Properties Of Aptamers Research Articles

New Insights into Aptamers: An Alternative to Antibodies in the Detection of Molecular Biomarkers.

Aptamers are short oligonucleotides with single-stranded regions or peptides that recently started to transform the field of diagnostics. Their unique ability to bind to specific target molecules with high affinity and specificity is at least comparable to many traditional biorecognition elements. Aptamers are synthetically produced, with a compact size that facilitates deeper tissue penetration and improved cellular targeting. Furthermore, they can be easily modified with various labels or functional groups, tailoring them for diverse applications. Even more uniquely, aptamers can be regenerated after use, making aptasensors a cost-effective and sustainable alternative compared to disposable biosensors. This review delves into the inherent properties of aptamers that make them advantageous in established diagnostic methods. Furthermore, we will examine some of the limitations of aptamers, such as the need to engage in bioinformatics procedures in order to understand the relationship between the structure of the aptamer and its binding abilities. The objective is to develop a targeted design for specific targets. We analyse the process of aptamer selection and design by exploring the current landscape of aptamer utilisation across various industries. Here, we illuminate the potential advantages and applications of aptamers in a range of diagnostic techniques, with a specific focus on quartz crystal microbalance (QCM) aptasensors and their integration into the well-established ELISA method. This review serves as a comprehensive resource, summarising the latest knowledge and applications of aptamers, particularly highlighting their potential to revolutionise diagnostic approaches.

RNA Condensate as a Versatile Platform for Improving Fluorogenic RNA Aptamer Properties and Cell Imaging.

Fluorogenic RNA aptamers are valuable tools for cell imaging, but they still suffer from shortcomings such as easy degradation, limited photostability, and low fluorescence enhancement. Molecular crowding conditions enable the stabilization of the structure, promotion of folding, and improvement of activity of functional RNA. Based on artificial RNA condensates, here we present a versatile platform to improve fluorogenic RNA aptamer properties and develop sensors for target analyte imaging in living cells. Using the CUG repeat as a general tag to drive phase separation, various fluorogenic aptamer-based RNA condensates (FLARE) were prepared. We show that the molecular crowding of FLARE can improve the enzymatic resistance, thermostability, photostability, and binding affinity of fluorogenic RNA aptamers. Moreover, the FLARE systems can be modularly engineered into sensors (FLARES), which demonstrate enhanced brightness and sensitivity compared to free sensors dispersed in homogeneous solution. This scalable design principle provides new insights into RNA aptamer property regulation and cellular imaging.

Aptamer Clicked Poly(ferrocenylsilanes) at Au Nanoparticles as Platforms with Multiple Function[†

Aptamers are widely used in biosensing due to their specific sensitivity toward many targets. Thus, gold nanoparticle (AuNP) aptasensors are subject to intense research due to the complementary properties of aptamers as sensing elements and AuNPs as transducers. We present herein a novel method for the functional coupling of thrombin-specific aptamers to AuNPs via an anionic, redox-active poly(ferrocenylsilane) (PFS) polyelectroyte. The polymer acts as a co-reductant and stabilizer for the AuNPs, provides grafting sites for the aptamer, and can be used as a redox sensing element, making the aptamer-PFS-AuNP composite (aptamer-AuNP) a promising model system for future multifunctional sensors. The aptamer-AuNPs exhibit excellent colloidal stability in high ionic strength environments owing to the combined electrosteric stabilizing effects of the aptamer and the PFS. The synthesis of each assembly element is described, and the colloidal stability and redox responsiveness are studied. As an example to illustrate applications, we present results for thrombin sensitivity and specificity using the specific aptamer.

A background correctable fluorescence sensing strategy for adenosine triphosphate evaluation based on aptamer configuration alteration

Ratiometric sensors with background correction are expected to get around the interference from complicated systems. However, there is currently a scarcity of viable carriers for quickly and easily assembling fluorophores with varying fluorescence emission. In this work, we developed a novel aptamer-based ratiometric fluorescence sensor for analysis of adenosine triphosphate (ATP) based on the properties of aptamer that enable conformational transformations upon binding to the target. The carbon dots (CDs) and thioflavin T (ThT) are employed as the reference and detection signal for the ratio-dependent sensor, which are both integrated with the aptamer. The green fluorescence of ThT was initially turn-on after binding with aptamer and then quenched in the presence of ATP, whereas the red fluorescence of CDs remained practically steady. This approach exhibits remarkable selectivity and can detect ATP in the range of 0.5–60 μM with a detection limit of 0.167 μM. Moreover, the method was successfully applied to the determination of ATP in human serum as well as in A549 cells, demonstrating the potential for medical diagnostic applications.

CRISPR/Cas12a-powered CLASA towards OTA ultrasensitive detection in cereal samples

Ochratoxin A (OTA) is classified as a Class 2B carcinogens and poses a significant risk bto the liver, kidney and other organs, potentially leading to carcinogenic and teratogenic effects. Accurate monitoring of OTA content in food is crucial for safeguarding human and animal health. Here, a novel approach called the CRISPR/Cas12a enzyme-linked aptamer-sorbent assay (CLASA) was developed, which integrates the high-performance trans-cleavage ability of CRISPR/Cas12a with the cost-effective and high-affinity properties of aptamer for ultra-sensitive OTA detection. In this strategy, we incubated aptamers on microwell plates, and used the fluorescence intensity changes caused by OTA and crRNA competitively bind OTA-specific aptamer to achieve the quantitative detection. Our sensitive CLASA method not only solves the problems of poor repeatability and relatively low sensitivity of fluorescence based-CRISPR/Cas12a reaction in solution, but also solves the defects of expensive antibody and false positive result in traditional ELISA method, achieving a limit of detection (LOD) as low as 171 fg·mL−1 with a broad linear range from 1 pg·mL−1 to 1000 pg·mL−1. The presented CLASA approach was validated successfully by using commercial ELISA kit on rice and oat samples as well. Furthermore, by strategically modifying the sequences of aptamer and crRNA, our CLASA platform can be flexibly customized to detect a wide range of small molecular targets. This adaptability allows for the extension of our framework beyond nucleic acid substances, enabling the detection of non-nucleic acid compounds using the CRISPR-Cas12a system. As a result, our CLASA approach holds great promise as a versatile sensing kit with applications in various fields requiring sensitive and specific detection of diverse analytes.

Enhanced molecular dynamic simulation studies unravel long-range effects caused by sequence variations and partner binding in RNA aptamers

Intrinsic flexibility and structural modularity are two common features of RNA molecules. Although functionally crucial, RNA plasticity often represents a major complication in high-resolution structural studies. To overcome this problem, RNAs may be rigidified through the complexation with high-affinity partners such as Fab molecules. This approach has been previously used to characterize the DIR2-aptamer. However, possible perturbations induced by the insertion of the Fab binding site on the DIR2-aptamer conformational properties were not investigated. Here, using enhanced molecular dynamics simulations, we compared the dynamics of the DIR2 aptamer holding the Fab binding site with that of the parental sequence. Our results suggest that the L2-loop modification for the Fab recognition leads to a significant increase in local flexibility that also affects the mobility of distant regions. The trajectories provide clear indications of the groups and the interactions mediating the dynamics transfer in DIR2. The effectiveness of our approach in addressing RNA flexibility was further corroborated by showing its ability to reproduce the most important events affecting the NF-κB RNA aptamer upon dissociation from the partner. Therefore, REMD analyses, a rarely adopted technique to unravel the structural/dynamical properties of aptamers, could efficiently complement experimental data guiding the rational design of nucleic acid therapeutics.

Paper-Based Aptasensors: Working Principles, Detection Modes, and Applications.

Aptamers are short oligonucleotides designed to possess high binding affinity towards specific target compounds (ions, molecules, or cells). Due to their function and unique advantages, aptamers are considered viable alternatives to antibodies as biorecognition elements in bioassays and biosensors. On the other hand, paper-based devices (PADs) have emerged as a promising and powerful technology for the fabrication of low-cost analytical tools, mainly intended for on-site and point-of-care applications. The present work aims to provide a comprehensive overview of paper-based aptasensors. The review describes the fabrication methods and working principles of paper-based devices, the properties of aptamers as bioreceptors, the different modes of detection used in conjunction with aptasensing PADs, and representative applications for the detection of ions, small molecules, proteins, and cells. The future challenges and prospects of these devices are also discussed.

Current Trends in the Use of Semiconducting Materials for Electrochemical Aptasensing

Aptamers are synthetic single-stranded oligonucleotides that exhibit selective binding properties to specific targets, thereby providing a powerful basis for the development of selective and sensitive (bio)chemical assays. Electrochemical biosensors utilizing aptamers as biological recognition elements, namely aptasensors, are at the forefront of current research. They exploit the combination of the unique properties of aptamers with the advantages of electrochemical detection with the view to fabricate inexpensive and portable analytical platforms for rapid detection in point-of-care (POC) applications or for on-site monitoring. The immobilization of aptamers on suitable substrates is of paramount importance in order to preserve their functionality and optimize the sensors’ sensitivity. This work describes different immobilization strategies for aptamers on the surface of semiconductor-based working electrodes, including metal oxides, conductive polymers, and carbon allotropes. These are presented as platforms with tunable band gaps and various surface morphologies for the preparation of low cost, highly versatile aptasensor devices in analytical chemistry. A survey of the current literature is provided, discussing each analytical method. Future trends are outlined which envisage aptamer-based biosensing using semiconductors.

Terminal Alkyne-Modified DNA Aptamers with Enhanced Protein Binding Affinities.

Nucleic acid-based receptors, known as aptamers, are relatively fast to discover and manufacture but lack the diverse functional groups of protein receptors (e.g., antibodies). The binding properties of DNA aptamers can be enhanced by attaching abiotic functional groups; for example, aromatic groups such as naphthalene slow dissociation from proteins. Although the terminal alkyne is a π-electron-rich functional group that has been used in small molecule drugs to enhance binding to proteins through noncovalent interactions, it remains unexplored for enhancing DNA aptamer binding affinity. Here, we demonstrate the utility of the terminal alkyne for improving the binding of DNA to proteins. We prepared a library of 256 terminal-alkyne-bearing variants of HD22, a DNA aptamer that binds the protein thrombin with nanomolar affinity. After a one-step thrombin-binding selection, a high-affinity aptamer containing two alkynes was discovered, exhibiting 3.2-fold tighter thrombin binding than the corresponding unmodified sequence. The tighter binding was attributable to a slower rate of dissociation from thrombin (5.2-fold slower than HD22). Molecular dynamics simulations with enhanced sampling by Replica Exchange with Solute Tempering (REST2) suggest that the π-electron-rich alkyne interacts with an asparagine side chain N-H group on thrombin, forming a noncovalent interaction that stabilizes the aptamer-protein interface. Overall, this work represents the first case of terminal alkynes enhancing the binding properties of an aptamer and underscores the utility of the terminal alkyne as an atom economical π-electron-rich functional group to enhance binding affinity with minimal steric perturbation.

Study of the influence of pH on the conformation of a modified aptamer to nucleolin protein

The search for agents for targeted delivery of anticancer drugs remains a crucial challenge for medicine. For this reason, DNA aptamers targeting cancer cell-specific proteins have medical use. At the same time, the additional fine-tuning of aptamer properties to lower affinity of aptamers for target cells that are not surrounding cancerous tissues makes the aptamers promising tools in clinical applications due to reduced immunogenicity and fewer side effects. One of the approaches is to use acidity disruption in cancer cells and tissues. In this work, aptamer AS1411 was modified to increase affinity for nucleolin by attaching a nucleotide sequence to its binding site to create a pH-sensitive linker. UV melting and fluorescence methods were employed to demonstrate that the conformation of the new aptamer depends on pH of the medium.

Aptamer‐based extracellular vesicle isolation, analysis and therapeutics

AbstractExtracellular vesicles (EVs) play an important role in many physiological processes. Thus, EV analysis has a great value for the understanding of mechanisms underlying disease progress or diagnosis, prognosis and therapy. The overlapped physical and immune properties between EVs and events in body fluids, as well as the phenotypic heterogeneity of EVs, require efficient isolation and analysis methods. The unique properties of aptamers, such as facile modification and programmability, make them easily assembled as powerful platforms for EV isolation and analysis. EVs can also be used as vehicles for drug delivery, benefiting from the properties of homing ability, hypo‐immunogenicity, and strong tolerance. The affinity recognition ability to targets and the feature of single stranded DNA of aptamers make them useful in promoting the targetability of EVs and delivery of nucleic acid drugs. This review summarizes recent progress in aptamer‐based EV isolation, analysis, and aptamer‐functionalized EVs for therapeutics.

Chemically modified aptamers for improving binding affinity to the target proteins via enhanced non-covalent bonding.

Nucleic acid aptamers are ssDNA or ssRNA fragments that specifically recognize targets. However, the pharmacodynamic properties of natural aptamers consisting of 4 naturally occurring nucleosides (A, G, C, T/U) are generally restricted for inferior binding affinity than the cognate antibodies. The development of high-affinity modification strategies has attracted extensive attention in aptamer applications. Chemically modified aptamers with stable three-dimensional shapes can tightly interact with the target proteins via enhanced non-covalent bonding, possibly resulting in hundreds of affinity enhancements. This review overviewed high-affinity modification strategies used in aptamers, including nucleobase modifications, fluorine modifications (2'-fluoro nucleic acid, 2'-fluoro arabino nucleic acid, 2',2'-difluoro nucleic acid), structural alteration modifications (locked nucleic acid, unlocked nucleic acid), phosphate modifications (phosphorothioates, phosphorodithioates), and extended alphabets. The review emphasized how these high-affinity modifications function in effect as the interactions with target proteins, thereby refining the pharmacodynamic properties of aptamers.

Selection of optimised ligands by fluorescence-activated bead sorting.

The chemistry of aptamers is largely limited to natural nucleotides, and although modifications of nucleic acids can enhance target aptamer affinity, there has not yet been a technology for selecting the right modifications in the right locations out of the vast number of possibilities, because enzymatic amplification does not transmit sequence-specific modification information. Here we show the first method for the selection of specific nucleoside modifications that increase aptamer binding efficacy, using the oncoprotein EGFR as a model target. Using fluorescence-activated bead sorting (FABS), we have successfully selected optimized aptamers from a library of >65 000 variations. Hits were identified by tandem mass spectrometry and validated by using an EGFR binding assay and computational docking studies. Our results provide proof of concept for this novel strategy for the selection of chemically optimised aptamers and offer a new method for rapidly synthesising and screening large aptamer libraries to accelerate diagnostic and drug discovery.

The Therapeutic Applications Of Aptamers And Aptamer-Conjugated Nanoparticles In Cancer, Inflammatory And Viral Diseases

Health care modalities that are more predictable and effective are the result of advances in early diagnosis and precise treatment options. Aptamers are three-dimensional structures made of nucleic acids that selectively bind to a target site. In the creation of aptamer-based drug delivery systems, the physicochemical properties of aptamers, their conjugation with nanoparticles (NPs) in theranostics, and their internalization have been found to be of interest.As a result, our goal was to provide an overview of the structure and production of aptamers, followed by the benefits of aptamer-conjugated nanoparticles and their applications as theranostics in oncology, inflammatory, and viral diseases. After that, a number of reports on aptamer internalization strategies, the efficiency of aptamers in comparison to their analogs, and the implications of aptamers for clinical trials were discussed. Last but not least, we talked about the problems that actively targeted aptamers face right now and what the future holds for their use in medicine. In conclusion, aptamer-based therapeutic platform development in clinical trials may benefit greatly from this review.

Therapeutic Potential of Aptamer-Protein Interactions.

Aptamers are single-stranded oligonucleotides (RNA or DNA) with a typical length between 25 and 100 nucleotides which fold into three-dimensional structures capable of binding to target molecules. Specific aptamers can be isolated against a large variety of targets through efficient and relatively cheap methods, and they demonstrate target-binding affinities that sometimes surpass those of antibodies. Consequently, interest in aptamers has surged over the past three decades, and their application has shown promise in advancing knowledge in target analysis, designing therapeutic interventions, and bioengineering. With emphasis on their therapeutic applications, aptamers are emerging as a new innovative class of therapeutic agents with promising biochemical and biological properties. Aptamers have the potential of providing a feasible alternative to antibody- and small-molecule-based therapeutics given their binding specificity, stability, low toxicity, and apparent non-immunogenicity. This Review examines the general properties of aptamers and aptamer-protein interactions that help to understand their binding characteristics and make them important therapeutic candidates.

Photoelectrochemical aptasensors for detection of viruses.

Photoelectrochemistry (PEC) is a dynamic discipline studying the effect of light on photoelectrode or photosensitive material, and the conversion from solar energy into electrical power. The basic PEC process refers to the oxidation or reduction reactions between electrochemical active species in solution and photoactive materials that occurred at the electrode/electrolyte interface during illumination. In recent years, the PEC biosensing approaches have also been developed by the combination of the PEC technique with bioanalysis, where the interaction between biological recognition element and analyte influences a photocurrent signal. This involves the charge and energy transfer of PEC reaction between electron donor/acceptor and photoactive material upon light irradiation. Coupling the advantages of PEC bioanalysis and aptamers has provided new concepts for highly selective and sensitive biosensors development, applicable in human health monitoring and environmental protection. In a typical assay, a photoactive material converts the affinity binding properties of aptamers into a detectable electrical signal, presenting an innovative method for probing numerous aptamer–analyte interactions. Using different aptamer probes aiming for specific purposes, more sensing strategies with rational design and exquisite signaling mechanisms have been proposed. This review concentrated on the current topic of PEC aptasensors that are used for the detection of viruses. The prospects in this area are also discussed.Graphical abstract

A review on the therapeutic applications of aptamers and aptamer-conjugated nanoparticles in cancer, inflammatory and viral diseases

The advancement in early diagnosis and precise treatments options result in more predictable and powerful health care modalities. Aptamers are known as nucleic acid structures with three-dimensional conformation to selectively bind a target site. Physicochemical properties of aptamers, their conjugation with nanoparticles (NPs) in theranostics applications and their internalization have been found to be of interest in development of aptamer-based drug delivery systems. Therefore, we aimed to present an overview on the structure and generation of aptamers followed by advantages of aptamers-conjugated NPs and their theranostics applications in various diseases such as oncology, inflammatory diseases and viral diseases. Afterward, we discussed several reports on the internalization approaches of aptamers, efficiency of aptamers vs. their analogous, and implications of aptamers in clinical trials. Finally, we discussed the current challenges and future perspectives of actively targeted aptamers for clinical application. In conclusion, this review may hold a great promise for development of aptamer-based therapeutic platforms in clinical trials.

Aptamers-Diagnostic and Therapeutic Solution in SARS-CoV-2.

The SARS-CoV-2 virus is currently the most serious challenge to global public health. Its emergence has severely disrupted the functioning of health services and the economic and social situation worldwide. Therefore, new diagnostic and therapeutic tools are urgently needed to allow for the early detection of the SARS-CoV-2 virus and appropriate treatment, which is crucial for the effective control of the COVID-19 disease. The ideal solution seems to be the use of aptamers—short fragments of nucleic acids, DNA or RNA—that can bind selected proteins with high specificity and affinity. They can be used in methods that base the reading of the test result on fluorescence phenomena, chemiluminescence, and electrochemical changes. Exploiting the properties of aptamers will enable the introduction of rapid, sensitive, specific, and low-cost tests for the routine diagnosis of SARS-CoV-2. Aptamers are excellent candidates for the development of point-of-care diagnostic devices and are potential therapeutic tools for the treatment of COVID-19. They can effectively block coronavirus activity in multiple fields by binding viral proteins and acting as carriers of therapeutic substances. In this review, we present recent developments in the design of various types of aptasensors to detect and treat the SARS-CoV-2 infection.

Highly performing graphene-based field effect transistor for the differentiation between mild-moderate-severe myocardial injury

Cardiovascular diseases result in millions of deaths around the globe, many of which could have been avoided if identified at an early stage. Preventive cardiovascular disease diagnostics plays a vital role reducing the critical fatality rate by allowing to take timely necessary precautions. Here, we demonstrate the exceptional properties of aptamer modified graphene-based FET (GFET) for cardiac troponin I (cTnI) specific sensing in different bodily fluids. Concentration-dependent measurements made with cardiac troponin I (cTnI) on an aptamer/polyethylene glycol (PEG) modified GFET exhibited real-time detection of cTnI from 1 to 400 pg mL−1 in 0.01 × phosphate-buffered saline (PBS) solution. The sensor performance is within the clinical important window of 10 pg mL−1 to 500 pg mL−1, allowing the differentiation between healthy, and people with low and high risk for myocardial infraction (AMI). Even more important is the very reproducible shift and neglectable leakage current of the individual IDSVGS-curves upon contact with cTnI solutions. To further evaluate the applicability of the developed point of care device for troponin I sensing in serum, samples of 15 patients with different troponin levels were analysed for their troponin I levels. The 15 patients were grouped according to the magnitude of peri-operative myocardial injury as assessed by the clinical hs-cTnT assay into mild (cTnT<15 pg mL−1), moderate (15 pg mL−1>c-TnT<500 mL−1) and severe (cTnT>500 pg mL−1), cases. The GFET sensor allowed to categorise all the 15 samples correctly and corresponding to 3 troponin zones (respectively, low, medium and high). While indeed a larger patient sample collection is required for clinical assessments, these data seem highly promising.

Aptamer Sandwich Assay for the Detection of SARS-CoV-2 Spike Protein Antigen.

The novel severe acute respiratory syndrome coronavirus (SARS-CoV-2) emerged at the end of 2019, resulting in the ongoing COVID-19 pandemic. The high transmissibility of the virus and the substantial number of asymptomatic individuals have led to an exponential rise in infections worldwide, urgently requiring global containment strategies. Reverse transcription-polymerase chain reaction is the gold standard for the detection of SARS-CoV-2 infections. Antigen tests, targeting the spike (S) or nucleocapsid (N) viral proteins, are considered as complementary tools. Despite their shortcomings in terms of sensitivity and specificity, antigen tests could be deployed for the detection of potentially contagious individuals with high viral loads. In this work, we sought to develop a sandwich aptamer-based assay for the detection of the S protein of SARS-CoV-2. A detailed study on the binding properties of aptamers to the receptor-binding domain of the S protein in search of aptamer pairs forming a sandwich is presented. Screening of aptamer pairs and optimization of assay conditions led to the development of a laboratory-based sandwich assay able to detect 21 ng/mL (270 pM) of the protein with negligible cross-reactivity with the other known human coronaviruses. The detection of 375 pg of the protein in viral transport medium demonstrates the compatibility of the assay with clinical specimens. Finally, successful detection of the S antigen in nasopharyngeal swab samples collected from suspected patients further establishes the suitability of the assay for screening purposes as a complementary tool to assist in the control of the pandemic.