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The "labeling" of nucleic acids (cell genetic information such as RNA or DNA) is not a new technology for monitoring them. However, current capabilities do not provide a complete picture of how tumor cells develop. What this platform, known as Clon Mapper, can do that was not possible before, is to go back in time and track how tumor cells change over time. This gives researchers the ability to see which cells "win" over fewer resistant cells, continue to clone themselves, and make the tumor more dangerous. By isolating these cells, researchers can better test which therapies work against them. Monitoring changes over time is the key to successful transmission therapies. Tumor cells adapt and become resistant to therapies, which is why patients can recover but experience a relapse later. This is one of the reasons why cancer treatment is so challenging; we do not have very good methods for early detection of cells sensitive to a drug and measuring their resistance. This resistance is the main cause of treatment failure in many cancer patients. CLL is a low-grade B cancer that is often monitored for months or even years before active treatment is needed. This treatment depends a lot on the patient's close supervision. In this study, Clon Mapper focuses on identifying cells that mimic themselves, the speed at which this process takes place, and its effect on the growth rate of surrounding cells over time. This allows for a more accurate analysis of the cell population and may lead to more customized treatment plans for patients. Keywords:Cancer; Cells; Tissues; Tumors; Prevention; Prognosis; Diagnosis; Imaging; Screening; Treatment; Management https://www.raftpubs.com/ijho-hematology-and-oncology/articles/ijho_raft1023.php

The discovery and identification of biomarkers promote the rational and fast development of medical diagnosis and therapeutics. Clinically, the application of ideal biomarkers still is limited due to the suboptimal technology in biomarker discovery. Aptamers are single-stranded deoxyribonucleic acid or ribonucleic acid molecules and can selectively bind to varied targets with high affinity and specificity. Compared with antibody, aptamers have desirable advantages, such as flexible design, easy synthesis and convenient modification with different functional groups. Currently, different aptamer-based technologies have been developed to facilitate biomarker discovery, especially CELL-SELEX and SOMAScan technology. CELL-SELEX technology is mainly used to identify cell membrane surface biomarkers of various cells. SOMAScan technology is an unbiased biomarker detection method that can analyze numerous and even thousands of proteins in complex biological samples at the same time. It has now become a large-scale multi-protein biomarker discovery platform. In this review, we introduce the aptamer-based biomarker discovery technologies, and summarize and highlight the discovered emerging biomarkers recently in several diseases.

Increased interest and insights gained by researchers on the roles of endothelial cells in the pathophysiology of cancer, inflammatory, and cardiovascular diseases have led to the design of pharmacological interventions aimed at the endothelium lining in the diseased sites. Toward this end, we used established brain microvascular endothelial cell lines mouse (bEND3), human (hCMEC/D3), and Toggle Cell-SELEX to identify a species cross-reactive, endothelial cell-internalizing aptamer R11-3. This 2'F-modified RNA aptamer is specific for endothelial cells as no internalization was seen with cells of nonendothelial origin. R11-3 was truncated in size, and its potential in endothelial targeted therapeutics was established using VEGFR2 targeting long interfering RNA (liRNA) aptamer chimera. Due to its specificity for both mouse and human endothelial cells, we believe that this aptamer not only fits for development of endothelial targeted drug development for human diseases but is also suitable for preclinical evaluation in mice.

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.

The sensitive and specific detection of pathogenic cells is essential in clinical diagnostics. To achieve this, molecular tools are required that unequivocally recognise appropriate cell surface molecules, such as biomarkers that come along with disease onset and progression. Aptamers are short single-stranded oligonucleotides that interact with cognate target molecules with high affinity and specificity. Within the last years they have gained an increased attention as cell-recognition tools. Here, we report a systematic analysis of a cell-SELEX procedure, for the identification of aptamers that recognise breast cancer cells. Besides a comparison of conventional (Sanger) with high-throughput sequencing techniques (next-generation sequencing), three different screening techniques have been applied to characterise the binding properties of selected aptamer candidates. This method has been found to be beneficial in finding DNA aptamers, rarely enriched in the libraries. Finally, four DNA aptamers were identified that exhibit broad-spectrum interaction patterns to different cancer cell lines derived from solid tumours.

Background: The objective of this study was to screen and identify a new biomarker for the detection of early gastric cancer. Aptamers are a group of promising probe that can specifically recognize and bind to target molecules and cells. Methods: Using an aptamer library, we selected high-specificity and high-affinity aptamers through 12 rounds of Cell-SELEX. The target protein could be extracted from early gastric cancer cells with streptavidin magnetic beads method and aptamers; before purification, the target protein was analyzed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The fluorescein-labeled aptamer was used for establishment and analysis of a new detection method for biomarkers. Then we used the ELISA (enzyme-linked immuno sorbent assay) to verify the new detection method. Results: Ultimately, aptamer 7 (Ap7) was selected as the aptamer with the highest specificity and affinity in the screen, which targeted the early gastric cancer protein named early gastric cancer 7 (EGA7). Based on comparison of sequences in the NCBI database, EGA7 was identified as Peroxiredoxin-4 (Prx4). Further analysis which contained the ELISA results showed a significant difference in EGA7 expression between early gastric cancer tissues and the normal gastric mucosa or advance gastric cancer tissues, confirming its utility as a marker for early gastric cancer. This is the first report using a high-specificity and high-affinity aptamer (Ap7) to screen for an early gastric cancer protein; using this method, a new biomarker, EGA7, was identified. Conclusions: These results provide the experimental foundation for future research on the use of aptamer libraries and biomarkers for the diagnosis and identification of drug targets in early gastric cancer. And Prx4 has the potential as a biomarker of early gastric cancer.

Aptamers are short single-stranded oligonucleotide (DNA or RNA) molecules with the ability to bind to other molecules with high affinity and specificity. Nowadays, aptamers are used as biosensing molecules with broader applications, such as ribozymes (RNA enzyme molecules with catalytic activities); riboswitches (modulating translational activities) and ligands, recognizing specific target binding. Aptamers are folded, directed by their sequences, acquiring specific 3D conformations. This 3D conformation property has been widely applied in order to recognize cell structures. Aptamers evolve from random oligonucleotide pools by a process called Systematic Evolution of Ligands by Exponential enrichment (SELEX). Conceptually, the SELEX process is controlled by the ability of these small oligonucleotides to fold into unique 3D structures that can interact with a specific target with high specificity and affinity [1]. This is a dynamic process, repeating exposures and elutions, allowing to screen in a heterogenic pool in low concentration ranges. A standard SELEX method has four major steps: 1.- Exposure of random sequence single stranded nucleotide oligo library to a target. 2.- Binding of oligos to the target molecule. 3.- Selection of binders and removal of non-binding oligos. 4.- Amplification of the binder fraction and portioning of the amplicon to single strand. These steps are iteratively performed till a pool of high binding aptamer is screened out from the library. The oligonucleotide library consists of a random base-sequence flanked on both ends by primer binding sites, which aids in amplification and enrichment (Safeh et al., 2010; Kim et al., 2013). Here is described how SELEX have been applied to develop aptamers, recognizing specific outer membrane structures, as LamB.

An aptamer-based strategy was developed for qualitative and quantitative analysis of viable Lactobacillus casei in dairy products. Three highly specific aptamers for L. casei were obtained using systematic evolution of ligands by exponential enrichment protocol using the whole bacterium cell as the target (cell-SELEX) facilitated by polyethyleneglycol and chitosan modified graphene oxide and complementary ring-mediated rolling circle amplification. Two aptamers, one for separating and enriching the L. casei cells and the other for generating fluorescence signals, were employed to develop an aptamer-based strategy, which was demonstrated for the selective detection of L. casei in commercial dairy drinks, with a dynamic range of 105 to 109 cfu/mL. Viable and nonviable L. casei cells could be discriminated based on the significant difference in fluorescence intensity. This established strategy is of high selectivity and sensitivity, and can be used for rapid analysis of viable L. casei in quality control and food surveillance areas.

Cell-SELEX is a live cell-based in vitro selection method that generates functional oligonucleotides, or aptamers. Often referenced as the chemist's antibody, aptamers bind to select targets with high affinity and can be utilized in a number of applications, including biomedicine, bioimaging, and biosensing. Here we describe the cell-SELEX technique and discuss this methodology's unique merit(s)-namely the ability to isolate highly selective aptamer panels with no prior knowledge of cellular signatures. This strategy thus presents as a technology that has the potential to enhance the precision of molecular medicine and targeted therapeutics.

Escherichia coli are important indicator organisms, used routinely for the monitoring of water and food safety. For quick, sensitive and real-time detection of E. coli we developed a 2′F modified RNA aptamer Ec3, by Cell-SELEX. The 31 nucleotide truncated Ec3 demonstrated improved binding and low nano-molar affinity to E. coli. The aptamer developed by us out-performs the commercial antibody and aptamer used for E. coli detection. Ec3(31) aptamer based E. coli detection was done using three different detection formats and the assay sensitivities were determined. Conventional Ec3(31)-biotin-streptavidin magnetic separation could detect E. coli with a limit of detection of 1.3 × 106 CFU/ml. Although, optical analytic technique, biolayer interferometry, did not improve the sensitivity of detection for whole cells, a very significant improvement in the detection was seen with the E. coli cell lysate (5 × 104 CFU/ml). Finally we developed Electrochemical Impedance Spectroscopy (EIS) gap capacitance biosensor that has detection limits of 2 × 104 CFU/mL of E. coli cells, without any labeling and signal amplification techniques. We believe that our developed method can step towards more complex and real sample application.

Detecting and understanding changes in cell conditions on the molecular level is of great importance for the accurate diagnosis and timely therapy of diseases. Cell-based SELEX (Systematic Evolution of Ligands by EXponential enrichment), a foundational technology used to generate highly-specific, cell-targeting aptamers, has been increasingly employed in studies of molecular medicine, including biomarker discovery and early diagnosis/targeting therapy of cancer. In this review, we begin with a mechanical description of the cell-SELEX process, covering aptamer selection, identification and identification, and aptamer characterization; following this introduction is a comprehensive discussion of the potential for aptamers as targeting moieties in the construction of various nanotheranostics. Challenges and prospects for cell-SELEX and aptamer-based nanotheranostic are also discussed.

Aptamers are a class of short artificial single-stranded oligo(deoxy) nucleotides that can bind to different targets, which generated by Systematic Evolution of Ligands by Exponential Enrichment (SELEX). Due to excellent selectivity and high affinity to targets, aptamers hold considerable potential as molecular probe in diverse applications ranging from ensuring food safety, monitoring environment, disease diagnosis to therapy. This review highlights recent development and challenges about aptamers screened by Cell-SELEX, and its application about cancer diagnostics and therapeutics. Advances about some operation methods such as seperation method and culture method in aptamers selection procedure were summarized in this paper. Some common challenges and technological difficulties such as nonspecific binding and biostability were discussed. Up to now, the recent endeavors about cancer diagnostic and therapeutic applications of aptamers are summarized and expatiated. Most of aptamers screened by Cell-SELEX took tumor cells as target cells, and such aptamers have been assembled to various aptasensor for cancer diagnosis. Aptamers conjugated various drugs or nanomaterials are functioned for cancer target therapy to improve drugs delivery efficiency and reduce side effects. Furthermore, the duplexed aptamer is discussed to be applied for cancer cells detection and some conflicts of theories about duplexed aptamer designs are analyzed.

Background SELEX technique is employed to select aptamers against wide range of targets. The in vitro method of aptamer selection using live cells as the target is referred as cell-SELEX. Scope of the review The review provides a comprehensive description on the development of aptamers through various cell-SELEX methods and list of aptamers identified through this method. In addition, it pinpoints the advantages and limitations of the cell-SELEX process and its variants. Major conclusions The use of aptamers as therapeutic and diagnostic agents is rapidly evolving, selection techniques such as Cell-SELEX could be beneficial in identifying aptamers when the target is in its native conformation and without prior information of the cognate target, thereby bringing the aptamer development one step closer to the clinic. General significance The information in this review can serve as a database for the design and development of futuristic oligonucleotide based diagnostics and therapeutics work.

Aptamers are often compared with antibodies since both types of molecules function as targeting ligands for specific cancer cell recognition. However, aptamers offer several advantages, including small size, facile chemical modification, high chemical stability, low immunogenicity, rapid tissue penetration, and engineering simplicity. Despite these advantages, several crucial factors have delayed their clinical translation, such as concerns over inherent physicochemical stability and safety. Meanwhile, steps have been taken to make aptamer-drug conjugates, or ApDCs, a clinically practical tool. In this review, we highlight the development of ApDCs and discuss how researchers are solving some problems associated with their clinical application for targeted therapy.

Aptamers have emerged as a novel category in the field of bioreceptors due to their wide applications ranging from biosensing to therapeutics. Several variations of their screening process, called SELEX have been reported which can yield sequences with desired properties needed for their final use. We report a facile microtiter plate-based Cell-SELEX method for a gram-negative bacteria E. coli. The optimized protocol allows the reduction of number of rounds for SELEX by offering higher surface area and longer retention times. In addition, this protocol can be modified for other prokaryotic and eukaryotic cells, and glycan moieties as target for generation of high affinity bio-receptors in a short course of time in-vitro.

Conventional diagnostics for cancer rely primarily on anatomical techniques. However, these techniques cannot monitor the changes at the molecular level in normal cells, which possibly signal the onset of cancer at its very earliest stages. For accurate prediction of the carcinogenesis at the molecular level, targeting ligands have been used in combination with imaging probes to monitor this biological process. Among these targeting ligands, aptamers have high binding affinity to various targets ranging from small molecules to whole organisms, and, hence, exceptional recognition ability. Many recent studies have been reported on aptamer-based molecular imaging, clearly indicating its clinical and diagnostic utility. In this review, we will discuss some key results of these studies.

Nucleic acid aptamers, which are generated by a novel technique called SELEX (systematic evolution of ligands by exponential enrichment), have recently attracted significant attention in the field of early detection and treatment of cancer based on their numerous merits, such as high affinity, high specificity, small size, little immunogenicity, stable structures, and ease of chemical modification. Furthermore, aptamers can gain more flexibility as cancer cell targeting tools when conjugated to nanomaterials, including metallic nanoparticles, carbon nanomaterials, DNA nanodevices, and polymeric nanoparticles. We discuss the progress achieved in cancer diagnosis and therapy through the conjugation of cell-SELEX-based aptamers with different nanomaterials.

Aptamers are short single stranded nucleic acid molecules, isolated from a pool of nucleic acid molecules by the method called SELEX (Systematic Evolution of Ligands by EXponential Enrichment) that can bind to the target with high affinity and specificity. A modified SELEX method where the targets are living whole cells is referred as cell-SELEX, and is of great interest because it opens up a simple and effective method for disease diagnostics and therapeutics. The cell-SELEX based approach and recent developments of the technique for identifying aptamers as well as their application in cancer diagnostics and therapeutics are summarized here.

Pseudomonas aeruginosa is an opportunistic pathogen that causes significant morbidity and mortality in immunocompromised patients, particular cystic fibrosis sufferers, burns victims, diabetics and neonates. It thrives in moist places where it forms biofilms that are exceedingly difficult to eradicate on hospital surfaces, in water supplies and implanted biomaterials. Using a live cell SELEX approach we selected DNA aptamers to P. aeruginosa grown as biofilms in microfluidic cells. From a pool of aptamer candidates showing tight binding a stem-loop structure was identified as being important for binding. Enhanced binding and increased specificity was achieved by truncating structures and generating chimeric aptamers from the pool of top candidates. The top candidates have low nanomolar binding constants and high discrimination for P. aeruginosa over other Gram-negative bacteria. The aptamers bind both planktonic grown and biofilm grown cells. They do not have intrinsic bacteriostatic or bactericidal activity, but are ideal candidates for modification for use as aptamer-drug conjugates and in biosensors.

Aptamers are single-stranded RNA or DNA, which bind to their target with high affinity and specificity. Method of isolating aptamers against cell surface protein is called cell-SELEX. Common approach for monitoring cell-SELEX developed aptamers is flow cytometry. Since flow cytometry is costly and requires sophisticated equipments, we suggested implementing easy access, high throughput enzyme-link apta-sorbent assay test (ELASA) to confirm the specificity of aptamers selected through cell-SELEX process. In this regard, we compared ELASA and flow cytometry techniques in order to screen potent candidate aptamers against A2780 Rcis cell line, which were selected by cell-SELEX. The obtained results demonstrated that both ELASA and flow cytometry are identical in terms of sensivity and precision for aptamers selection. Then it could be concluded that ELASA method could be used as a versatile, inexpensive procedure for in vito evaluation of isolated aptamers from cell-SELEX based process.

Aptamer selection protocols, named cell-SELEX, have been developed to target trans-membrane proteins using whole living cells as target. This technique presents several advantages. (1) It does not necessitate the use of purified proteins. (2) Aptamers are selected against membrane proteins in their native conformation. (3) Cell-SELEX can be performed to identify aptamers against biomarkers differentially expressed between different cell lines without prior knowledge of the targets. (4) Aptamers identified by cell-SELEX can be further used to purify their targets and to identify new biomarkers. Here, we provide a protocol of cell-SELEX including the preparation of an oligonucleotide library, next-generation sequencing and radioactive binding assays. Furthermore, we also provide a protocol to purify and identify the target of these aptamers. These protocols could be useful for the discovery of lead therapeutic compounds and diagnostic cell-surface biomarkers.

In order to better control nosocomial infections, and facilitate the most prudent and effective use of antibiotics, improved strategies for the rapid detection and identification of problematic bacterial pathogens are required. DNA aptamers have much potential in the development of diagnostic assays and biosensors to address this important healthcare need, but further development of aptamers targeting common pathogens, and the strategies used to obtain specific aptamers are required. Here we demonstrate the application of a quantitative PCR (qPCR) controlled Cell-SELEX process, coupled with downstream secondary-conformation-based aptamer profiling. We used this approach to identify and select DNA aptamers targeted against uropathogenic Escherichia coli, for which specific aptamers are currently lacking, despite the prevalence of these infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation of standard PCR amplification of recovered aptamer pools at each round. Identification of useful aptamer candidates was also facilitated by profiling of secondary conformations and selection based on putative aptamer secondary structure. One aptamer selected this way (designated EcA5-27), displaying a guanine-quadruplex sequence motif, was shown to have high affinity and specificity for target cells, and the potential to discriminate between distinct strains of E. coli, highlighting the possibility for development of aptamers selectively recognising pathogenic strains. Overall, the identified aptamers hold much potential for the development of rapid diagnostic assays for nosocomial urinary tract infections caused by E. coli.

Cell-SELEX can not only generate aptamers for specific cell isolation/detection, diagnosis, and therapy, but also lead to the discovery of biomarkers and unexpected molecular events. However, most cell-SELEX research is concentrated on aptamer generation and applications. In this progress report, recent research progress with cell-SELEX in terms of the discovery of biomarkers and unexpected molecular events is highlighted. In particular, the key technical challenges for cell-SELEX-based biomarker discovery, namely, the methods for identification and validation of target proteins of aptamers, are discussed in detail. Finally, the prospects of the applications of cell-SELEX in this field now and in the near future are described. It is expected that this report will attract attention to the benefit of cell-SELEX and provide a practical reference for biomedical researchers.

The development of molecular probes for Hepatic Carcinoma recognition with high sensitivity and specificity would be of great importance for the early diagnosis of liver cancer. In this work, with a cholangiocarcinoma cell line QBC-939 as counter cell, a hepatocellular carcinoma cell line Bel-7404 was used as target cells for the selection of aptamers based on Cell-SELEX. Four aptamers were selected which all could bind to target cells with an equilibrium dissociation constant ( K d) in the nanomolar range. Furthermore, these aptamers could recognize the target Bel-7404 cells with high specificity. These aptamers would serve as effective tools for early diagnosis of hepatocellular carcinoma and liver pathological classification.

Early detection and treatment of cancer depends on developing highly sensitive and specific methods for targeting cancer cells. To do this, aptamers, which are generated by a novel technique called cell-SELEX (systematic evolution of ligands by exponential enrichment), have been widely applied in cancer cell targeting based on such merits as high target affinity and specificity, small size, minimal immunogenicity, and ease of chemical modification. Furthermore, aptamers can gain more flexibility as cancer cell targeting tools when conjugated to nanomaterials, including metallic nanoparticles, quantum dots, silica nanoparticles, and carbon nanomaterials, among others. In this review, we discuss the use of cell-SELEX-based aptamer-nanomaterials conjugates as novel molecular tools for enhanced targeting of cancer cells.

Key Points Aptamers are single-stranded oligonucleotides that fold into defined architectures and bind to targets such as proteins.In binding proteins they often inhibit protein–protein interactions and thereby may elicit therapeutic effects such as antagonism.Aptamers are discovered using SELEX (systematic evolution of ligands by exponential enrichment), a directed in vitro evolution technique in which large libraries of degenerate oligonucleotides are iteratively and alternately partitioned for target binding. They are then amplified enzymatically until functional sequences are identified by the sequencing of cloned individuals.For most therapeutic purposes, aptamers are truncated to reduce synthesis costs, modified at the sugars and capped at their termini to increase nuclease resistance, and conjugated to polyethylene glycol or another entity to reduce renal filtration rates.The first aptamer approved for a therapeutic application was pegaptanib sodium (Macugen; Pfizer/Eyetech), which was approved in 2004 by the US Food and Drug Administration for macular degeneration.Eight other aptamers are currently undergoing clinical evaluation for various haematology, oncology, ocular and inflammatory indications.Aptamers are ultimately chemically synthesized in a readily scalable process in which specific conjugation points are introduced with defined stereochemistry.Unlike some protein therapeutics, aptamers do not elicit antibodies, and because aptamers generally contain sugars modified at their 2′-positions, Toll-like receptor-mediated innate immune responses are also abrogated.As aptamers are oligonucleotides they can be readily assembled into supramolecular multi-component structures using hybridization.Owing to the fact that binding to appropriate cell-surface targets can lead to internalization, aptamers can also be used to deliver therapeutic cargoes such as small interfering RNA.Supramolecular assemblies of aptamers and delivery agents have already been demonstrated in vivo and may pave the way for further therapeutic strategies with this modality in the future., Aptamers are oligonucleotide sequences that are capable of recognizing target proteins with an affinity and specificity rivalling that of antibodies. In this article, Keefe and colleagues discuss the development, properties and therapeutic potential of aptamers, highlighting those currently in the clinic., Nucleic acid aptamers can be selected from pools of random-sequence oligonucleotides to bind a wide range of biomedically relevant proteins with affinities and specificities that are comparable to antibodies. Aptamers exhibit significant advantages relative to protein therapeutics in terms of size, synthetic accessibility and modification by medicinal chemistry. Despite these properties, aptamers have been slow to reach the marketplace, with only one aptamer-based drug receiving approval so far. A series of aptamers currently in development may change how nucleic acid therapeutics are perceived. It is likely that in the future, aptamers will increasingly find use in concert with other therapeutic molecules and modalities.

Aptamers, single stranded DNA or RNA molecules, generated by a method called SELEX (systematic evolution of ligands by exponential enrichment) have been widely used in various biomedical applications. The newly developed Cell-SELEX (cell based-SELEX) targeting whole living cells has raised great expectations for cancer biology, -therapy and regenerative medicine. Combining nanobiotechnology with aptamers, this technology opens the way to more sophisticated applications in molecular diagnosis. This paper gives a review of recent developments in SELEX technologies and new applications of aptamers.

Aptamers are short single-stranded DNA or RNA, which can be selected from random combinatorial library by SELEX in vitro. The SELEX technology has been modified over the years in different ways to become more efficient and less time-consuming, to reach higher affinities of the aptamers and for automation of the process. The multitude of different targets used in SELEX implicates aptamers are possible to be selected using any target theoretically. This paper presents the SELEX technology screening aptamers and its latest progress including modified selection methods (negative SELEX, counter SELEX and substractive SELEX) and efficient selection methods (CE-SELEX, non-SELEX, automated-SELEX and microfluidic SELEX). Additionally, cell SELEX using whole cells as targets is introduced. Varieties of live pathogenic organisms and many cancer cells have been used as targets for cell SELEX. Finally, an overview of biomedical applications of aptamers is given. Aptamers as a class of biorecognition elements that possess many advantages such as high specificity and binding affinity, easy synthesis, easy modification, small size, non-toxicity and good stability, have been increasingly applied in biomedical field. Especially, the combination of aptamers with nanomaterials will continuously play more and more important roles in many applications such as detection of targets, diagnosis and treatment of diseases, bioimaging, and drugs delivery.

Aptamers are functional oligonucleic acids that bind to specific targets with high affinity. They have demonstrated their unique advantages as chemical antibodies in molecular recognition for many applications in biomedicine and biotechnology such as biosensing, bioimaging, and drug. In particular, a recently developed method of live cell-based in vitro selection, termed cell-SELEX, has successfully isolated aptamers without prior knowledge of a cell’s molecular signature, making such aptamers promising for applications in molecular medicine. This chapter reviews the evolution of cell-SELEX technology and introduces the content of this book, which surveys the advancements in cell-SELEX DNA aptamer selection, characterization, modification, and application for theranostics.

Changes at the molecular level always occur at different stages in diseased cells. The detection of these changes is critical for understanding the molecular mechanisms underlying pathogenesis, as well as accurately diagnosing disease states and monitoring therapeutic modalities. Cell-SELEX is a foundational tool used to select probes able to recognize molecular signatures on the surface of diseased cells. This technology has been increasingly used in biomarker discovery, as well as cancer diagnosis and therapy. In this chapter, the whole cell-SELEX process is described, including aptamer selection, identification, and validation. In addition, we will explore the challenges and prospects for cell-SELEX now and in the coming years. It is anticipated that this chapter will guide readers toward a better understanding of the working principles underlying the cell-SELEX technology and serve as a practical reference for bench scientists engaged in cell and molecular biology.

Systematic evolution of ligands by exponential enrichment (SELEX) is an established procedure for developing short single-stranded nucleic acid ligands called aptamers against a target of choice. This approach has also been used for developing aptamers specific to whole cells named Cell-SELEX. Aptamers selected by Cell-SELEX have the potential to act as cell specific therapeutics, cell specific markers or cell specific drug delivery and imaging agents. However, aptamer development is a laborious and time-consuming process which is often challenging due to the requirement of frequent optimization of various steps involved in Cell-SELEX procedures. This review provides an insight into various procedures for selection, aptamer enrichment, regeneration and aptamer-binding analysis, in addition to a very recent update on all aptamers selected by Cell-SELEX procedures.

Separating the specific from the nonspecific bound single-strand DNA (ssDNA) is the most important step to improve the efficiency of selection procedure. However, most cell-SELEX protocols (where SELEX = systematic evolution of ligands by exponential enrichment) use simply washing only, which leads to incomplete separation. It is well-established that ssDNAs can be adsorbed on single-walled carbon nanotubes (SWCNTs). Based on this, herein, we developed a modified cell-SELEX approach termed "SWCNTs-assisted cell-SELEX". In our approach, SWCNTs are applied in the separation step, during which the unbound or the nonspecific ssDNAs are adsorbed onto SWCNTs, while the bound ssDNAs still remain on the cell surface, because of the stronger interaction between ssDNA and target. The cells can then be centrifuged to enrich the specifically binding aptamers. As a proof of concept, two nasopharyngeal carcinoma (NPC) cell lines-CNE2 cell and HONE cell-were used as the target cell and the negative cell, respectively. The result show that it takes only 6 cycles to enrich the aptamer pool through the SWCNTs-assisted cell-SELEX, which is much shorter than 15 cycles in the conventional cell-SELEX, thus improving the screening efficiency. Moreover, the achieved aptamers show high specificity and affinity with CNE2 cells, which are highly attractive for clinical diagnosis and biomedicine applications.

Aptamers are molecules identified from large combinatorial nucleic acid libraries by their high affinity to target molecules. Due to a variety of desired properties, aptamers are attractive alternatives to antibodies in molecular biology and medical applications. Aptamers are identified through an iterative selection–amplification process known as systematic evolution of ligands by exponential enrichment (SELEX). Although SELEX is typically carried out using purified target molecules, whole live cells are also employable as selection targets. This technology, Cell-SELEX, has several advantages. For example, generated aptamers are functional with a native conformation of the target molecule on live cells, and thus, cell surface transmembrane proteins would be targets even when their purifications in native conformations are difficult. In addition, cell-specific aptamers can be obtained without any knowledge about cell surface molecules on the target cells. Here, I review the progress of Cell-SELEX technology and discuss advantages of the technology.

A most able label: Labeled aptamers can be cross-linked to their target structures in a light-dependent and highly specific manner as a result of a new strategy termed aptamer-based affinity labeling (ABAL) of proteins. The aptamer-protein complexes can be enriched in vitro, from a cellular lysate and from the surface of living cells, opening new ways to study aptamer interactions in biological contexts.

Nucleic acid aptamers, generated by an in vitro selection procedure named SELEX (Systematic Evolution of Ligands by EXponential enrichment), have been popularly used in various biomedical implementations so far. An adaptation of the conventional SELEX practice to whole living cells was referred to as cell-SELEX, which is very promising in ample sophisticated analytical, therapeutic and diagnostic applications. The current review article would like to provide a survey of contemporary developments in cell-specific aptamers, including methodology, applications and optimization of cell-SELEX.

Biomarker discovery is essential to the advancement of our knowledge about diseases and to the development of new strategies for treatment and therapy. Among the newest technologies in this rapidly growing field is a process termed Cell SELEX by which a panel of aptamers can be generated to specifically target disease cells. Thus, Cell SELEX, together with the in vitro selection of aptamers, brings a promising approach to accelerate the progress of biomarker discovery. In this review, the Cell SELEX method and potential uses of selected aptamers are discussed. In addition, identification of potential biomarkers by using cell-binding aptamers is addressed. We believe that using the Cell SELEX strategy represents a significant improvement over current approaches to biomarker identification by the simplicity, efficiency and specificity of its application.

Molecular medicine is an emerging field focused on understanding the molecular basis of diseases and translating this information into strategies for diagnosis and therapy. This approach could lead to personalized medical treatments. Currently, our ability to understand human diseases at the molecular level is limited by the lack of molecular tools to identify and characterize the distinct molecular features of the disease state, especially for diseases such as cancer. Among the new tools being developed by researchers including chemists, engineers, and other scientists is a new class of nucleic acid probes called aptamers, which are ssDNA/RNA molecules selected to target a wide range of molecules and even cells. In this Account, we will focus on the use of aptamers, generated from cell-based selections, as a novel molecular tool for cancer research. Cancers originate from mutations of human genes. These genetic alterations result in molecular changes to diseased cells, which, in turn, lead to changes in cell morphology and physiology. For decades, clinicians have diagnosed cancers primarily based on the morphology of tumor cells or tissues. However, this method does not always give an accurate diagnosis and does not allow clinicians to effectively assess the complex molecular alterations that are predictive of cancer progression. As genomics and proteomics do not yet allow a full access to this molecular knowledge, aptamer probes represent one effective and practical avenue toward this goal. One special feature of aptamers is that we can isolate them by selection against cancer cells without prior knowledge of the number and arrangement of proteins on the cellular surface. These probes can identify molecular differences between normal and tumor cells and can discriminate among tumor cells of different classifications, at different disease stages, or from different patients. This Account summarizes our recent efforts to develop aptamers through cell-SELEX for the study of cancer and apply those aptamers in cancer diagnosis and therapy. We first discuss how we select aptamers against live cancer cells. We then describe uses of these aptamers. Aptamers can serve as agents for molecular profiling of specific cancer types. They can also be used to modify therapeutic reagents to develop targeted cancer therapies. Aptamers are also aiding the discovery of new cancer biomarkers through the recognition of membrane protein targets. Importantly, we demonstrate how molecular assemblies can integrate the properties of aptamers and, for example, nanoparticles or microfluidic devices, to improve cancer cell enrichment, detection and therapy.

Here we introduce a technology for biomarker discovery in which (i) DNA aptamers to biomarkers differentially expressed on the surfaces of cells being in different states are selected; (ii) aptamers are used to isolate biomarkers from the cells; and (iii) the isolated biomarkers are identified by means of mass spectrometry. The technology is termed aptamer-facilitated biomarker discovery (AptaBiD). AptaBiD was used to discover surface biomarkers that distinguish live mature and immature dendritic cells. We selected in vitro two DNA aptamer pools that specifically bind to mature and immature dendritic cells with a difference in strength of approximately 100 times. The aptamer pools were proven to be highly efficient in flow- and magnetic-bead-assisted separation of mature cells from immature cells. The two aptamer pools were then used to isolate biomarkers from the cells. The subsequent mass spectrometry analysis of the isolated proteins revealed unknown biomarkers of immature and mature dendritic cells.