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PubMed2003

Microscale separation and analysis.

There is a recent and growing interest in microscale separation and analysis, a result of advantages of miniaturization such as rapid separation times, high performance and throughput, reduced costs, and the possibility of system integration and multiplexing. Adopting the concepts of conventional capillary electrophoresis, capillary electrochromatography, micellar electrokinetic chromatography and various sample preparation techniques to microchip format, in conjunction with the integration of different analysis steps into a monolithic system, have opened new levels in performance, functionality and throughput. This review summarizes the recent advances in the field of microfabricated separation devices for genomics, proteomics and high-throughput screening applications, also addressing system integration and micropreparative functionalities.

DNA↗

PubMed2005

High-throughput RNA interference strategies for target discovery and validation by using synthetic short interfering RNAs: functional genomics investigations of biological pathways.

During the past five years, RNA interference (RNAi) has emerged as arguably the best functional genomics tool available to date, providing direct, causal links between individual genes and loss-of-function phenotypes through robust, broadly applicable, and readily upscalable methodologies. Originally applied experimentally in C. elegans and Drosophila, RNAi is now widely used in mammalian cell systems also. The development of commercially available libraries of short interfering RNAs (siRNAs) and other RNAi silencing reagents targeting entire classes of human genes provide the opportunity to carry out genome-scale screens to discover and characterize gene functions directly in human cells. A key challenge of these studies, also faced by earlier genomics or proteomics approaches, resides in reaching an optimal balance between the necessarily high throughput and the desire to achieve the same level of detailed analysis that is routine in conventional small-scale studies. This chapter discusses technical aspects of how to perform such screens, what parameters to monitor, and which readouts to apply. Examples of homogenous assays and multiplexed high-content microscopy-based screens are demonstrated.

Apoptosis↗

PubMed2004

Applications of eTag trade mark assay platform to systems biology approaches in molecular oncology and toxicology studies.

We have developed a universal eTag trade mark multiplex assay platform that can be uniquely applied to survey the molecule profiles of biologic systems in sub-global large-scale analyses. The effectiveness of eTag trade mark assays when applied to focused system biology studies in molecular oncology and predictive toxicology is herein described while reviewing the current methods commonly used. The multi-analyte and multi-parameter assay approach for parallel analysis will form the basis of an emerging paradigm of multiplexed molecular profiling for signaling pathway networks and various aspects of drug development processes.

Gene Expression Profiling↗

PubMed2006

Cytokine assay of the epidural space lavage in patients with lumbar intervertebral disk herniation and radiculopathy.

INTRODUCTION: Lumbar disk herniation may result in a radiculopathic pattern of symptoms. Consideration for a primary biochemical inducement of pain over a mechanical mechanism is a contemporary topic of spinal research. However, the exact pathomechanism by which a degenerative intervertebral disk leads to neural inflammation and pain has not been determined. Using modern techniques of chemical analysis, biochemical markers can be identified which participate in the degenerative cascade, and possibly with the onset of pain. The purpose of this research is to identify potential biochemical markers through a novel technique of epidural space lavage that may be helpful in understanding the pathogeneses of pain in the presence of intervertebral disk degeneration and herniation. METHODS: Fifty consecutive patients with acute radiculopathy secondary to a symptomatic herniated lumbar intervertebral disk or spinal stenosis, and who were indicated for epidural steroid injection were identified. Additionally, 3 volunteers with no history of back pain or radiculopathy volunteered to undergo epidural lavage. After needle insertion, a lavage followed by fluid aspiration of the epidural space at the level of the disc herniation, in the case of the symptomatic patients, was performed using normal saline, before the instillation of corticosteroids. The fluid samples were frozen at -20 degrees C until analysis. A biochemical evaluation for a battery of cytokines was undertaken (IL-1beta, IL-1ra, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p40, IL-13, IL-15, IL-17, TNF-alpha, IFN-alpha, IFN-gamma, GM-CSF, MIP-1alpha, MIP-1beta, IP-10, MIG, Eotaxin, RANTES, and MCP-1, and neuropeptides) using high-resolution multiplex bead immunoassays and enzyme-linked immunosorbent assay (ELISA). Additionally, polyacrylamide gel electrophoresis was carried out to verify the presence of serum proteins. RESULTS: Despite the presence of amino acids/serum proteins in the epidural lavage fluid, none of the aforementioned mediators were isolated in a quantifiable concentration using the ELISA techniques with >5 pg/mL resolution. DISCUSSION: The current proteomics array technology was not able to detect critical levels of biochemical markers present in the epidural space through the mentioned lavage technique. This lack of detection could be due to the absence of the factors in this environment or the inability of the technique to obtain or detect factors which may be present. CONCLUSION: Although a novel approach, the current study was unable to identify the presence of a series of inflammatory peptides in the epidural lavage of patients with symptomatic radicular pain due to herniated disc disease. We recommend alternative experimental designs than the one we pursued for definitively identifying potential sources of pain generators.

Acute Disease↗

PubMed2002

Mass spectrometry in coupling with affinity capture-release and isotope-coded affinity tags for quantitative protein analysis.

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) and isotope-coded affinity tags (ICAT) are two recently introduced techniques for the quantitation of protein activity and content with applications to clinical enzymology and functional proteomics, respectively. One common feature of these methods is that they use biotinylated tags that function as molecular handles for highly selective and reversible affinity capture of conjugates from complex biological mixtures such as cell homogenates and sub-cellular organelles. ACESIMS uses synthetic substrate conjugates specifically to target cellular enzymes that, when deficient, are the cause of genetic diseases. Multiplex determination of enzyme activities is used for the diagnosis of lysosomal storage diseases. The ICAT method relies on selective conjugation of cysteine thiol groups in proteins, followed by enzymatic digestion and quantitative analysis of peptide conjugates by mass spectrometry. Another common feature of the ACESIMS and ICAT approaches is that both use conjugates labeled with stable heavy isotopes as internal standards for quantitation. Selected applications of the ACESIMS and ICAT techniques are presented that include molecular-level diagnosis of genetic diseases in children and quantitative determination of protein expression in cells.

Affinity Labels↗

PubMed2025

Comprehensive proteomic and pathological profiling identifies PRAS40 as a novel biomarker and mediator of primary immune checkpoint blockade resistance in non-small cell lung cancer.

BACKGROUND: Immune checkpoint blockade (ICB) has revolutionized the treatment landscape of non-small cell lung cancer (NSCLC), yet primary resistance remains a significant clinical challenge. Recent evidence implicates PRAS40 (AKT1S1) in regulating cellular survival and immune responses, but its role in immunotherapy resistance is not fully understood. METHODS: Transcriptomic data from TCGA and GTEx cohorts were analyzed to assess PRAS40 expression. Prognostic value was evaluated using Cox regression. Immune microenvironment features were characterized with CIBERSORT and TIMER. Predictive efficacy for ICB response was examined using TIDE and IPS. Plasma PRAS40 levels in 66 NSCLC patients receiving ICB were quantified by proximity extension assay (PEA), and multiplex immunohistochemistry assessed associations among PRAS40, PD-L1, and CD8+ T cells in tumor tissues. RESULTS: High PRAS40 expression was associated with poor prognosis, reduced CD8+ T cell infiltration, and downregulation of immune checkpoint genes. Elevated circulating PRAS40 predicted primary ICB resistance and shorter progression-free survival, independent of PD-L1 or CD8+ T cell status. CONCLUSION: PRAS40 is strongly associated with primary ICB resistance in NSCLC and may serve as a novel predictive biomarker. These findings support its potential to guide personalized immunotherapy in lung cancer.

Humans↗

PubMed2001

High-throughput genomic and proteomic analysis using microarray technology.

BACKGROUND: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication. METHODS: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000-24,000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel. RESULTS: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82-0.99 (depending on analyte) compared with ELISAs. CONCLUSIONS: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.

Arylamine N-Acetyltransferase↗

PubMed2005

Subnanoliter enzymatic assays on microarrays.

Many areas of research today are based on enzymatic assays most of which are still performed as enzyme-linked immunosorbent assays in microtiter plates. The demand for highly parallel screening of thousands of samples eventually led to a miniaturization and automation of these assays. However, the final transfer of enzymatic assays from a microtiter-based technology to microarrays has proven to be difficult for various reasons, such as the inability to maintain unbound reaction products on the spot of reaction or the missing capability of multiplexing. Here, we have conducted multiplex enzymatic assays in subnanoliter volumes on a single microarray using the multiple spotting technology. We were able to measure enzymatic activity with a sensitivity down to 35 enzyme molecules, applying only conventional flat microarray surfaces and standard microarray hardware. We have performed assays of inhibition and applied this format for the detection of prognostic markers, such as cathepsin D. The new approach allows the rapid and multiplex screening of thousands of samples on a single microarray with applications in drug screening, metagenomics, and high-throughput enzyme assays.

Alkaline Phosphatase↗

PubMed2003

Profiling the global tyrosine phosphorylation state.

Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Functional profiling of the tyrosine phosphoproteome is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel molecular diagnostic approaches. The ultimate aim of current mass spectrometry-based phosphoproteomic approaches is the comprehensive characterization of the phosphoproteome. However, current methods are not yet sensitive enough for routine detection of a large percentage of tyrosine-phosphorylated proteins, which are generally of low abundance. In this article, we discuss alternative methods that exploit Src homology 2 (SH2) domains for profiling the tyrosine phosphoproteome. SH2 domains are small protein modules that bind specifically to tyrosine-phosphorylated peptides; there are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of tyrosine-phosphorylated ligands. These domains play a critical role in the propagation of signals in the cell, mediating the relocalization and complex formation of proteins in response to changes in tyrosine phosphorylation. We have developed an SH2 profiling method based on far-Western blotting, in which a battery of SH2 domains is used to probe the global state of tyrosine phosphorylation. Application to the classification of human malignancies suggests that this approach has potential as a molecular diagnostic tool. We also describe ongoing efforts to modify and improve SH2 profiling, including the development of a multiplexed assay system that will allow high-throughput functional profiling of the tyrosine phosphoproteome.

Blotting, Far-Western↗

PubMed2006

Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioisotope detection.

The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR- tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR- mammary tumor specimens. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4-9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels ( approximately 3.6 microg protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR- providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.

Antineoplastic Agents, Hormonal↗

PubMed2005

Concanavalin A-captured glycoproteins in healthy human urine.

Both the urinary proteome and its glycoproteome can reflect human health status, and more directly, functions of kidney and urinary tracts. Because the high abundance protein albumin is not N-glycosylated, the urine N-glycoprotein enrichment procedure could deplete it, and urine proteome could thus provide a more detailed protein profile in addition to glycosylation information especially when albuminuria occurs in some kidney diseases. In terms of describing the details of urinary proteins, the urine glycoproteome is even a better choice than the proteome itself. Pooled urine samples from healthy volunteers were collected and acetone-precipitated for proteins. N-Linked glycoproteins enriched with concanavalin A affinity purification were separated and analyzed by SDS-PAGE-reverse phase LC/MS/MS or two-dimensional LC/MS/MS. A total of 225 urinary proteins were identified based on two-hit criteria with reliability over 97% for each peptide. Among these proteins, 94 were identified in previous urine proteome works, 150 were annotated as glycoproteins in Swiss-Prot, and 43 were predicted as glycoproteins by NetNGlyc 1.0. A number of known biomarkers and disease-related glycoproteins were identified. Because changes in protein quantity or the glycosylation status can lead to changes in the concanavalin A-captured glycoprotein profile, specific urine glycoproteome patterns might be observed for specific pathological conditions as multiplex urinary biomarkers. Knowledge of the urine glycoproteome is important in understanding kidney and body function.

Concanavalin A↗

PubMed2024

Integrative Multi-PTM Proteomics Reveals Dynamic Global, Redox, Phosphorylation, and Acetylation Regulation in Cytokine-Treated Pancreatic Beta Cells.

Studying regulation of protein function at a systems level necessitates an understanding of the interplay among diverse posttranslational modifications (PTMs). A variety of proteomics sample processing workflows are currently used to study specific PTMs but rarely characterize multiple types of PTMs from the same sample inputs. Method incompatibilities and laborious sample preparation steps complicate large-scale physiological investigations and can lead to variations in results. The single-pot, solid-phase-enhanced sample preparation (SP3) method for sample cleanup is compatible with different lysis buffers and amenable to automation, making it attractive for high-throughput multi-PTM profiling. Herein, we describe an integrative SP3 workflow for multiplexed quantification of protein abundance, cysteine thiol oxidation, phosphorylation, and acetylation. The broad applicability of this approach is demonstrated using cell and tissue samples, and its utility for studying interacting regulatory networks is highlighted in a time-course experiment of cytokine-treated β-cells. We observed a swift response in the global regulation of protein abundances consistent with rapid activation of JAK-STAT and NF-κB signaling pathways. Regulators of these pathways as well as proteins involved in their target processes displayed multi-PTM dynamics indicative of complex cellular response stages: acute, adaptation, and chronic (prolonged stress). PARP14, a negative regulator of JAK-STAT, had multiple colocalized PTMs that may be involved in intraprotein regulatory crosstalk. Our workflow provides a high-throughput platform that can profile multi-PTMomes from the same sample set, which is valuable in unraveling the functional roles of PTMs and their co-regulation.

Proteomics↗

PubMed1999

Laser-assisted microdissection: applications in molecular pathology.

Tissue microdissection is potentially one of the most useful techniques in molecular pathology. Laser-assisted microdissection has been developed to procure precisely the cells of interest in a tissue specimen, in a rapid and practical manner. Together with multiplex molecular approaches, it is now feasible to study genetic alterations and isolate genes and proteins in defined cell populations from complex normal and diseased tissues. The fundamental advantage of this technique is the possibility of capturing single cells from which high-quality DNA and mRNA can be isolated for analysis of sequence and quantitation of expression. Moreover, the integration of laser-assisted microdissection and proteomic analysis could identify novel protein markers for disease. The advent of laser-assisted microdissection is likely to have a profound impact on molecular pathology.

Cell Separation↗

PubMed2025

The FIBRILLIN multigene family in tomato, their roles in plastoglobuli structure and metabolism.

Plastoglobuli (PG) are plant lipoprotein compartments, present in plastid organelles. They are involved in the formation and/or storage of lipophilic metabolites. FIBRILLINs (FBNs) are one of the main PG-associated proteins and are particularly abundant in carotenoid-enriched chromoplasts found in ripe fruits and flowers. To address the contribution of different FBNs, independently and in combination, to isoprenoid formation and sequestration, a multiplex gene editing approach was undertaken in tomato. This approach generated a suite of single and high-order fbn mutants that were shown to lack transcripts and respective protein products. The major PG-related FBNs in tomato chosen for this study are SlFBN1, SlFBN2a, SlFBN4 and SlFBN7a. When knocked out independently, functional redundancy was revealed. However, paralog-specific roles were detected regulating specific isoprenoids (e.g. plastochromanol 8) or plastidial esterification capability. In addition, high-order fbn mutants displayed altered isoprenoid chromoplast sequestration patterns, notably with a significant reduction in carotenes (phytoene and phytofluene) in the PG fraction. Proteomic analysis confirmed the absence of PG-core associated proteins, including NAD(P)H-ubiquinone oxidoreductase C1, tocopherol cyclase (VTE1) and phytol esterase (PES1/PYP). Perturbations to the ultrastructure of the plastid were revealed, with aberrant PG formation and morphology predominating in high-order mutants. Global lipidome profiles also highlighted broader changes directly affecting storage and plastid membrane lipids, for example, tri- and diacylglycerides and galactolipid species. Collectively, these results support both structural and metabolic roles of SlFBNs in PGs. The findings expose fundamental aspects of metabolic compartmentalisation in plant cells and the importance of lipoprotein particles for plastid integrity and functionality.

Solanum lycopersicum↗

PubMed2004

Development of a sensitive microarray immunoassay and comparison with standard enzyme-linked immunoassay for cytokine analysis.

Cytokine and cytokine inhibitors represent important components of the inflammatory response in patients with trauma, shock, and sepsis. Many investigators wish to quantify cytokines and it would be advantageous to measure multiple cytokines in a multiplex manner to obtain an inflammatory profile rather than a single value. Using the well-accepted standard enzyme-linked immunoassay (ELISA) as a basis, a microarray immunoassay (MI) was designed to measure 16 different human cytokines simultaneously. The MI was performed by spotting antibodies on nitrocellulose pads affixed to glass slides. Detection of the mediators was performed with biotin-conjugated antibodies followed by fluorescently labeled streptavidin. All antibodies and other reagents were purchased commercially. The MI achieved a lower limit of detection that was generally similar to traditional ELISAs (approximately 4-12 pg/mL) and also had a similar coefficient of variation. In the multiplexed MI, there was no cross reactivity between mediators. To verify the utility of the MI, cytokines and cytokine inhibitors were measured in endotoxin stimulated human blood by both ELISA and MI. Virtually identical cytokine concentrations were measured by both methods. These results describe the development of a sensitive, specific and cost-effective multiplexed microarray immunoassay that produces values similar to traditional ELISAs.

Antibodies↗

PubMedDate not supplied

Dissection of proteolytic 18O labeling: endoprotease-catalyzed 16O-to-18O exchange of truncated peptide substrates.

Proteolytic labeling in H2(18)O has been recently revived as a versatile method for proteomics research. To understand the molecular basis of the labeling process, we have dissected the process into two separate events: cleavage of the peptide amide bonds and exchange of the terminal carboxyl oxygens. It was demonstrated that both carboxyl oxygens can be catalytically labeled, independent of the cleavage step. Reaction kinetics of the tryptic 16O-to-18O exchange of YGGFMR, YGGFMK, and the tryptic digest of apomyoglobin were studied by matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry. A larger KM for the Lys-peptide (4400 +/- 700 microM), when compared to that of the Arg-peptide (KM 1300 +/- 300 microM), was mainly responsible for the slower reaction with YGGFMK (kcat/KM 0.64 +/- 0.14 microM(-1)min(-1)) compared to YGGFMR (kcat/KM 2.6 +/- 0.9 microM(-1)min(-1)). Multiplexed kinetic studies showed that endoprotease-catalyzed oxygen exchange is a general phenomenon, allowing homogeneous 18O2-coding of a variety of peptides. It was demonstrated for the first time that chymotrypsin 18O2-codes peptides during proteolysis. On the basis of the analyses reported here, we propose that proteolytic 18O labeling can be advantageously decoupled from protein digestion, and endoproteases can be used in a separate step to 18O2-code peptides for comparative studies after proteolysis has taken place.

Animals↗

PubMed2026

Spatial proteomics reveals four-stage molecular evolution in cancer immunotherapy-related gastritis.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have transformed cancer treatment, yet immune-related adverse events (irAEs) including immunotherapy-related gastritis (IRAEG) pose significant clinical challenges-often necessitating treatment interruption that may compromise antitumor efficacy. IRAEG presents with atypical symptoms, lacks specific biomarkers, and shows histopathological overlap with other forms of gastritis, complicating diagnosis and management. Despite increasing clinical recognition, a systematic understanding of spatial molecular alterations across the full disease course remains limited. Here, we used spatial proteomics to map the molecular landscape of IRAEG during disease progression and to define stage-specific patterns of molecular evolution relevant to cancer immunotherapy management. METHODS: We analyzed tissue samples from seven patients, including four non-immunotherapy-related gastritis controls and three cancer patients who developed IRAEG following ICI therapy for solid tumors, sampled longitudinally across four disease stages: baseline (G1), acute severe inflammation (G2), early recovery (G3), and complete recovery (G4). Using laser capture microdissection coupled with data-independent acquisition mass spectrometry, we profiled 177 spatially resolved gastric tissue regions. Multiplex immunohistochemistry and immunofluorescence characterized features of the immune microenvironment, while Gene Ontology, KEGG pathway analysis, Gene Set Variation Analysis, and xCell inference enabled functional, metabolic, and immune profiling. Key immune and NET-related findings were further validated by multiplex immunofluorescence in an independent, expanded cohort of IRAEG and non-immunotherapy-related gastritis samples. RESULTS: IRAEG was characterized by widespread HLA molecule activation and enhanced antigen processing, resembling the immune phenotype observed in organ transplant rejection. The acute G2 stage exhibited excessive neutrophil extracellular trap formation, profound metabolic suppression, and collapse of immune homeostasis-features that may inform early intervention strategies to preserve ICI treatment continuity. During early recovery (G3), inflammatory injury transitioned toward repair, marked by activation of fatty acid metabolism and PPAR signaling. Notably, even at complete clinical recovery (G4), more than 1,000 proteins remained differentially expressed, reflecting sustained enhancement of metabolic and immune functions and establishing a distinct molecular "memory" state with implications for ICI rechallenge decisions. CONCLUSIONS: These findings define four molecularly distinct stages of IRAEG progression and recovery. The stage-specific signatures identified here serve as candidate biomarkers for diagnosis, disease staging, and therapeutic response assessment, and may guide clinical decisions regarding irAE management, treatment modification, and safe ICI rechallenge to support continued antitumor therapy.

Humans↗

PubMed2005

Production and processing of aptamer microarrays.

Aptamers are nucleic acid species that are selected in vitro for their specific binding properties. We describe methods for the production and processing of aptamer microarrays, including detailed procedures for the high-throughput, enzymatic synthesis of 5' RNA biotinylated aptamers and for arraying them onto streptavidin-coated glass slides. Also presented are methods for processing the aptamer microarrays, including blocking, washing, drying, and scanning. Examples are shown for the specific capture of fluorescently labeled target proteins either alone in binding buffer or in competition with labeled intracellular proteins from cell lysates. Consideration is given to the challenges involved in producing multiplex aptamer chips composed of aptamers taken from disparate literature sources, and to the development of standardized methods for characterizing the performance of capture reagents used in biosensors.

Aptamers, Nucleotide↗