Search Results (57)

Fluorescence Anisotropy (FA) is a sensitive and efficient technique for quantifying biomolecular interactions, offering advantages such as minimal sample requirements and elimination of separation of bound from unbound species. Thus, it is well suited for aptamer–protein binding affinity studies. However, accurately determining equilibrium dissociation constants (K_D) in FA requires low concentrations of fluorescently labeled aptamers to prevent ligand depletion. A significant challenge arises at low aptamer concentrations due to an unexpected and physically nonmeaningful increase in apparent anisotropy, which impairs accurate data fitting. This anomalous increase in apparent anisotropy may arise from non-specific adsorption of aptamers to surfaces. In this study, we investigated the use of non-ionic surfactants to mitigate these effects and stabilize the anisotropy signal at low aptamer concentrations using the thrombin aptamer as a model system. We evaluated the impact of varying concentrations of two surfactants (Tween 20 and Triton X-100) on plots of anisotropy as a function of aptamer concentration and determined aptamer–protein binding affinities. Addition of 0.1% Tween 20 corrects the anomalous increase in anisotropy at low aptamer concentrations, enabling the use of aptamer concentrations as low as 5 nM in binding assays. Triton X-100 was less effective. By incorporating optimized concentrations of Tween 20, we demonstrated improved assay reproducibility and accuracy in K_D determination, expanding the dynamic range of usable aptamer concentrations in FA-based binding affinity studies. Similar benefits were observed with the clinically relevant aptamer s10yh2 and human serum albumin. These findings provide a practical strategy for enhancing the robustness of FA measurements and may be applicable to other aptamer–target systems and high-throughput assay formats. Full article

The use of DNA as a scaffold for nanoclusters is particularly interesting due to its structural versatility and easy integration with aptamers. In their structure, aptamers often contain non-canonical forms of DNA, i.e., G-quadruplexes (GQs). Four-stranded GQs are used to construct nanomachines and biosensors for monitoring changes in the concentration of potassium ions. In the present study, we continue our work related to the synthesis of silver nanoclusters formed on a bifunctional DNA template. By attaching a cytosine-rich domain (C12) to a G-quadruplex-forming sequence—human telomeric (Tel22) or thrombin-binding aptamer (TBA)—we constructed bifunctional templates for fluorescent silver nanoclusters (C12) with the ability to detect potassium ions (GQs). The changing localization of the C12 domain from the 3′ to 5′ end of the oligonucleotide was a successful way to improve the fluorescence properties of the obtained fluorescent probes. The best performance as a probe for potassium ions was exhibited by C12Tel22-AgNCs, with an LOD of 0.68 mM in PBS. The introduction of the fluorescent cytosine analog tC leads to an LOD of 0.68 mM in PBS and 0.46 mM in Tris-acetate. Additionally, we performed AFM, TEM, DLS analysis, and cellular studies to further investigate the structural properties and behavior of the Tel22C12-AgNCs in biological contexts. Full article

Since lead can cause severe effects on living organisms’ health and life, the regular monitoring of Pb levels in water and soil is of particular significance. Recently, it was shown that lead ions can also be detected using affinity-based biosensors, namely, using aptamers as recognition elements. In most cases, thrombin binding aptamer (TBA) was utilized; however, there are more examples of DNA aptamers which could also serve that purpose. Herein, we present studies on the electrochemical detection of lead ions using PS2M aptamer, which contains several guanine nucleotides, as the receptor element. Firstly, the method of aptamer-based layer fabrication was optimized along with the choice of a redox active indicator, which was a source of current signal. The experiments revealed the possibility of lead ion detection from 50 to 600 nM, which covers the range below and above the maximum accepted limit stated by US EPA (72 nM). Moreover, the sensing layer exhibited high selectivity towards lead ions and was successfully applied both for the analysis of tap water spiked with Pb²⁺ ions and as a miniaturized sensor. Finally, stability and regeneration studies on the aptamer-based receptor layer were executed to confirm the utility of the elaborated tool. Full article

Thrombin binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers that efficiently binds to thrombin, resulting in anticoagulant effects. TBA also possesses promising antiproliferative properties. As with most therapeutic oligonucleotides, chemical modifications are critical for therapeutic applications, particularly to improve thermodynamic stability, resistance in biological environment, and target affinity. To evaluate the effects of nucleobase and/or sugar moiety chemical modifications, five TBA analogues have been designed and synthesized considering that the chair-like G4 structure is crucial for biological activity. Their structural and biological properties have been investigated by Circular Dichroism (CD), Nuclear Magnetic Resonance (NMR), native polyacrylamide gel electrophoresis (PAGE) techniques, and PT and MTT assays. The analogue TBAB contains 8-bromo-2′-deoxyguanosine (B) in G-syn glycosidic positions, while TBAL and TBAM contain locked nucleic acid guanosine (L) or 2′-O-methylguanosine (M) in G-anti positions, respectively. Instead, both the two types of modifications have been introduced in TBABL and TBABM with the aim of obtaining synergistic effects. In fact, both derivatives include B in syn positions, exhibiting in turn L and M in the anti ones. The most appealing results have been obtained for TBABM, which revealed an interesting cytotoxic activity against breast and prostate cancer cell lines, while in the case of TBAB, extraordinary thermal stability (T_m approximately 30 °C higher than that of TBA) and an anticoagulant activity higher than original aptamer were observed, as expected. These data indicate TBAB as the best TBA anticoagulant analogue here investigated and TBABM as a promising antiproliferative derivative. Full article

Biosensors play an important role in numerous research fields. Quartz crystal microbalances with dissipation monitoring (QCM-Ds) are sensitive devices, and binding events can be observed in real-time. In combination with aptamers, they have great potential for selective and label-free detection of various targets. In this study, an alternative surface functionalization for a QCM-D-based aptasensor was developed, which mimics an artificial cell membrane and thus creates a physiologically close environment for the binding of the target to the sensor. Vesicle spreading was used to form a supported lipid bilayer (SLB) of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphethanolamine-N-(cap biotinyl) (biotin-PE). The SLB was then coated with streptavidin followed by applying a biotinylated aptamer against thrombin. SLB formation was investigated in terms of temperature and composition. Temperatures of 25 °C and below led to incomplete SLB formation, whereas a full bilayer was built at higher temperatures. We observed only a small influence of the content of biotinylated lipids in the mixture on the further binding of streptavidin. The functionalization of the sensor surface with the thrombin aptamer and the subsequent thrombin binding were investigated at different concentrations. The sensor could be reconstituted by incubation with a 5 M urea solution, which resulted in the release of the thrombin from the sensor surface. Thereafter, it was possible to rebind thrombin. Thrombin in spiked samples of human serum was successfully detected. The developed system can be easily applied to other target analytes using the desired aptamers. Full article

Liquid biopsy is expected to become widespread in the coming years thanks to point of care devices, which can include label-free biosensors. The surface functionalization of biosensors is a crucial aspect that influences their overall performance, resulting in the accurate, sensitive, and specific detection of target molecules. Here, the surface of a microring resonator (MRR)-based biosensor was functionalized for the detection of protein biomarkers. Among the several existing functionalization methods, a strategy based on aptamers and mercaptosilanes was selected as the most highly performing approach. All steps of the functionalization protocol were carefully characterized and optimized to obtain a suitable protocol to be transferred to the final biosensor. The functionalization protocol comprised a preliminary plasma treatment aimed at cleaning and activating the surface for the subsequent silanization step. Different plasma treatments as well as different silanes were tested in order to covalently bind aptamers specific to different biomarker targets, i.e., C-reactive protein, SARS-CoV-2 spike protein, and thrombin. Argon plasma and 1% v/v mercaptosilane were found as the most suitable for obtaining a homogeneous layer apt to aptamer conjugation. The aptamer concentration and time for immobilization were optimized, resulting in 1 µM and 3 h, respectively. A final passivation step based on mercaptohexanol was also implemented. The functionalization protocol was then evaluated for the detection of thrombin with a photonic biosensor based on microring resonators. The preliminary results identified the successful recognition of the correct target as well as some limitations of the developed protocol in real measurement conditions. Full article

The high affinity of the biotin–streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin–streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer–protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag–streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin–streptavidin interaction while increasing throughput and improving assay efficiency. Full article

Thrombin-binding aptamer (TBA) is one of the best-known G-quadruplex (G4)-forming aptamers. By adopting its peculiar chair-like G4 structure, TBA can efficiently bind to thrombin, thus producing an anticoagulant effect. The major limit to its therapeutic application is represented by its poor thermal and biological resistance. Therefore, numerous research studies have been focused on the design of TBA analogues with chemical modifications to improve its pharmacokinetic and pharmacodynamic properties. To maintain the functional recognition to protein surface on which TBA anticoagulant activity depends, it is essential to preserve the canonical antiparallel topology of the TBA quadruplex core. In this paper, we have designed three TBA variants with modified G-tetrads to evaluate the effects of nucleobase and sugar moiety chemical modifications on biological properties of TBA, preserving its chair-like G-quadruplex structure. All derivatives contain 8-bromo-2′-deoxyguanosine (G^Br) in syn positions, while in the anti-positions, locked nucleic acid guanosine (G^LNA) in the analogue TBABL, 2’-O-methylguanosine (G^OMe) in TBABM, and 2’-F-riboguanosine (G^F) in TBABF is present. CD (Circular Dichroism), CD melting, 1H-NMR (Nuclear Magnetic Resonance), and non-denaturing PAGE (Polyacrylamide Gel Electrophoresis), nuclease stability, prothrombin time (PT) and fibrinogen-clotting assays have been performed to investigate the structural and biological properties of these TBA analogues. The most interesting results have been obtained with TBABF, which revealed extraordinary thermal stability (T_m approximately 40 °C higher than that of TBA), anticoagulant activity almost doubled compared to the original aptamer, and, above all, a never-observed resistance to nucleases, as 50% of its G4 species was still present in 50% FBS at 24 h. These data indicate TBABF as one of the best TBA analogue ever designed and investigated, to the best of our knowledge, overcoming the main limitations to therapeutic applications of this aptamer. Full article

The surface functionalisation of self-assembled colloidal quantum dot supraparticle lasers with a thrombin binding aptamer (TBA-15) has been demonstrated. The self-assembly of CdSSe/ZnS alloyed core/shell microsphere-shape CQD supraparticles emitting at 630 nm was carried out using an oil-in-water emulsion technique, yielding microspheres with an oleic acid surface and an average diameter of 7.3 ± 5.3 µm. Surface modification of the microspheres was achieved through a ligand exchange with mercaptopropionic acid and the subsequent attachment of TBA-15 using EDC/NHS coupling, confirmed by zeta potential and Fourier transform IR spectroscopy. Lasing functionality between 627 nm and 635 nm was retained post-functionalisation, with oleic acid- and TBA-coated microspheres exhibiting laser oscillation with thresholds as low as 4.10 ± 0.37 mJ·cm⁻² and 7.23 ± 0.78 mJ·cm⁻², respectively. Full article

Modification of DNA aptamers is aimed at increasing their thermodynamic stability, and improving affinity and resistance to biodegradation. G-quadruplex DNA aptamers are a family of affinity ligands that form non-canonical DNA assemblies based on a G-tetrads stack. Modification of the quadruplex core is challenging since it can cause complete loss of affinity of the aptamer. On the other hand, increased thermodynamic stability could be a worthy reward. In the current paper, we developed new three- and four-layer modified analogues of the thrombin binding aptamer with high thermal stability, which retain anticoagulant activity against alpha-thrombin. In the modified aptamers, one or two G-tetrads contained non-natural anti-preferred alpha-deoxyguanosines at specific positions. The use of this nucleotide analogue made it possible to control the topology of the modified structures. Due to the presence of non-natural tetrads, we observed some decrease in the anticoagulant activity of the modified aptamers compared to the natural prototype. This negative effect was completely compensated by conjugation of the aptamers with optimized tripeptide sequences. Full article

The complexity of genetic circuits has not seen a significant increase over the last decades, even with the rapid development of synthetic biology tools. One of the bottlenecks is the limited number of orthogonal transcription factor–operator pairs. Researchers have tried to use aptamer–ligand pairs as genetic parts to regulate transcription. However, most aptamers selected using traditional methods cannot be directly applied in gene circuits for transcriptional regulation. To that end, we report a new method called CIVT-SELEX to select DNA aptamers that can not only bind to macromolecule ligands but also undergo significant conformational changes, thus affecting transcription. The single-stranded DNA library with affinity to our example ligand human thrombin protein is first selected and enriched. Then, these ssDNAs are inserted into a genetic circuit and tested in the in vitro transcription screening to obtain the ones with significant inhibitory effects on downstream gene transcription when thrombins are present. These aptamer–thrombin pairs can inhibit the transcription of downstream genes, demonstrating the feasibility and robustness of their use as genetic parts in both linear DNAs and plasmids. We believe that this method can be applied to select aptamers of any target ligands and vastly expand the genetic part library for transcriptional regulation. Full article

In this paper, we study the biological properties of two TBA analogs containing one and two extra G-tetrads, namely TBAG3 and TBAG4, respectively, and two further derivatives in which one of the small loops at the bottom (TBAG41S) or the large loop at the top (TBAG4GS) of the TBAG4 structure has been completely modified by replacing all loop residues with abasic site mimics. The therapeutical development of the TBA was hindered by its low thermodynamic and nuclease stability, while its potential as an anticancer/antiproliferative molecule is also affected by the anticoagulant activity, being a side effect in this case. In order to obtain suitable TBA analogs and to explore the involvement of specific aptamer regions in biological activity, the antiproliferative capability against DU 145 and MDAMB 231 cancer cell lines (MTT), the anticoagulant properties (PT), the biological degradability (nuclease stability assay) and nucleolin (NCL) binding ability (SPR) of the above described TBA derivatives have been tested. Interestingly, none of the TBA analogs exhibits an anticoagulant activity, while all of them show antiproliferative properties to the same extent. Furthermore, TBAG4 displays extraordinary nuclease stability and promising antiproliferative properties against breast cancer cells binding NCL efficiently. These results expand the range of G4-structures targeting NCL and the possibility of developing novel anticancer and antiviral drugs. Full article

In recent years, the classic geometries of sensors based on surface plasmon resonance (SPR) have been adapted for use in optical fibers (both extrinsic and intrinsic configurations), thus providing a simple approach to low-cost plasmonic sensing. For instance, polymer optical fibers (POFs) are particularly advantageous due to their excellent flexibility, ease of manipulation, great numerical aperture, large diameter and, last but not least, the fact that plastic can withstand smaller bend radii than glass. In bio-chemical applications, a very specific medium (receptor layer) for the selective binding of the considered analyte is deposited on a gold layer of the SPR platform. A simple and low-cost experimental setup, consisting of a halogen lamp and a spectrometer, can be arranged to measure the light spectrum transmitted through the SPR-POF sensors. Interesting applications have been devised and successfully implemented by exploiting these low-cost plasmonic POF platforms combined with different receptors, such as molecularly imprinted polymers (MIPs), chemical receptors, and bio-receptors (aptamers and antibodies). For example, by exploiting SPR in a D-shaped POF probe with different receptors, interesting results have been achieved in medical diagnostics for cancer bio-markers detection, the monitoring of antigens in celiac disease, L-nicotine detection, thrombin detection, and SARS-CoV-2 virus and pancreatic amylase detection. A survey of these medical applications is presented, highlighting the advantages and limitations of each application and revealing possible future implementations of the platform as a point-of-care device. Full article

A major obstacle to the therapeutic application of an aptamer is its susceptibility to nuclease digestion. Here, we confirmed the acquisition of relative nuclease resistance of a DNA-type thrombin binding aptamer with a warhead (TBA₃) by covalent binding to a target protein in the presence of serum/various nucleases. When the thrombin-inhibitory activity of TBA₃ on thrombin was reversed by the addition of the complementary strand, the aptamer was instantly degraded by the nucleases, showing that the properly folded/bound aptamer conferred the resistance. Covalently binding aptamers possessing both a prolonged drug effect and relative nuclease resistance would be beneficial for in vivo translational applications. Full article

An impedance technique-based aptasensor for the detection of thrombin was developed using a single-walled carbon nanotube (SWCNT)-modified screen-printed carbon electrode (SPCE). In this work, a thrombin-binding aptamer (TBA) as probe was used for the determination of thrombin, and that was immobilized on SWCNT through π–π interaction. In the presence of thrombin, the TBA on SWCNT binds with target thrombin, and the amount of TBA on the SWCNT surface decreases. The detachment of TBA from SWCNT will be affected by the concentration of thrombin and the remaining TBA on the SWCNT surface can be monitored by electrochemical methods. The TBA-modified SWCNT/SPCE sensing layer was characterized by cyclic voltammetry (CV). For the measurement of thrombin, the change in charge-transfer resistance (R_ct) of the sensing interface was investigated using electrochemical impedance spectroscopy (EIS) with a target thrombin and [Fe(CN)₆]³⁻ as redox maker. Upon incubation with thrombin, a decrease of R_ct change was observed due to the decrease in the repulsive interaction between the redox marker and the electrode surface without any label. A plot of R_ct changes vs. the logarithm of thrombin concentration provides the linear detection ranges from 0.1 nM to 1 µM, with a ~0.02 nM detection limit. Full article