Arrest of Rolling Circle Amplification by Protein‐Binding DNA Aptamers

Certain DNA polymerases, such as ?29 DNA polymerase, can isothermally copy the sequence of a circular template round by round in a process known as rolling circle amplification (RCA), which results in super‐long single‐stranded (ss) DNA molecules made of tandem repeats. The power of RCA reflects the high processivity and the strand‐displacement ability of these polymerases. In this work, the ability of ?29DNAP to carry out RCA over circular templates containing a protein‐binding DNA aptamer sequence was investigated. It was found that protein–aptamer interactions can prevent this DNA polymerase from reading through the aptameric domain. This finding indicates that protein‐binding DNA aptamers can form highly stable complexes with their targets in solution. This novel observation was exploited by translating RCA arrest into a simple and convenient colorimetric assay for the detection of specific protein targets, which continues to showcase the versatility of aptamers as molecular recognition elements for biosensing applications.     

Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes

Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33 nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins.     

Target‐Induced and Equipment‐Free DNA Amplification with a Simple Paper Device

We report on a paper device capable of carrying out target‐induced rolling circle amplification (RCA) to produce massive DNA amplicons that can be easily visualized. Interestingly, we observed that RCA was more proficient on paper than in solution, which we attribute to a significantly higher localized concentration of immobilized DNA. Furthermore, we have successfully engineered a fully functional paper device for sensitive DNA or microRNA detection via printing of all RCA‐enabling molecules within a polymeric sugar film formed from pullulan, which was integrated with the paper device. This encapsulation not only stabilizes the entrapped reagents at room temperature but also enables colorimetric bioassays with minimal steps.     

Target-Dependent Protection of DNA Aptamers against Nucleolytic Digestion Enables Signal-On Biosensing with Toehold-Mediated Rolling Circle Amplification

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5′-end protects it from 3′-exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration-dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn-on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real-time fluorescence detection of two human proteins: platelet-derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay.     

A Universal Strategy for Aptamer‐Based Nanopore Sensing through Host–Guest Interactions inside α‐Hemolysin

Nanopore emerged as a powerful single‐molecule technique over the past two decades, and has shown applications in the stochastic sensing and biophysical studies of individual molecules. Here, we report a versatile strategy for nanopore sensing by employing the combination of aptamers and host–guest interactions. An aptamer is first hybridized with a DNA probe which is modified with a ferrocene?cucurbit[7]uril complex. The presence of analytes causes the aptamer–probe duplex to unwind and release the DNA probe which can quantitatively produce signature current events when translocated through an α‐hemolysin nanopore. The integrated use of magnetic beads can further lower the detection limit by approximately two to three orders of magnitude. Because aptamers have shown robust binding affinities with a wide variety of target molecules, our proposed strategy should be universally applicable for sensing different types of analytes with nanopore sensors.     

In Vitro Selection of a DNA Aptamer Targeting Degraded Protein Fragments for Biosensing

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper‐based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer‐selection and paper‐sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.     

G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection

Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.     

Optimal DNA Templates for Rolling Circle Amplification Revealed by In Vitro Selection

Rolling circle amplification (RCA) has been widely used as an isothermal DNA amplification technique for diagnostic and bioanalytical applications. Because RCA involves repeated copying of the same circular DNA template by a DNA polymerase thousands of times, we hypothesized there exist DNA sequences that can function as optimal templates and produce more DNA amplicons within an allocated time. Herein we describe an in vitro selection effort conducted to search from a random sequence DNA pool for such templates for phi29 DNA polymerase, a frequently used polymerase for RCA. Diverse DNA molecules were isolated and they were characterized by richness in adenosine (A) and cytidine (C) nucleotides. The top ranked sequences exhibit superior RCA efficiency and the use of these templates for RCA results in significantly improved detection sensitivity. AC‐rich sequences are expected to find useful applications for setting up effective RCA assays for biological sensing.     

Rolling circle amplification: applications in nanotechnology and biodetection with functional nucleic acids

Rolling circle amplification (RCA) is an isothermal, enzymatic process mediated by certain DNA polymerases in which long single-stranded (ss) DNA molecules are synthesized on a short circular ssDNA template by using a single DNA primer. A method traditionally used for ultrasensitive DNA detection in areas of genomics and diagnostics, RCA has been used more recently to generate large-scale DNA templates for the creation of periodic nanoassemblies. Various RCA strategies have also been developed for the production of repetitive sequences of DNA aptamers and DNAzymes as detection platforms for small molecules and proteins. In this way, RCA is rapidly becoming a highly versatile DNA amplification tool with wide-ranging applications in genomics, proteomics, diagnosis, biosensing, drug discovery, and nanotechnology.     

An electrochemiluminescent assay for high sensitive detection of mercury (II) based on isothermal rolling circular amplification

In this study, we firstly demonstrated that Bst DNA polymerase shows specific recognition and function on the T–Hg²⁺–T biomimetic structure. Based on this, a novel available electrochemiluminescence (ECL) sensor for Hg²⁺ has been developed. In this strategy, magnet beads tagged primer was designed to complementary to the region of the circular padlock probe but with two T–T mismatches at the 3′ end. The mismatched primers cannot be extended by Bst DNA polymerase in the absence of Hg²⁺. Stable T–Hg²⁺–T can be formed in the presence of Hg²⁺, thus induces the elongation and amplification reaction by DNA polymerase with a rolling circular amplification (RCA) mechanism. Subsequently, the resulted RCA products are hybridized with the tris (bipyridine) ruthenium (TBR)-tagged probes and detected by ECL platform. Current method shows a sub-nanomolar sensitivity and excellent selectivity over a spectrum of interference metal ions.     

基于磁珠分离的酶联适配体检测痕量蛋白质的新型比色分析方法

以核酸适配体作为高效专一的识别/传感元件, 构建了一种新型的磁性分离和特异性捕获的检测方法. 两个适配体通过简单的生物素化修饰, 利用其与凝血酶不同位点的高亲和力形成夹心结构, 其中连接适配体的磁珠可捕获蛋白质, 加入另一个适配体及链霉亲和素标记的辣根过氧化物酶后, 通过比色法实现靶蛋白检测. 该法操作简单, 分析时间短, 对凝血酶的线性响应范围为 10~80 nmol/L, 检出限为 10 nmol/L.     

Fluorogenic assays for activated protein C using aptamer modified magnetic beads

We describe a fluorogenic assay for activated protein C (APC) by using magnetic beads modified with DNA aptamers, taking advantage of strong binding affinity of aptamer, facile magnetic separation, and signal amplification via an enzymatic reaction. APC is specifically captured from a sample by the DNA aptamers on magnetic beads, and the concentrated APC then catalyzes the conversion of a fluorogenic substrate of APC to a fluorescent product. Detection of APC is achieved by measuring the generated product. This method is simple, sensitive, and specific. APC can be detected at 0.4 pM concentration level in a sample volume of 250 μL, corresponding to 0.1 femtomole of APC, when 2-h enzymatic reaction is employed. The proteins thrombin, trypsin, proteinase K, chymotrypsin, and elastase do not interfere.

Nuclease cleavage-assisted target recycling for signal amplification of free-label impedimetric aptasensors

A simple, novel, label-free impedimetric aptasensor with signal amplification is developed for ultrasensitive detection of small molecules on a carbon nanotubes-based sensing platform by combining target-induced release of the aptamers and nuclease cleavage-assisted target recycling.     

Rapid Colorimetric Determination of Procalcitonin Using Magnetic Separation and Enzymatic Catalysis

Human procalcitonin is an early diagnostic biomarker for sepsis and bacterial infections and can be used in distinguishing bacterial infections from viral infections. In this study, a colorimetric sensing platform for the rapid determination of procalcitonin was developed. The approach involves the capture of procalcitonin by immunomagnetic beads, and a detection antibody labeled with horseradish peroxidase to perform sandwich format, where it catalyzes the oxidation of 3,3′,5,5′–tetramethylbenzidine to produce the colorimetric signal. Under the optimal conditions, a detection limit of 0.04?ng/mL (3σ) was obtained within the calibration range 0.1–10?ng/mL. The proposed method was performed in less than 90?min and exhibited good specificity without interferences from other biomarkers including C-reactive protein and human serum albumin. Overall, the proposed method provided a new alternative strategy for procalcitonin detection due to its sensitive, rapid, specific, and simple characteristics. This method is suitable for rapid screening of various biomedical targets.     

Virus-Like Particle-Induced cGAS-STING Activation and AIM2 Inflammasome-Mediated Pyroptosis for Robust Cancer Immunotherapy

cGAS-STING-mediated DNA sensing is demonstrated to be critical for launching antitumor immunity. However, DNA-based cGAS-STING agonists are rarely reported owing to low cell permeability, poor biostability and, especially, limited length of exogenous DNA. Here, we present a virus-like particle which is self-assembled from long DNA building blocks generated through rolling-circle amplification (RCA) and covered with cationic liposomes. Based on long and densely packed DNA structure, it could efficiently induce liquid phase condensation of cGAS and activate STING signaling to produce inflammatory cytokines. Moreover, this virus-like particle could also trigger the formation of AIM2 inflammasome to induce gasdermin D-mediated pyroptosis, boosting antitumor immunity. Thus, this study provides a simple and robust strategy for cancer immunotherapy for clinical application. This is the first study to report the intrinsic immunogenicity of RCA products, thus facilitating their biomedical applications.     

Translating Bacterial Detection by DNAzymes into a Litmus Test

Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front‐line preventative care, particularly in resource‐limited regions. Thus, there is a significant need to develop low‐cost and easy‐to‐use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria‐specific RNA‐cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pH value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world.     

Aptamer-barcode based immunoassay for the instantaneous derivatization chemiluminescence detection of IgE coupled to magnetic beads

We report on a highly sensitive aptameric assay system for the determination of IgE, where a special chemiluminescence (CL) reagent, 3,4,5-trimethoxylphenylglyoxal (TMPG), acts as the signaling molecule and polystyrene beads as the amplification platform. Briefly, a "sandwich-type" detection strategy is employed in our design, where magnetic beads functionalized with a capture antibody were reacted with the target protein IgE, and then sandwiched with the aptamer-barcodes which were prepared by assembling polystyrene beads with IgE aptamer. The target immunoreaction event could be sensitively detected via an instantaneous derivatization reaction between TMPG and the guanine (G) nucleotides within the aptamer-barcodes to form an unstable CL intermediate for the generation of light. Further signal amplification is achieved by extending the G nucleotide-rich domain on the aptamer backbone for second amplification. Such simple amplified CL transduction allows the detection of IgE down to the 4.6 pM level, which is better than most previous aptameric methods for IgE detection. This new protocol also provides a good capability in discriminating IgE from nontarget proteins such as IgG, IgA, IgM, interferon and thrombin. The practical application of the proposed aptamer-barcode based immunoassay was successfully carried out for the determination of IgE in 20 human serum samples. It is straightforward to adapt this strategy to detect a spectrum of other proteins by using different aptamers, thus this method may offer a new direction in designing high-performance CL aptasensors for early diagnoses of diseases.     

Stabilizing structure-switching signaling RNA aptamers by entrapment in sol-gel derived materials for solid-phase assays

Structure-switching, fluorescence-signaling DNA and RNA aptamers have been reported as highly versatile molecular recognition elements for biosensor development. While structure-switching DNA aptamers have been utilized for solid-phase sensing, equivalent RNA aptamers have yet to be successfully utilized in solid-phase sensors due to their lack of chemical stability and susceptibility to nuclease attack. In this study, we examined entrapment into sol-gel derived organic-inorganic composite materials as a platform for immobilization of structure-switching fluorescence-signaling RNA aptamer reporters, using both the synthetic theophylline- and naturally occurring thiamine pyrophosphate-binding RNA aptamers as test cases. Structure-switching versions of both aptamers were entrapped into a series of sol-gel derived composites, ranging from highly polar silica to hydrophobic methylsilsesquioxane-based materials, and the target-binding and signaling capabilities of these immobilized aptamers were assessed relative to solution. Both immobilized aptamers demonstrated sensitivity and selectivity similar to that of free aptamers when entrapped in a composite material derived from 40% (v/v) methyltrimethoxysilane/tetramethoxysilane. Importantly, this material also conferred protection from nuclease degradation and imparted long-term chemical stability to the RNA reporter systems. Given the versatility of sol-gel entrapment for development of biosensors, microarrays, bioaffinity columns, and other devices, this entrapment method should provide a useful platform for numerous solid-phase RNA aptamer-based devices.     

In Vitro Selection of a DNA Aptamer Targeting Degraded Protein Fragments for Biosensing

Protein biomarkers often exist as degradation fragments in biological samples, and affinity agents derived using a purified protein may not recognize them, limiting their value for clinical diagnosis. Herein, we present a method to overcome this issue, by selecting aptamers against a degraded form of the toxin B protein, which is a marker for diagnosing toxigenic Clostridium difficile infections. This approach has led to isolation of a DNA aptamer that recognizes degraded toxin B, fresh toxin B, and toxin B spiked into human stool samples. DNA aptamers selected using intact recombinant toxin B failed to recognize degraded toxin B, which is the form present in stored stool samples. Using this new aptamer, we produced a simple paper-based analytical device for colorimetric detection of toxin B in stool samples, or in the NAP1 strain of Clostridium difficile. The combined aptamer-selection and paper-sensing strategy can expand the practical utility of DNA aptamers in clinical diagnosis.     

A Genosensor for Point Mutation Detection of P53 Gene PCR Product Using Magnetic Particles

The development of an electrochemical genosensor involving DNA biotinylated capture probe immobilized on streptavidin coated paramagnetic beads and microfluidic based platform for the detection of P53 gene PCR product is reported. The novelty of this work is the combination of a sensitive electrochemical platform and a proper microfluidic system with a simple and effective enzyme signal amplification technology, ELISA, for detection of target DNA sequence and single nucleotide mutation in p53 tumor suppressor gene sequence. The biosensor has been applied to detect the PCR amplified samples and the results shows that it can discriminate successfully perfect matched DNA from mutant form.