackgroundAptamer-based methods for detecting pesticides are more efficient than antibody-based methods as aptamers have high thermal stability, low molecular weight, easy modification, and low cost. However, only few studies on SELEX performed in combination with next generation sequencing (NGS) to screen aptamers specific for pesticides have been reported. Therefore, this study aimed to develop the systematic evolution of ligands by exponential enrichment (SELEX) process, combined with NGS, to select aptamers specific to the pesticide, diazinon, which was fixed on a sol-gel-coated nanoporous anodized aluminum oxide membrane. Methods and resultsThe frequency of specific nucleotide sequences obtained after SELEX rounds was analyzed using NGS. Nine sequences with the highest frequency after SELEX round 10 followed by NGS were selected and tested to derive their binding affinity with the target, diazinon, through circular dichroism (CD) spectrophotometry. The CD signal difference of the aptamer candidates ranged from 0.13 to 2.242 mdeg between diazinon-only treated and diazinon-aptamer-treated samples at a wavelength near 270 nm. Aptamer D-4, which had the highest binding affinity from CD spectrophotometry analysis, showed no cross-reactivity with non-target pesticides, such as baycarb, bifenthrin, and pyridaben, but interacted with the other pesticides, fipronil and 2-phenylphenol.ConclusionsThe proposed SELEX process combined with NGS for the discovery of aptamers for new targets can shorten the SELEX cycle by reducing the number of SELEX rounds to 10 or less. Therefore, an efficiently selected aptamer is highly expected to be used for the detection of pesticides in foods in the field.

Selection of a Nuclease-Resistant RNA Aptamer Targeting CD19

The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell–specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as ‘chemical antibodies’, they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2′-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies.

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.

Applications of Aptamers in the Diagnosis and Treatment of Ovarian Cancer: Progress From 2016 to 2020

Nucleic acid aptamers are short single-stranded DNA or RNA oligonucleotides selected from a random single-stranded nucleic acid library using systematic evolution of ligands by exponential enrichment technology. To allow them to bind to molecular targets with the same specificity and precision as that of antibodies, aptamers are folded into secondary or tertiary structures. However, compared to antibodies, aptamers are not immunogenic and are easier to synthesize. Furthermore, they are chemically modified, which protects them from degradation by nucleases. Hence, due to their stability and favorable targeting ability, aptamers are promising for the diagnosis and treatment of diseases. Ovarian cancer has the worst prognosis among all gynecological diseases and is usually diagnosed at the medium and advanced stages due to its nonspecific symptoms. Relapse is common, even if patients receive a standard therapeutic regimen including surgery and chemotherapy; simultaneously, drug resistance and adverse effects are reported in a several patients. Therefore, the safer and more efficient diagnostic and treatment method for ovarian cancer is imperative. Scientists have been trying to utilize aptamer technology for the early diagnosis and accurate treatment of ovarian cancer and some progress has been made in this field. This review discusses the screening of nucleic acid aptamers by targeting ovarian cancer cells and the application of aptamers in the diagnosis and treatment of ovarian cancer.