Novel Aptamer-Based Small-Molecule Drug Screening Assay to Identify Potential Sclerostin Inhibitors against Osteoporosis.

A novel aptamer-based competitive drug screening platform for osteoporosis was devised in which fluorescence-labeled, sclerostin-specific aptamers compete with compounds from selected chemical libraries for the binding of immobilized recombinant human sclerostin to achieve high-throughput screening for potential small-molecule sclerostin inhibitors and to facilitate drug repurposing and drug discovery. Of the 96 selected inhibitors and FDA-approved drugs, six were shown to result in a significant decrease in the fluorescence intensity of the aptamer, suggesting a higher affinity toward sclerostin compared with that of the aptamer. The targets of these potential sclerostin inhibitors were correlated to lipid or bone metabolism, and several of the compounds have already been shown to be potential osteogenic activators, indicating that the aptamer-based competitive drug screening assay offered a potentially reliable strategy for the discovery of target-specific new drugs. The six potential sclerostin inhibitors suppressed the level of both intracellular and/or extracellular sclerostin in mouse osteocyte IDG-SW3 and increased alkaline phosphatase activity in IDG-SW3 cells, human bone marrow-derived mesenchymal stem cells and human fetal osteoblasts hFOB1.19. Potential small-molecule drug candidates obtained in this study are expected to provide new therapeutics for osteoporosis as well as insights into the structure-activity relationship of sclerostin inhibitors for rational drug design.

Target Affinity and Structural Analysis for a Selection of Norovirus Aptamers.

Aptamers, single-stranded oligonucleotides that specifically bind a molecule with high affinity, are used as ligands in analytical and therapeutic applications. For the foodborne pathogen norovirus, multiple aptamers exist but have not been thoroughly characterized. Consequently, there is little research on aptamer-mediated assay development. This study characterized seven previously described norovirus aptamers for target affinity, structure, and potential use in extraction and detection assays. Norovirus-aptamer affinities were determined by filter retention assays using norovirus genotype (G) I.1, GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney virus-like particles. Of the seven aptamers characterized, equilibrium dissociation constants for GI.7, GII.3, GII.4 New Orleans and GII.4 Sydney ranged from 71 ± 38 to 1777 ± 1021 nM. Four aptamers exhibited affinity to norovirus GII.4 strains; three aptamers additionally exhibited affinity toward GII.3 and GI.7. Aptamer affinity towards GI.1 was not observed. Aptamer structure analysis by circular dichroism (CD) spectroscopy showed that six aptamers exhibit B-DNA structure, and one aptamer displays parallel/antiparallel G-quadruplex hybrid structure. CD studies also showed that biotinylated aptamer structures were unchanged from non-biotinylated aptamers. Finally, norovirus aptamer assay feasibility was demonstrated in dot-blot and pull-down assays. This characterization of existing aptamers provides a knowledge base for future aptamer-based norovirus detection and extraction assay development and aptamer modification.

Advances in aptamer screening and aptasensors' detection of heavy metal ions.

Heavy metal pollution has become more and more serious with industrial development and resource exploitation. Because heavy metal ions are difficult to be biodegraded, they accumulate in the human body and cause serious threat to human health. However, the conventional methods to detect heavy metal ions are more strictly to the requirements by detection equipment, sample pretreatment, experimental environment, etc. Aptasensor has the advantages of strong specificity, high sensitivity and simple preparation to detect small molecules, which provides a new direction platform in the detection of heavy metal ions. This paper reviews the selection of aptamers as target for heavy metal ions since the 21th century and aptasensors application for detection of heavy metal ions that were reported in the past five years. Firstly, the selection methods for aptamers with high specificity and high affinity are introduced. Construction methods and research progress on sensor based aptamers as recognition element are also introduced systematically. Finally, the challenges and future opportunities of aptasensors in detecting heavy metal ions are discussed.

A human epidermal growth factor receptor 3/heregulin interaction inhibitor aptamer discovered using SELEX.

The interaction of human epidermal growth factor receptor 3 (HER3) and heregulin (HRG) is involved in resistance to human epidermal growth factor receptor 2 (HER2)-targeted cancer treatment, such as therapies using anti-HER2 monoclonal antibody. Therefore, inhibition of the HER3/HRG interaction is potentially valuable therapeutic target for cancer treatment. In this study, we used in vitro selection, also known as systematic evolution of ligands by exponential enrichment (SELEX) against the extracellular domain of human HER3, and discovered a novel RNA aptamer. Pull-down and bio-layer interferometry assays showed that RNA aptamer discovered specifically bound to HER3 with a dissociation constant (KD) of 700 nM. Pull-down assays using chemiluminescence detection also revealed that the HER3-binding RNA aptamer inhibited interactions between HER3 and human HRG. These results indicated that the novel HER3-binding RNA aptamer has potential to be used as basic tool in a range of applications involving HER3/HRG interactions, including research, therapeutic, and diagnostic applications.

Advances and Challenges in Small-Molecule DNA Aptamer Isolation, Characterization, and Sensor Development.

Aptamers are short oligonucleotides isolated in vitro from randomized libraries that can bind to specific molecules with high affinity, and offer a number of advantages relative to antibodies as biorecognition elements in biosensors. However, it remains difficult and labor-intensive to develop aptamer-based sensors for small-molecule detection. Here, we review the challenges and advances in the isolation and characterization of small-molecule-binding DNA aptamers and their use in sensors. First, we discuss in vitro methodologies for the isolation of aptamers, and provide guidance on selecting the appropriate strategy for generating aptamers with optimal binding properties for a given application. We next examine techniques for characterizing aptamer-target binding and structure. Afterwards, we discuss various small-molecule sensing platforms based on original or engineered aptamers, and their detection applications. Finally, we conclude with a general workflow to develop aptamer-based small-molecule sensors for real-world applications.

Terminal deoxynucleotidyl transferase-mediated formation of protein binding polynucleotides.

Terminal deoxynucleotidyl transferase (TdT) enzyme plays an integral part in the V(D)J recombination, allowing for the huge diversity in expression of immunoglobulins and T-cell receptors within lymphocytes, through their unique ability to incorporate single nucleotides into oligonucleotides without the need of a template. The role played by TdT in lymphocytes precursors found in early vertebrates is not known. In this paper, we demonstrated a new screening method that utilises TdT to form libraries of variable sized (vsDNA) libraries of polynucleotides that displayed binding towards protein targets. The extent of binding and size distribution of each vsDNA library towards their respective protein target can be controlled through the alteration of different reaction conditions such as time of reaction, nucleotide ratio and initiator concentration raising the possibility for the rational design of aptamers prior to screening. The new approach, allows for the screening of aptamers based on size as well as sequence in a single round, which minimises PCR bias. We converted the protein bound sequences to dsDNA using rapid amplification of variable ends assays (RAVE) and sequenced them using next generation sequencing. The resultant aptamers demonstrated low nanomolar binding and high selectivity towards their respective targets.

Cloning and Detection of Aptamer-Ribozyme Conjugations.

RNA aptamers can be used to target proteins or nucleic acids for therapeutic purposes and are candidates for RNA-mediated gene therapy. Like other small therapeutic RNAs, they can be expressed in cells from DNA templates that include a cellular promoter upstream of the RNA coding sequence. Secondary structures flanking aptamers can be used to enhance the activity or stability of these molecules. Notably, flanking self-cleaving ribozymes to remove extraneous nucleotides included during transcription as well as flanking hairpins to improve RNA stability have been used to increase the effect of therapeutic aptamers. Here we describe the cloning procedure of aptamers containing different flanking secondary structures and methods to compare their expression levels by a northern blot protocol optimized for the detection of small RNA molecules.

Application of Aptamer-Based Assays to the Diagnosis of Arboviruses Important for Public Health in Brazil.

Arbovirus infections represent a global public health problem, and recent epidemics of yellow fever, dengue, and Zika have shown their critical importance in Brazil and worldwide. Whilst a major effort for vaccination programs has been in the spotlight, a number of aptamer approaches have been proposed in a complementary manner, offering the possibility of differential diagnosis between these arboviruses, which often present similar clinical symptoms, as well as the potential for a treatment option when no other alternative is available. In this review, we aim to provide a background on arbovirus, with a basic description of the main viral classes and the disease they cause, using the Brazilian context to build a comprehensive understanding of their role on a global scale. Subsequently, we offer an exhaustive revision of the diagnostic and therapeutic approaches offered by aptamers against arboviruses. We demonstrate how these promising reagents could help in the clinical diagnosis of this group of viruses, their use in a range of diagnostic formats, from biosensors to serological testing, and we give a short review on the potential approaches for novel aptamer-based antiviral treatment options against different arboviral diseases.

Selection of High-Affinity RNA Aptamers That Distinguish between Doxycycline and Tetracycline.

Oligonucleotide aptamers are found in prokaryotes and eukaryotes, and they can be selected from large synthetic libraries to bind protein or small-molecule ligands with high affinities and specificities. Aptamers can function as biosensors, as protein recognition elements, and as components of riboswitches allowing ligand-dependent control of gene expression. One of the best studied laboratory-selected aptamers binds the antibiotic tetracycline, but it binds with a much lower affinity to the closely related but more bioavailable antibiotic doxycycline. Here we report enrichment of doxycycline binding aptamers from a selectively randomized library of tetracycline aptamer variants over four selection rounds. Selected aptamers distinguish between doxycycline, which they bind with dissociation constants of approximately 7 nM, and tetracycline, which they bind undetectably. They thus function as orthogonal complements to the original tetracycline aptamer. Unexpectedly, doxycycline aptamers adopt a conformation distinct from that of the tetracycline aptamer and depend on constant regions originally installed as primer binding sites. We show that the fluorescence emission intensity of doxycycline increases upon aptamer binding, permitting their use as biosensors. This new class of aptamers can be used in multiple contexts where doxycycline detection, or doxycycline-mediated regulation, is necessary.

Recent Progress in the Identification of Aptamers Against Bacterial Origins and Their Diagnostic Applications.

Aptamers have gained an increasing role as the molecular recognition element (MRE) in diagnostic assay development, since their first conception thirty years ago. The process to screen for nucleic acid-based binding elements (aptamers) was first described in 1990 by the Gold Laboratory. In the last three decades, many aptamers have been identified for a wide array of targets. In particular, the number of reports on investigating single-stranded DNA (ssDNA) aptamer applications in biosensing and diagnostic platforms have increased significantly in recent years. This review article summarizes the recent (2015 to 2020) progress of ssDNA aptamer research on bacteria, proteins, and lipids of bacterial origins that have implications for human infections. The basic process of aptamer selection, the principles of aptamer-based biosensors, and future perspectives will also be discussed.

RaptRanker: in silico RNA aptamer selection from HT-SELEX experiment based on local sequence and structure information.

Aptamers are short single-stranded RNA/DNA molecules that bind to specific target molecules. Aptamers with high binding-affinity and target specificity are identified using an in vitro procedure called high throughput systematic evolution of ligands by exponential enrichment (HT-SELEX). However, the development of aptamer affinity reagents takes a considerable amount of time and is costly because HT-SELEX produces a large dataset of candidate sequences, some of which have insufficient binding-affinity. Here, we present RNA aptamer Ranker (RaptRanker), a novel in silico method for identifying high binding-affinity aptamers from HT-SELEX data by scoring and ranking. RaptRanker analyzes HT-SELEX data by evaluating the nucleotide sequence and secondary structure simultaneously, and by ranking according to scores reflecting local structure and sequence frequencies. To evaluate the performance of RaptRanker, we performed two new HT-SELEX experiments, and evaluated binding affinities of a part of sequences that include aptamers with low binding-affinity. In both datasets, the performance of RaptRanker was superior to Frequency, Enrichment and MPBind. We also confirmed that the consideration of secondary structures is effective in HT-SELEX data analysis, and that RaptRanker successfully predicted the essential subsequence motifs in each identified sequence.

Rapid isolation of bacteria-specific aptamers with a non-SELEX-based method.

Usually, isolation of bacteria-specific aptamers by SELEX is a time-consuming process due to the required repeated rounds of binding, separation, and amplification of the probes to target bacteria. Here, we show that it is possible to isolate bacteria-specific DNA aptamers omitting the repeated rounds of binding incubation, separation, and amplification that are indispensable for SELEX. The serial removal of unbound DNAs to the bacterial cells from an initial mixture of bacteria and DNA libraries through serial centrifugation, one-time separation, and further one-time amplification of DNA bound to the target bacterial cells applied in this non-SELEX-based method allows successful aptamer isolation.

Nucleic Acid Aptamers as Emerging Tools for Diagnostics and Theranostics.

Aptamers are ssDNA or RNA sequences (20-80 nucleotides) generated in vitro by SELEX (Systematic Evolution of Ligands using EXponential enrichment) against diverse range of targets from small molecules to bacteria, viruses, and even eukaryotic cells. Aptamers, also known as chemical bodies, bind to their respective targets with tunable affinity and specificity, making aptamers as potent probes for diagnostics and excellent ligands for drug delivery in therapeutics. In this chapter, we have described the methods for generating DNA aptamers against proteins and their use in theranostics.

Aptamer Selection for Detecting Molecular Target Using Cell-SELEX (Systematic Evolution of Ligands by Exponential Enrichment) Technology.

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.

Co-SELECT reveals sequence non-specific contribution of DNA shape to transcription factor binding in vitro.

Understanding the principles of DNA binding by transcription factors (TFs) is of primary importance for studying gene regulation. Recently, several lines of evidence suggested that both DNA sequence and shape contribute to TF binding. However, the following compelling question is yet to be considered: in the absence of any sequence similarity to the binding motif, can DNA shape still increase binding probability? To address this challenge, we developed Co-SELECT, a computational approach to analyze the results of in vitro HT-SELEX experiments for TF-DNA binding. Specifically, Co-SELECT leverages the presence of motif-free sequences in late HT-SELEX rounds and their enrichment in weak binders allows Co-SELECT to detect an evidence for the role of DNA shape features in TF binding. Our approach revealed that, even in the absence of the sequence motif, TFs have propensity to bind to DNA molecules of the shape consistent with the motif specific binding. This provides the first direct evidence that shape features that accompany the preferred sequence motifs also bestow an advantage for weak, sequence non-specific binding.

A Novel TNF-α-Targeting Aptamer for TNF-α-Mediated Acute Lung Injury and Acute Liver Failure.

Rationale: The TNF-α pathway plays as a double-edged sword that simultaneously regulates cell apoptosis and proliferation. The dysregulated TNF-α signaling can trigger cytokine storms that lead to profound cell death during the phase of acute tissue injury. On the other hand, an optimal level of TNF-α signaling is required for tissue repair following the acute injury phase. The TNF-α pathway is commonly upregulated in acute lung injury (ALI) and acute liver failure (ALF). Previous studies investigated the feasibility of adopting protein-based TNF-α blockers as disease modifiers in ALI and ALF, but none of these came out with a positive result. One of the potential reasons that resides behind the failure of the trials might be the long half-life of these inhibitors that led to undesired side effects. Developing alternative TNF-α blockers with manageable half-lives remain an unmet need in this regard. Methods: In the current study, we developed a novel TNF-α-targeting aptamer (aptTNF-α) and its PEG-derivate (aptTNF-α-PEG) with antagonistic functions. We investigated the in vivo antagonistic effects using mouse ALI and ALF models. Results: Our data showed that aptTNF-α possessed good in vitro binding affinity towards human/mouse TNF-α and successfully targeted TNF-α in vivo. In the mouse ALI model, aptTNF-α/aptTNF-α-PEG treatment attenuated the severity of LPS-induced ALI, as indicated by the improvement of oxygen saturation and lung injury scores, the reduction of protein-rich fluid leakage and neutrophil infiltration in the alveolar spaces, and the suppression of pro-inflammatory cytokines/chemokines expressions in the lung tissues. In the mouse ALF model, we further showed that aptTNF-α/aptTNF-α-PEG treatment not only attenuated the degree of hepatocyte damage upon acute injury but also potentiated early regeneration of the liver tissues. Conclusion: The results implicated potential roles of aptTNF-α/aptTNF-α-PEG in ALI and ALF. The data also suggested their translational potential as a new category of TNF-α blocking agent.

High Enrichment of Nucleobase-modified Aptamers in Early Selection Rounds by Microbeads-assisted Capillary Electrophoresis SELEX.

Nucleobase-modified aptamers are attractive candidates for diagnostic and therapeutic agents due to the high affinity, stability and functionality. However, since even conventional SELEX requires many selection rounds, acquisition of modified aptamers is much more laborious. Herein, microbeads-assisted capillary electrophoresis (MACE)-SELEX was applied against thrombin using the indole-modified DNA library. After only three selection rounds, we successfully enriched the modified aptamers and they showed slower off-rate than reported aptamers, suggesting MACE-SELEX is a promising approach for rapid identification of modified aptamers.

DNA-Nanoscaffold-Assisted Selection of Femtomolar Bivalent Human α-Thrombin Aptamers with Potent Anticoagulant Activity.

Multivalent aptamers that interact with their target proteins through multiple sites exhibit much stronger binding strengths than their monovalent counterparts. In this work, we have designed a single-stranded DNA (ssDNA) library (1015 molecules, each 145 nt) based on a predefined DNA nanostructure designed to present two random-loop sites for bivalent aptamer evolution. From this library, a group of ultra-strong bivalent aptamers against human α-thrombin (with apparent KD values of ≈340 fm) were easily identified through a simple seven-round conventional systematic evolution of ligands by exponential enrichment (SELEX) procedure. The dominant bivalent aptamers consist of two components, one binding to exosite I and the other to exosite II. The best of these bivalent aptamers show strong allosteric attenuation of the thrombin cleavage activity and also display an extremely potent anticoagulation effect in human plasma, demonstrating their great potential in therapeutic applications. The method developed here can easily be adapted to conventional SELEX techniques, opening a new route for fast selection of multivalent aptamers with superior binding affinity for other targets.

In vitro isolation of class-specific oligonucleotide-based small-molecule receptors.

Class-specific bioreceptors are highly desirable for recognizing structurally similar small molecules, but the generation of such affinity elements has proven challenging. We here develop a novel 'parallel-and-serial' selection strategy for isolating class-specific oligonucleotide-based receptors (aptamers) in vitro. This strategy first entails parallel selection to selectively enrich cross-reactive binding sequences, followed by serial selection that enriches aptamers binding to a designated target family. As a demonstration, we isolate a class-specific DNA aptamer against a family of designer drugs known as synthetic cathinones. The aptamer binds to 12 diverse synthetic cathinones with nanomolar affinity and does not respond to 11 structurally similar non-target compounds, some of which differ from the cathinone targets by a single atom. This is the first account of an aptamer exhibiting a combination of broad target cross-reactivity, high affinity and remarkable specificity. Leveraging the qualities of this aptamer, instantaneous colorimetric detection of synthetic cathinones at nanomolar concentrations in biological samples is achieved. Our findings significantly expand the binding capabilities of aptamers as class-specific bioreceptors and further demonstrate the power of rationally designed selection strategies for isolating customized aptamers with desired binding profiles. We believe that our aptamer isolation approach can be broadly applied to isolate class-specific aptamers for various small molecule families.

Anti-Idiotype DNA Aptamer Affinity Purification⁻High-Temperature Reversed-Phase Liquid Chromatography: A Simple, Accurate, and Selective Bioanalysis of Bevacizumab.

This study presents a simple, accurate, and selective bioanalytical method of bevacizumab detection from plasma samples based on aptamer affinity purification⁻high-temperature reversed-phased liquid chromatography (HT-RPLC) with fluorescence detection. Bevacizumab in plasma samples was purified using magnetic beads immobilized with an anti-idiotype DNA aptamer for bevacizumab. The purified bevacizumab was separated with HT-RPLC and detected with its native fluorescence. Using aptamer affinity beads, bevacizumab was selectively purified and detected as a single peak in the chromatogram. HT-RPLC achieved good separation for bevacizumab with a sharp peak within 10 min. The calibration curves of the two monoclonal antibodies ranged from 1 to 50 µg/mL and showed good correlation coefficients (r² > 0.999). The limit of detection (LOD) and lower limit of quantification (LLOQ) values for bevacizumab were 0.15 and 0.51 µg/mL, respectively. The proposed method was successfully applied to the bioanalysis of the plasma samples obtained from the patients with lung cancer and may be extended to plan optimal therapeutic programs and for the evaluation of biological equivalencies in the development of biosimilars.