Aptamer Scaffold Research Articles

Versatile MOF@COF catalyzed and magnetically multivalent aptamer assisted biosensing platform for rapid and ultrasensitive dual-mode detection of multidrug-resistant bacteria

Given the frequent occurrence of multidrug-resistant bacteria (MDRB), which pose a severe threat towards public hygiene, it is important to construct facile and sensitive strategies for the detection of MDRB. We developed a fluorescent/colorimetric dual-mode biosensing platform for the analysis of MDRB by integrating the remarkable peroxidase-like activity of boric acid-decorated fluorescent framework hybrid (MOF@COF) nanozyme and enhanced bacterial affinity of DNA programmable multivalent aptamer scaffold. The efficiency and sensitivity of the proposed analysis approach were dramatically increased due to richer aptamers and high-performance MOF@COF nanozyme. Compared with commonly used single recognition methods, multivalent aptamers and bacteria-affinity boric acid groups on the nanozyme surface could identify multiple sites of MDRBs to improve site utilization for ultrasensitive biosensing. The platform realized selective detection of Acinetobacter baumannii (AB) and Pseudomonas aeruginosa (PA) in two modes with consistent results. In colorimetry and fluorescence modes, the linear ranges of AB were 102-108 and 10–108 CFU/mL with corresponding detection limits of 12 and 2 CFU/mL, and the linear ranges of PA were 102-108 and 10–108 CFU/mL with corresponding detection limits of 10 and 3 CFU/mL, respectively. More importantly, the biosensing platform could be applied for MDRB quantification in biological specimens, including human serum, cerebrospinal fluid, and urine with remarkable recoveries (96.8–105.0%) in both modes, indicating the bright prospect of the biosensing platform for MDRB determination in clinical diagnosis.

Polyvalent aptamer scaffold coordinating light-responsive oxidase-like nanozyme for sensitive detection of zearalenone

An efficient aptasensor was developed for the colorimetric determination of zearalenone (ZEN) based on polyvalent aptamer scaffold and light-responsive oxidase-like nanozyme. The sensitivity and efficiency of the development method were significantly improved owing to rich aptamers and signal labels (3, 4-dihydroxybenzoic acid, PCA) packed in the scaffold. The scaffold integrated functions of target recognition, surface immobilization and signal transduction. The photoresponsive nanoenzyme of TiO2-PCA was formed by PCA coordinated with Ti (IV) on the surface of TiO2. TiO2-PCA catalyzed dissolved oxygen rather than H2O2 to generate colorimetric signal by stimulating the chromogenic substrate, which made the assay greener and safer. The detection limit of colorimetric mode was 0.0087 ng/mL and the satisfactory recoveries 92.00 %−111.00 % were achieved in spiked food samples. This strategy opens new horizons for sensitive detection of small molecule hazards and promises to be a powerful tool for safeguarding food safety.

Rational Design of Trident Aptamer Scaffold for Rapid and Accurate Monitoring of 25-Hydroxyvitamin D3 Metabolism in Living Cells.

The level of 25-hydroxyvitamin D3 [25(OH)VD3] in human blood is considered as the best indicator of vitamin D status, and its deficiency or excess can lead to various health problems. Current methods for monitoring 25(OH)VD3 metabolism in living cells have limitations in terms of sensitivity and specificity and are often expensive and time-consuming. To address these issues, an innovative trident scaffold-assisted aptasensor (TSA) system has been developed for the online quantitative monitoring of 25(OH)VD3 in complex biological environments. Through the computer-aided design, the TSA system includes an aptamer molecule recognition layer that is uniformly oriented, maximizing binding site availability, and enhancing sensitivity. The TSA system achieved the direct, highly sensitive, and selective detection of 25(OH)VD3 over a wide concentration range (17.4-12,800 nM), with a limit of detection of 17.4 nM. Moreover, we evaluated the efficacy of the system in monitoring the biotransformation of 25(OH)VD3 in human liver cancer cells (HepG2) and normal liver cells (L-02), demonstrating its potential as a platform for drug-drug interaction studies and candidate drug screening.

DNA-templated electrodeposition of silver nanoparticles for direct and label-free aptasensing of ochratoxin A

We present in this work, an aptasensing strategy based on the DNA-templated electrodeposition of silver nanoparticles (AgNPs). The homogeneous electro-deposition of AgNPs on screen printed carbon electrode (SPCE) surface was achieved based on a unique aptamer scaffold. This was constructed by immobilizing a DNA aptamer on SPCE by electrochemical oxidation of its amine groups. The electrodeposition of AgNPs was investigated before and after the addition of the aptamer's specific target; the mycotoxin, ochratoxin A (OTA). Electrochemical characterization by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) showed the effect of the scaffold layer on the electrodeposition of AgNPs. The conformational change induced by aptamer after binding its targeted molecule affects AgNPs electrodeposition and the electron transfer thus allowing OTA detection by cyclic voltammetry. The voltammograms showed a good proportionality between the analyte concentration and the current response. The constructed platform allowed the quantitative aptasensing of OTA within the range of (1.56–400 ng/mL) and the detection limit of 0.6 ng/mL. In term of aptasensor applicability, the proposed strategy showed excellent performance in rice samples.

Polyvalent Biotinylated Aptamer Scaffold for Rapid and Sensitive Detection of Tau Proteins.

Biomimetic construction of artificial scaffolds has attracted increasing attention. However, the construction methods usually require redundant materials and procedures, which is inconvenient for further application. Herein, inspired by the polyvalent multifunctional structure in nature, we have designed a polyvalent biotinylated aptamer scaffold (PBAS) which can conduct analytical performance with high sensitivity and simplified procedures. To construct a PBAS, the aptamers are designed to hybridize with prepared linker probes to form polyvalent biotinylated scaffolds, which contain both multiple aptamers and signal labels. Therefore, multifunctional scaffolds can be constructed with high recognition and capture efficiency as well as significant signal amplification. Furthermore, the scaffold can be used for the assay of some disease marker proteins. By taking tau proteins as an example, the proposed aptasensor can exhibit excellent performance with a low detection limit of 153 pg mL-1 and a short assay time of 50 min, which is much better than most of the previous methods. By assays of tau proteins in both serum and artificial cerebro spinal fluid, the PBAS-based aptasensor can work well. Therefore, the scaffold may be expected to be a powerful analytical tool which may have wide applications in the detection of a variety of analytes.

Highly Specific Recognition of Guanosine Using Engineered Base‐Excised Aptamers

Purines and their derivatives are highly important molecules in biology for nucleic acid synthesis, energy storage, and signaling. Although many DNA aptamers have been obtained for binding adenine derivatives such as adenosine, adenosine monophosphate, and adenosine triphosphate, success for the specific binding of guanosine has been limited. Instead of performing new aptamer selections, we report herein a base-excision strategy to engineer existing aptamers to bind guanosine. Both a Na+ -binding aptamer and the classical adenosine aptamer have been manipulated as base-excising scaffolds. A total of seven guanosine aptamers were designed, of which the G16-deleted Na+ aptamer showed the highest bindng specificity and affinity for guanosine with an apparent dissociation constant of 0.78 mm. Single monophosphate difference in the target molecule was also recognizable. The generality of both the aptamer scaffold and excised site were systematically studied. Overall, this work provides a few guanosine binding aptamers by using a non-SELEX method. It also provides deeper insights into the engineering of aptamers for molecular recognition.

Improving Spinach2-and Broccoli-based biosensors for single and double analytes

The use of “Spinach”-based RNA sensors for the detection of small metabolites and proteins has received growing interest in the recent years. While this approach can be used in vivo for cell sensing and in vitro for microfluidic assays, their overall utility is limited as a result of a high magnesium ion dependence. Here, we alleviate this limitation through incorporating a human tRNALys3 or an engineered viral F30 (a three-way junction RNA motif) scaffold to facilitate aptamer folding in vitro and improve the performance of selected streptavidin, tyrosine, and thrombin aptamers as exemplary cases. Furthermore, we demonstrate the use of a Broccoli aptamer scaffold in conjunction with the viral F30 to enable simultaneous sensing of two molecules in a logic gate type fashion. Our proof-of-concept results demonstrate the ability to redesign these aptamer sensors for improved brightness as well as signal stability without the need for high magnesium—both traits that can further enhance downstream screening applications.

Flavin Binding Allosteric Aptamer with Noncovalent Labeling for miR Sensing.

Modular allosteric aptamers with discrete recognition and signaling regions provide a facile method of carrying out label-free detection by forgoing complex target labeling requirements. Herein, we describe the design and function of an aptamer scaffold capable of forming a hairpin loop in the presence of FAD (the signaling trigger). The aptamer includes a recognition region for the microRNA (miR) Let-7i. Upon selective miR hybridization, the aptamer undergoes a conformational shift to release FAD and thus produce a measurable response. As a result, the described method can sensitively and selectively detect miR Let-7i with a wide linear range of 0.1 pM to 1 μM and a detection limit of 150 fM. Additionally, this strategy was able to selectively discriminate between sequences with 1- and 2-nucleotide (nt) differences.

Redox-Switchable Binding Properties of the ATP-Aptamer.

In this study, we report ona redox-controllable and reversible complete "ON"/"OFF"-switchable aptamer binding to ATP.Aseries of methylene blue-modified ATP-aptamers was synthesized, revealing improved binding affinities toward ATP as compared to the nonmodified aptamer. These binding affinities were dependent on the conjugation site of the redox label on the aptamer scaffold. Importantly, we find that the oxidized methylene blue-modified aptamers bind to ATPwith micromolar affinity, while the reduced form lacks binding affinity toward ATP, resulting in an unprecedented complete "ON"/"OFF" redox-controllable aptamer switch. We demonstrate the cyclic "ON"/"OFF" binding of ATP to the methylene blue-functionalized aptamer through cyclic oxidation and reduction of the redox label using both chemical and electrochemical means. Molecular dynamics and docking simulations were performed to account for the redox-switchable properties of the conjugated aptamers and to rationalize the enhanced binding affinities of the different aptamer designs.

Nucleoapzymes: catalyst-aptamer conjugates as enzyme-mimicking structures

The conjugation of catalytic sites to sequence-specific, ligand-binding nucleic acid aptamers yields functional catalytic ensembles mimicking the catalytic/binding properties of native enzymes. These catalyst-aptamer conjugates termed 'nucleoapzymes' reveal structural diversity, and thus, vary in their catalytic activity, due to the different modes of conjugation of the catalytic units to the nucleic acid aptamer scaffold. The concept of nucleoapzymes is introduced with the assembly of a set of catalysts consisting of the hemin/G-quadruplex DNAzyme (hGQ) conjugated to the dopamine aptamer. The nucleoapzymes catalyze the oxidation of dopamine by H2O2 to yield aminochrome. The catalytic processes are controlled by the structures of the nucleoapzymes, and chiroselective oxidation of l-DOPA and d-DOPA by the nucleoapzymes is demonstrated. In addition, the conjugation of a Fe(III)-terpyridine complex to the dopamine aptamer and of a bis-Zn(II)-pyridyl-salen-type complex to the ATP-aptamer yields hybrid nucleoapzymes (conjugates where the catalytic site is not a biomolecule) that catalyze the oxidation of dopamine to aminochrome by H2O2 and the hydrolysis of ATP to ADP, respectively. Variable, structure-controlled catalytic activities of the different nucleoapzymes are demonstrated. Molecular dynamic simulations are applied to rationalize the structure-catalytic function relationships of the different nucleoapzymes. The challenges and perspectives of the research field are discussed.

Electrochemical Aptamer Scaffold Biosensors for Detection of Botulism and Ricin Proteins.

Electrochemical DNA (E-DNA) biosensors enable the detection and quantification of a variety of molecular targets, including oligonucleotides, small molecules, heavy metals, antibodies, and proteins. Here we describe the design, electrode preparation and sensor attachment, and voltammetry conditions needed to generate and perform measurements using E-DNA biosensors against two protein targets, the biological toxins ricin and botulinum neurotoxin. This method can be applied to generate E-DNA biosensors for the detection of many other protein targets, with potential advantages over other systems including sensitive detection limits typically in the nanomolar range, real-time monitoring, and reusable biosensors.

Highly Sensitive Colorimetric Cancer Cell Detection Based on Dual Signal Amplification.

Facile and efficient detection of cancer cells at their preclinical stages is one of the central challenges in cancer diagnostics. A direct, rapid, highly sensitive and specific biosensor for detection of cancer biomarkers is desirable in early diagnosis and prognosis of cancer. In this work, we developed, for the first time, an easy and intuitive dispersion-dominated colorimetric strategy for cancer cell detection based on combining multi-DNA released from an aptamer scaffold with cyclic enzymatic amplification, which was triggered by aptamer DNA conformational switch and demonstrated by non-cross-linking gold nanoparticles (Au NPs) aggregation. First, five kinds of messenger DNAs (mDNAs) were aligned on the cancer cell aptamers modified on magnetic beads (MBs) to form mDNAs-Apt-MBs biocompatible nanosensors. In the presence of target cells, the aptamer would bind to the receptors on the cell membranes, and mDNAs would be released, resulting in the first amplification that one biological binding event would cause the release of multiple kinds of mDNAs simultaneously. After magnetic separation, the released mDNAs were introduced into the cyclic enzymatic amplification to cleave more single strand DNA (ssDNA) fragments. Instead of modification of Au NPs, these fragments and mDNAs could be adsorbed on the surface of Au NPs to prevent particle aggregation and ensure the stability and color of solution in high salt environments. The linear response for HL-60 cells in a concentration range from 10 to 10(4) cells was obtained with a detection limit of four cells in buffer solution. Moreover, the feasibility of the proposed strategy was demonstrated in a diluted serum sample. This dual signal amplification method can be extended to other types of cancer cells, which has potential application in point-of-care cancer diagnosis.

Non-helical DNA Triplex Forms a Unique Aptamer Scaffold for High Affinity Recognition of Nerve Growth Factor

Discerning the structural building blocks of macromolecules is essential for understanding their folding and function. For a new generation of modified nucleic acid ligands (called slow off-rate modified aptamers or SOMAmers), we previously observed essential functions of hydrophobic aromatic side chains in the context of well-known nucleic acid motifs. Here we report a 2.45-Å resolution crystal structure of a SOMAmer complexed with nerve growth factor that lacks any known nucleic acid motifs, instead adopting a configuration akin to a triangular prism. The SOMAmer utilizes extensive hydrophobic stacking interactions, non-canonical base pairing and irregular purine glycosidic bond angles to adopt a completely non-helical, compact S-shaped structure. Aromatic side chains contribute to folding by creating an unprecedented intercalating zipper-like motif and a prominent hydrophobic core. The structure provides compelling rationale for potent inhibitory activity of the SOMAmer and adds entirely novel motifs to the repertoire of structural elements uniquely available to SOMAmers.

Electrochemical aptamer scaffold biosensors for detection of botulism and ricin toxins

Protein toxins present considerable health risks, but detection often requires laborious analysis. Here, we developed electrochemical aptamer biosensors for ricin and botulinum neurotoxins, which display robust and specific signal at nanomolar concentrations and function in dilute serum. These biosensors may aid future efforts for the rapid diagnosis of toxins.

The use of a neutral peptide aptamer scaffold to anchor BH3 peptides constitutes a viable approach to studying their function

The B-cell CLL/lymphoma-2 (Bcl-2) family of proteins are important regulators of the intrinsic pathway of apoptosis, and their interactions, driven by Bcl-2 homology (BH) domains, are of great interest in cancer research. Particularly, the BH3 domain is of clinical relevance, as it promotes apoptosis through activation of Bcl-2-associated x protein (Bax) and Bcl-2 antagonist killer (Bak), as well as by antagonising the anti-apoptotic Bcl-2 family members. Although investigated extensively in vitro, the study of the BH3 domain alone inside cells is more problematic because of diminished secondary structure of the unconstrained peptide and a lack of stability. In this study, we report the successful use of a novel peptide aptamer scaffold – Stefin A quadruple mutant – to anchor and present the BH3 domains from Bcl-2-interacting mediator of cell death (Bim), p53 upregulated modulator of apoptosis (Puma), Bcl-2-associated death promoter (Bad) and Noxa, and demonstrate its usefulness in the study of the BH3 domains in vivo. When expressed intracellularly, anchored BH3 peptides exhibit much the same binding specificities previously established in vitro, however, we find that, at endogenous expression levels, Bcl-2 does not bind to any of the anchored BH3 domains tested. Nonetheless, when expressed inside cells the anchored PUMA and Bim BH3 α-helices powerfully induce cell death in the absence of efficient targeting to the mitochondrial membrane, whereas the Noxa helix requires a membrane insertion domain in order to kill Mcl-1-dependent myeloma cells. Finally, the binding of the Bim BH3 peptide to Bax was the only interaction with a pro-apoptotic effector protein observed in this study.

Optimisation of a multivalent Strep tag for protein detection

The Strep tag is a peptide sequence that is able to mimic biotin's ability to bind to streptavidin. Sequences of Strep tags from 0 to 5 have been appended to the N-terminus of a model protein, the Stefin A Quadruple Mutant (SQM) peptide aptamer scaffold, and the recombinant fusion proteins expressed. The affinities of the proteins for streptavidin have been assessed as a function of the number of tags inserted using a variety of labelled and label-free bioanalytical and surface based methods (Western blots, microarray assays and surface plasmon resonance spectroscopy). The binding affinity increases with the number of tags across all assays, reaching nanomolar levels with 5 inserts, an observation assigned to a progressive increase in the probability of a binding interaction occurring. In addition a novel interfacial FRET based assay has been developed for generic Strep tag interactions, which utilises a conventional microarray scanner and bypasses the requirement for expensive lifetime imaging equipment. By labelling both the tagged StrepX-SQM 2 and streptavidin targets, the conjugate is primed for label-free FRET based displacement assays.

Design and Validation of a Neutral Protein Scaffold for the Presentation of Peptide Aptamers

Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein-protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold. Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets.