Generation Of Aptamers Research Articles

Riboswitch Mechanisms for Regulation of P1 Helix Stability.

Riboswitches are highly structured RNA regulators of gene expression. Although found in all three domains of life, they are particularly abundant and widespread in bacteria, including many human pathogens, thus making them an attractive target for antimicrobial development. Moreover, the functional versatility of riboswitches to recognize a myriad of ligands, including ions, amino acids, and diverse small-molecule metabolites, has enabled the generation of synthetic aptamers that have been used as molecular probes, sensors, and regulatory RNA devices. Generally speaking, a riboswitch consists of a ligand-sensing aptamer domain and an expression platform, whose genetic control is achieved through the formation of mutually exclusive secondary structures in a ligand-dependent manner. For most riboswitches, this involves formation of the aptamer's P1 helix and the regulation of its stability, whose competing structure turns gene expression ON/OFF at the level of transcription or translation. Structural knowledge of the conformational changes involving the P1 regulatory helix, therefore, is essential in understanding the structural basis for ligand-induced conformational switching. This review provides a summary of riboswitch cases for which ligand-free and ligand-bound structures have been determined. Comparative analyses of these structures illustrate the uniqueness of these riboswitches, not only in ligand sensing but also in the various structural mechanisms used to achieve the same end of regulating switch helix stability. In all cases, the ligand stabilizes the P1 helix primarily through coaxial stacking interactions that promote helical continuity.

AptaDiff: de novo design and optimization of aptamers based on diffusion models.

Aptamers are single-stranded nucleic acid ligands, featuring high affinity and specificity to target molecules. Traditionally they are identified from large DNA/RNA libraries using $in vitro$ methods, like Systematic Evolution of Ligands by Exponential Enrichment (SELEX). However, these libraries capture only a small fraction of theoretical sequence space, and various aptamer candidates are constrained by actual sequencing capabilities from the experiment. Addressing this, we proposed AptaDiff, the first in silico aptamer design and optimization method based on the diffusion model. Our Aptadiff can generate aptamers beyond the constraints of high-throughput sequencing data, leveraging motif-dependent latent embeddings from variational autoencoder, and can optimize aptamers by affinity-guided aptamer generation according to Bayesian optimization. Comparative evaluations revealed AptaDiff's superiority over existing aptamer generation methods in terms of quality and fidelity across four high-throughput screening data targeting distinct proteins. Moreover, surface plasmon resonance experiments were conducted to validate the binding affinity of aptamers generated through Bayesian optimization for two target proteins. The results unveiled a significant boost of $87.9\%$ and $60.2\%$ in RU values, along with a 3.6-fold and 2.4-fold decrease in KD values for the respective target proteins. Notably, the optimized aptamers demonstrated superior binding affinity compared to top experimental candidates selected through SELEX, underscoring the promising outcomes of our AptaDiff in accelerating the discovery of superior aptamers.

Novel antibody- and aptamer-based approaches for sensitive detection of mycotoxin fusaric acid in cereal

This study presents the first successful generation of polyclonal antibodies (pAbs) and oligonucleotide aptamers specifically targeting fusaric acid (FA). Utilizing these pAbs and aptamers, three highly sensitive and specific assays were developed for the detection of FA in cereals with limits of detection (LOD) ranging from 5 to 50 ng/g: an antibody-based enzyme-linked immunosorbent assay (ELISA), an aptamer-based enzyme-linked aptamer-sorbent assay (ELASA), and a hybrid enzyme-linked aptamer-antibody sandwich assay (ELAAA). The recovery rates of FA in spiked cereal samples ranged from 87 % to 112 % across all assays. Analysis of 15 cereal feed samples revealed FA contamination levels of 459 to 1743 ng/g (ELISA), 427 to 1960 ng/g (ELASA), and 381 to 1987 ng/g (ELAAA). These results were further validated by HPLC analysis, confirming high consistency within developed assays. Overall, the ELISA, ELASA, and ELAAA are promising tools for the rapid detection of FA, significantly contributing to food safety monitoring.

Generation of specific aptamers for constructing CoOOH nanozymes-based colorimetric aptasensor for detection of fleroxacin in milk

The misuse or abuse of fleroxacin (FLE) in livestock and aquaculture can result in elevated levels of FLE in animal-derived foods, posing significant health risks to humans. Detection of FLE has been relying on instrumental analysis methods that need expensive equipment and longer analysis time. Herein we report the first isolation of two specificity aptamers against FLE (FLE-9 and FLE-51) by capture-SELEX, characterized them well and explored the binding mechanism. Furthermore, FLE-9 was selected to construct a CoOOH@Apt based biosensor. The catalytic activity of the biosensor was evaluated using the Michaelis equation, revealing a Vmax of 4.87×10−8 M s−1 and a Km of 0.11 mM. These results suggest that the affinity of CoOOH@Apt is higher than that of CoOOH nanozymes. In the present of FLE, the aptamer FLE-9 specifically binds to FLE, distancing itself from CoOOH nanozymes and thereby reducing the catalytic activity of the biosensor. Finally, the label-free colorimetric aptasensor was successfully applied to detect FLE, with a detection limit of 4.26 ng/mL. The recovery rates ranged from 96.70 % to 101.70 %, with a relative standard deviation (RSD) below 3.02 %. This study presents promising probes and a novel strategy for the determination of FLE in milk samples, contributing to improved food safety monitoring.

Surface‐Based Multimeric Aptamer Generation and Bio‐Functionalization for Electrochemical Biosensing Applications

AbstractMultimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface‐based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio‐receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS‐CoV‐2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10‐fold), as well as higher sensitivity (almost 4‐fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano‐/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples.

Surface-Based Multimeric Aptamer Generation and Bio-Functionalization for Electrochemical Biosensing Applications.

Multimeric aptamers have gained more attention than their monomeric counterparts due to providing more binding sites for target analytes, leading to increased affinity. This work attempted to engineer the surface-based generation of multimeric aptamers by employing the room temperature rolling circle amplification (RCA) technique and chemically modified primers for developing a highly sensitive and selective electrochemical aptasensor. The multimeric aptamers, generated through surface RCA, are hybridized to modified spacer primers, facilitating the positioning of the aptamers in the proximity of sensing surfaces. These multimeric aptamers can be used as bio-receptors for capturing specific targets. The surface amplification process was fully characterized, and the optimal amplification time for biosensing purposes was determined, using SARS-CoV-2 spike protein (SP). Interestingly, multimeric aptasensors produced considerably higher response signals and affinity (more than 10-fold), as well as higher sensitivity (almost 4-fold) compared to monomeric aptasensors. Furthermore, the impact of surface structures on the response signals was studied by utilizing both flat working electrodes (WEs) and nano-/microislands (NMIs) WEs. The NMIs multimeric aptasensors showed significantly higher sensitivity in buffer and saliva media with the limit of detection less than 2 fg/ml. Finally, the developed NMIs multimeric aptasensors were clinically challenged with several saliva patient samples.

Advancing Genetic Alphabet Expansion: Synthesis of 7-(2-Thienyl)-Imidazo[4,5-b]pyridine (Ds) and 4-(4-Pentyne-1,2-diol)-1-Propynyl-2-Nitropyrrole (Diol-Px) for Use in Replicable Unnatural Base Pairs for PCR Applications.

Expanding the genetic alphabet enhances DNA recombinant technologies by introducing unnatural base pairs (UBPs) beyond the standard A-T and G-C pairs, leading to biomaterials with novel and increased functionalities. Recent developments include UBPs that effectively function as a third base pair in replication, transcription, and/or translation processes. One such UBP, Ds-Px, demonstrates extremely high specificity in replication. Chemically synthesized DNA fragments containing Ds bases are amplified by PCR with the 5'-triphosphates of Ds and Px deoxyribonucleosides (dDsTP and dPxTP). The Ds-Px pair system has applications in enhanced DNA data storage, generation of high-affinity DNA aptamers, and incorporation of functional elements into RNA through transcription. This protocol describes the synthesis of the amidite derivative of Ds (dDs amidite), the triphosphate dDsTP, and the diol-modified dPxTP (Diol-dPxTP) for PCR amplifications involving the Ds-Px pair. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of Ds deoxyribonucleoside (dDs) Basic Protocol 2: Synthesis of dDs amidite Basic Protocol 3: Synthesis of dDs triphosphate (dDsTP) Basic Protocol 4: Synthesis of Pn deoxyribonucleoside (4-iodo-dPn) Basic Protocol 5: Synthesis of acetyl-protected diol-modified Px deoxyribonucleoside (Diol-dPx) Basic Protocol 6: Synthesis of Diol-dPx triphosphate (Diol-dPxTP) Basic Protocol 7: Purification of triphosphates Support Protocol 1: Synthesis of Hoffer's chlorosugar Support Protocol 2: Preparation of 0.5 M pyrophosphate in DMF Support Protocol 3: Preparation of 2 M TEAB buffer.

Generation of epitope-specific hCG aptamers through a novel targeted selection approach.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone used as a biomarker for several medical conditions, including pregnancy, trophoblastic and nontrophoblastic cancers. Most commercial hCG tests rely on a combination of antibodies, one of which is usually specific to the C-terminal peptide of the β-subunit. However, cleavage of this region in many hCG degradation variants prevents rapid diagnostic tests from quantifying all hCG variants in serum and urine samples. An epitope contained within the core fragment, β1, represents an under-researched opportunity for developing immunoassays specific to most variants of hCG. In the study described here, we report on a SELEX procedure tailored towards the identification of two pools of aptamers, one specific to the β-subunit of hCG and another to the β1 epitope within it. The described SELEX procedure utilized antibody-blocked targets, which is an underutilized strategy to exert negative selection pressure and in turn direct aptamer enrichment to a specific epitope. We report on the first aptamers, designated as R4_64 and R6_5, each capable of recognising two distinct sites of the hCG molecule-the β-subunit and the (presumably) β1-epitope, respectively. This study therefore presents a new SELEX approach and the generation of novel aptamer sequences that display potential hCG-specific biorecognition.

Generation of a specific aptamer for accurate detection of sarafloxacin based on fluorescent/colorimetric/SERS triple-readout sensor

Sarafloxacin (SAR), one of the most widely used fluoroquinolone antibiotics, is a serious threat to aquatic environments and human health due to its illegal abuse. Herein, we first screened an aptamer (SAR-1) that specifically binds to SAR using capture-SELEX technology. Based on molecular docking technology, SAR-1 was gradually truncated, and a short SAR-1a with better affinity and specificity was obtained. The optimal SAR-1a was further combined with a Pt nanoparticle (Pt NP)- decorated bimetallic Fe/Co-MOF to fabricate a multimode sensing platform for SAR determination. The Fe/Co-MOF@Pt NPs exhibited excellent peroxidase-like activity, which catalyzed the H2O2-mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), thereby enabling visual detection of SAR. Meanwhile, the generated oxTMB can also produce SERS responses and be used for the SERS detection of SAR. Moreover, the inherent fluorescence property of Fe/Co-MOF@Pt NPs enabled fluorescence detection of SAR. The designed triple-readout aptasensor showed good sensitivity for SAR detection with limits of detection of 0.125 ng/mL (fluorescent mode) and 0.05 ng/mL (colorimetric and SERS mode). The aptamer-based triple-mode sensing platform provided mutual verification of detection results in different output modes, effectively improving the assay accuracy and providing a promising tool for highly sensitive, selective, and accurate determination of SAR in daily life.

Label-free and highly sensitive detection of microRNA from cancer cells via target-induced cascade amplification generation of lighting-up RNA aptamers

The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.

Reverse transcription as key step in RNA in vitro evolution with unnatural base pairs

Unnatural base pairs (UBPs) augment the chemical diversity of artificial nucleic acids and can enable the generation of new aptamers and catalytic nucleic acids by in vitro selection. Reverse transcription of UBPs as key step during RNA in vitro selection is investigated.

Interaction of Amyloid Beta with Mitochondrial RNAs

AbstractBackgroundDespite mitochondrial RNA’s (mt‐RNA) polycistronic origin, research has shown that a number of post‐transcriptional mechanisms can control transcription. The amyloidogenic proteins such as prion and aSyn are known to bind RNA. However, the main peptide responsible for the pathogenesis of Alzheimer’s disease (AD), amyloid beta 1‐42 (Aβ1‐42), is not known to interact with RNA. It is well known that Aβ1‐42 is an alpha‐helix in monomer form. However, by attaching zinc, Aβ1‐42 achieves sheet folding. Aβ’s new conformation resembles zinc finger transcription factors. Furthermore, using randomly generated RNA molecules, it has been shown that the Aβ peptide domain that interacts with metals may also interact with RNA. As a result, Aβ has the potential to interact with mtRNAs, and this possible interaction may be linked to malfunctioning mitochondria in AD. Aβ1‐42, on the other hand, may affect the LRP130 protein, which is implicated in the mtRNA post‐transcriptional processes. In the current study, we seek to determine whether Aβ1‐42 may interact with mtRNA and whether Aβ1‐42 treatment affects LRP130’s ability to bind to mtRNA.MethodIn our study, the possible interaction of Aβ1‐42‐mtRNA and whether LRP130’s binding capacity to mtRNA was changed due to 0,1µM of Aβ1‐42 peptide treatment was investigated by RNA immunoprecipitation (RNA‐IP) and qRT‐PCR in differentiated LUHMES cells.ResultAβ – RNA IP experiment revealed that certain specific mtRNA binding signals were detected in untreated cells, albeit at a low level. Compared to untreated cells, the administration of Aβ1‐42 dramatically reduced LRP130’s affinity for mtRNAs.ConclusionAβ1‐42, the major peptide of AD, may be able to influence post‐transcriptional mitochondrial expression. When we consider the significance of RNA‐protein interactions in neurodegenerative processes, we determine that our research can offer a current strategy for AD biomarker research and the generation of therapeutic RNA aptamers. This study is supported by The Scientific and Technological Research Council of Turkey (TUBITAK) (Project ID:219Z179).

Computational generation and characterization of IsdA-binding aptamers with single-molecule FRET analysis.

Staphylococcus aureus is a major foodborne bacterial pathogen. Early detection of S. aureus is crucial to prevent infections and ensure food quality. The iron-regulated surface determinant protein A (IsdA) of S. aureus is a unique surface protein necessary for sourcing vital iron from host cells for the survival and colonization of the bacteria. The function, structure, and location of the IsdA protein make it an important protein for biosensing applications relating to the pathogen. Here, we report an in-silico approach to develop and validate high-affinity binding aptamers for the IsdA protein detection using custom-designed in-silico tools and small-molecule Fluorescence Resonance Energy Transfer (smFRET) measurements. We utilized in-silico oligonucleotide screening methods and metadynamics-based methods to generate 10 aptamer candidates and characterized them based on the Dissociation Free Energy (DFE) of the IsdA-aptamer complexes. Three of the aptamer candidates were shortlisted for smFRET experimental analysis of binding properties. Limits of detection in the low picomolar range were observed for the aptamers, and the results correlated well with the DFE calculations, indicating the potential of the in-silico approach to support aptamer discovery. This study showcasesa computational SELEXmethod in combination with single-molecule binding studies decipheringeffectiveaptamersagainst S. aureusIsdA, protein. Theestablished approach demonstrates the abilityto expediteaptamerdiscovery that has the potential to cutcosts and predict binding efficacy.The application can be extendedto designingaptamersfor various protein targets, enhancing molecular recognition, and facilitating the development of high-affinityaptamersfor multiple uses. This article is protected by copyright. All rights reserved.

Strict Interactions of Fifth Letters, Hydrophobic Unnatural Bases, in XenoAptamers with Target Proteins.

XenoAptamers are DNA fragments containing additional letters (unnatural bases, UBs) that bind specifically to their target proteins with high affinities (sub-nanomolar KD values). One of the UBs is the highly hydrophobic 7-(2-thienyl)imidazo[4,5-b]pyridine (Ds), which significantly increases XenoAptamers' affinities to targets. Originally, Ds was developed as a third base pair with a complementary UB, 2-nitro-4-propynylpyrrole (Px), for replication, and thus it can be used for aptamer generation by an evolutional engineering method involving PCR amplification. However, it is unclear whether the Ds base is the best component as the hydrophobic fifth-letter ligand for interactions with target proteins. To optimize the ligand structure of the fifth letter, we prepared 13 Ds variants and examined the affinities of XenoAptamers containing these variants to target proteins. The results obtained using four XenoAptamers prepared by the replacement of Ds bases with variants indicated that subtle changes in the chemical structure of Ds significantly affect the XenoAptamer affinities. Among the variants, placing either 4-(2-thienyl)pyrrolo[2,3-b]pyridine (Ys) or 4-(2-thienyl)benzimidazole (Bs) at specific Ds positions in each original XenoAptamer greatly improved their affinities to targets. The Ys and Bs bases are variants derived by replacing only one nitrogen with a carbon in the Ds base. These results demonstrate the strict intramolecular interactions, which are not simple hydrophobic contacts between UBs and targets, thus providing a method to mature XenoAptamers' affinities to targets.

Aptamers for mycotoxin recognition in food: Recent advances and future considerations

The contamination of mycotoxin in food has posed a serious threat to human health and safety. Accurate detection of mycotoxins provides a strong guarantee for food safety. Aptamers with excellent recognition capacity to mycotoxins have gained growing attention because of their high binding affinity, high specificity and low cost. They are commonly generated by an in vitro selection procedure called SELEX. However, the existing performance of mycotoxin aptamers are not good enough in complex food matrix, and the success rate of SELEX still need to be improved. The employment of efficient SELEX strategies for the generation of better mycotoxin aptamers is significantly important. This article has reviewed different SELEX approaches conducted for mycotoxin aptamer generation. Furthermore, the properties of various mycotoxin aptamers have been discussed. Finally, challenges and future considerations for mycotoxin aptamer selection are summarized, and their strengths and limitations in food safety application are highlighted.

Generation of DNA Aptamers with Functional Activity in Mammalian Cells by Mimicking Retroviruses.

DNA aptamers are single-stranded DNA oligonucleotide sequences that bind to specific targets with high affinity. Currently, DNA aptamers can be produced only by in vitro synthesis. It is difficult for DNA aptamers to have a sustained impact on intracellular protein activity, which limits their clinical application. In this study, we developed a DNA aptamer expression system to generate DNA aptamers with functional activity in mammalian cells by mimicking retroviruses. Using this system, DNA aptamers targeting intracellular Ras (Ra1) and membrane-bound CD71 (XQ2) were successfully generated in cells. In particular, the expressed Ra1 not only specifically bound to the intracellular Ras protein but also inhibited the phosphorylation of downstream ERK1/2 and AKT. Furthermore, by inserting the DNA aptamer expression system for Ra1 into a lentivirus vector, the system can be delivered into cells and stably produce Ra1 over time, resulting in the inhibition of lung cancer cell proliferation. Therefore, our study provides a novel strategy for the intracellular generation of DNA aptamers with functional activity and opens a new avenue for the clinical application of intracellular DNA aptamers in disease treatment.

FRET-Based Single-Molecule Detection of Pathogen Protein IsdA Using Computationally Selected Aptamers.

Iron-regulated surface determinant protein A (IsdA) is a key surface protein found in the foodborne bacteria─Staphylococcus aureus (S. aureus)─which is known to be critical for bacterial survival and colonization. S. aureus is pathogenic and has been linked to foodborne diseases; thus, early detection is critical to prevent diseases caused by this bacterium. Despite IsdA being a specific marker for S. aureus and several detection methods have been developed for sensitive detection of this bacteria such as cell culture, nucleic acids amplification, and other colorimetric and electrochemical methods, the detection of S. aureus through IsdA is underdeveloped. Here, by combining computational generation of target-guided aptamers and fluorescence resonance energy transfer (FRET)-based single-molecule analysis, we presented a widely applicable and robust detection method for IsdA. Three different RNA aptamers specific to the IsdA protein were identified and their ability to switch a FRET construct to a high-FRET state in the presence of protein was verified. The presented approach demonstrated the detection of IsdA down to picomolar levels (×10-12 M, equivalent to ∼1.1 femtomoles IsdA) with a dynamic range extending to ∼40 nM. The FRET-based single-molecule technique that we reported here is capable of detecting the foodborne pathogen protein IsdA with high sensitivity and specificity and has a broader application in the food industry and aptamer-based sensing field by enabling quantitative detection of a wide range of pathogen proteins.

A high-dimensional microfluidic approach for selection of aptamers with programmable binding affinities.

Aptamers are being applied as affinity reagents in analytical applications owing to their high stability, compact size and amenability to chemical modification. Generating aptamers with different binding affinities is desirable, but systematic evolution of ligands by exponential enrichment (SELEX), the standard for aptamer generation, is unable to quantitatively produce aptamers with desired binding affinities and requires multiple rounds of selection to eliminate false-positive hits. Here we introduce Pro-SELEX, an approach for the rapid discovery of aptamers with precisely defined binding affinities that combines efficient particle display, high-performance microfluidic sorting and high-content bioinformatics. Using the Pro-SELEX workflow, we were able to investigate the binding performance of individual aptamer candidates under different selective pressures in a single round of selection. Using human myeloperoxidase as a target, we demonstrate that aptamers with dissociation constants spanning a 20-fold range of affinities can be identified within one round of Pro-SELEX.

Formaldehyde Cross-Linking-Assisted Phase Separation for Protein Aptamer Selection.

With the merits of easy synthesis, strong modifiability, and high affinity, aptamers have been broadly applied for protein targeting in bioanalysis, diagnosis, and therapeutics. The selection of protein-targeted aptamers is currently largely dependent on solid-liquid separation by using different types of nano- or micro-beads. However, the use of beads inescapably introduces unwanted nonspecific binding and thus affects selection efficiency. In order to sidestep this obstacle, we herein report an integrated technique to facilitate the discovery and development of protein-targeting aptamers by incorporating formaldehyde cross-linking with phase separation (FCPS). The feasibility and universality of FCPS were confirmed by the successful selection of two aptamers that could target various antibodies. Unlike traditional approaches, the proposed technique avoids the use of beads and enables the rapid generation of aptamers after only one to three rounds of selection. The as-selected aptamers were further used to regulate and control antibody activity, showing potential applications in biomedicine.

Kanamycin triggered nanozyme for homogeneous and amplified colorimetric detection of T4 polynucleotide kinase

It is of significance to develop efficient methods for detecting the activity of T4 polynucleotide kinase (T4 PNK) due to its essential role in the modulation of different life activities. In this work, we constructed a novel nanozyme using Kanamycin (KANA) as a trigger for the [Fe(CN)6]3- coordinated Cu2(OH)3NO3 (Cu2(OH)3NO3/[Fe(CN)6]3-) nanorods, and designed an amplified colorimetric method to detect T4 PNK. That was, the free KANA efficiently triggered the peroxidase-like activity of Cu2(OH)3NO3/[Fe(CN)6]3-, while the bound KANA by its aptamer lost the stimulative capability for the nanomaterials. On the basis of the bioreaction regulated generation of the KANA aptamer, a highly sensitive colorimetric assay aided by the rolling circle amplification (RCA) reaction for the detection of T4 PNK was realized. Results showed that this assay can detect T4 PNK from 1.0 × 10−3 to 10.0 U/mL, with a limit of detection (LOD) of 1.42 × 10−4 U/mL. The assay also showed acceptable performance in the detection of T4 PNK in serum samples. In addition to the satisfactory sensitivity and selectivity, the displayed T4 PNK assay also presented merits of operational convenience, without labeling or immobilization process and did not require costly instrument. It is expected that the KANA as a stimulator would have extended biosensing applications by coupling various bioreactions that can produce the KANA aptamer.