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.     

Protein-Mediated Suppression of Rolling Circle Amplification for Biosensing with an Aptamer-Containing DNA Primer

We report a method to detect proteins via suppression of rolling circle amplification (RCA) by using an appropriate aptamer as the linear primer (denoted as an aptaprimer) to initiate RCA. In the absence of a protein target, the aptaprimer is free to initiate RCA, which can produce long DNA products that are detected via binding of a fluorescent intercalating dye. Introduction of a target causes the primer region within the aptamer to become unavailable for binding to the circular template, inhibiting RCA. Using SYBR Gold or QuantiFluor dyes as fluorescent probes to bind to the RCA reaction product, it is possible to produce a generic protein-modulated RCA assay system that does not require fluorophore- or biotin-modified DNA species, substantially reducing complexity and cost of reagents. Based on this modulation of RCA, we demonstrate the ability to produce both solution and paper-based assays for rapid and quantitative detection of proteins including platelet derived growth factor and thrombin.     

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.     

Self‐assembly of DNA Origami Using Rolling Circle Amplification Based DNA Nanoribbons

During the development of structural DNA nanotechnology, the emerging of scaffolded DNA origami is marvelous. It utilizes DNA double helix inherent specificity of Watson‐Crick base pairing and structural features to create self‐assembling structures at the nanometer scale exhibiting the addressable character. However, the assembly of DNA origami is disorderly and unpredictable. Herein, we present a novel strategy to assemble the DNA origami using rolling circle amplification based DNA nanoribbons as the linkers. Firstly, long single‐stranded DNA from Rolling Circle Amplification is annealed with several staples to form kinds of DNA nanoribbons with overhangs. Subsequently, the rectangle origami is formed with overhanged staple strands at any edge that would hybridize with the DNA nanoribbons. By mixing them up, we illustrate the one‐dimensional even two‐dimensional assembly of DNA origami with good orientation.     

Biosensing by Tandem Reactions of Structure Switching,Nucleolytic Digestion,and DNA Amplification of a DNA Assembly

?29 DNA polymerase (?29DP) is able to carry out repetitive rounds of DNA synthesis using a circular DNA template by rolling circle amplification (RCA). It also has the ability to execute 3′–5′ digestion of single‐stranded but not double‐stranded DNA. A biosensor engineering strategy is presented that takes advantage of these two properties of ?29DP coupled with structure‐switching DNA aptamers. The design employs a DNA assembly made of a circular DNA template, a DNA aptamer, and a pre‐primer. The DNA assembly is unable to undergo RCA in the absence of cognate target owing to the formation of duplex structures. The presence of the target, however, triggers a structure‐switching event that causes nucleolytic conversion of the pre‐primer by ?29DP into a mature primer to facilitate RCA. This method relays target detection by the aptamer to the production of massive DNA amplicons, giving rise to dramatically enhanced detection sensitivity.     

Colorimetric Sensing by Using Allosteric‐DNAzyme‐Coupled Rolling Circle Amplification and a Peptide Nucleic Acid–Organic Dye Probe

Target detection by the naked eye : The action of an RNA‐cleaving allosteric DNAzyme in response to ligand binding was coupled to a rolling circle amplification process to generate long single‐stranded DNA molecules for colorimetric sensing (see scheme). Upon hybridization of the resulting DNA with a complementary PNA sequence in the presence of a duplex‐binding dye, the color of the dye changed from blue to purple.

     

滚环扩增介导的核酸信号放大策略在生物传感器中的应用研究进展

滚环扩增(RCA)是一种等温核酸扩增技术,因其具有扩增效率高、保真度高等特点,在生物传感器领域得到了广泛的应用。该文介绍了RCA技术的基本原理、RCA环状模板环化方式和RCA的扩增类型,并对基于RCA技术的荧光、比色以及电化学生物传感器进行了介绍,旨在为RCA技术在生物传感器领域的进一步发展和应用提供参考。     

DNA的滚环扩增合成研究

滚环扩增(RCA)反应作为一种简单高效的等温酶促反应,现已发展为核酸扩增领域的新技术,其产物在组装体搭建和多功能材料的制备方面有着广泛的应用。本文采用琼脂糖凝胶、紫外和透射电镜(TEM)等手段,探究了时间、三磷酸脱氧核糖核苷(dNTPs)、酶以及引物的浓度等因素对脱氧核糖核酸(DNA)滚环扩增产物的影响。结果表明:在反应开始的前30 min,RCA产物的长度受时间的影响比较明显;随着dNTPs浓度的提高,RCA产物的链长增长,浓度也不断提高;酶和引物的浓度对滚环扩增产物的长度没有明显影响,但对RCA产物浓度的影响较大,过量的酶致使RCA产物的含量显著下降。     

In Vitro Selection of pH‐Activated DNA Nanostructures

We report the first in vitro selection of DNA nanostructures that switch their conformation when triggered by change in pH. Previously, most pH‐active nanostructures were designed using known pH‐active motifs, such as the i‐motif or the triplex structure. In contrast, we performed de novo selections starting from a random library and generated nanostructures that can sequester and release Mipomersen, a clinically approved antisense DNA drug, in response to pH change. We demonstrate extraordinary pH‐selectivity, releasing up to 714‐fold more Mipomersen at pH 5.2 compared to pH 7.5. Interestingly, none of our nanostructures showed significant sequence similarity to known pH‐sensitive motifs, suggesting that they may operate via novel structure‐switching mechanisms. We believe our selection scheme is general and could be adopted for generating DNA nanostructures for many applications including drug delivery, sensors and pH‐active surfaces.     

支化滚环扩增均相光散射检测单核苷酸多态性

结合简便、高效的支化滚环扩增反应与光散射技术,建立了一种均相、无标记检测单核苷酸多态性的方法.通过设计与突变型DNA完全匹配的锁式探针,突变型DNA可作为模板,使锁式探针在连接酶作用下于45℃连接成环,由Phi29 DNA聚合酶在33℃引发支化滚环扩增反应,生成大量的长链DNA产物.反应产物在0.2 mol/L HCl...     

Engineering Micrometer-Sized DNA Tracks for High-Speed DNA Synthesis and Biosensing

φ29 DNA polymerase (Polφ29) is capable of synthesizing long-chain single-stranded (ss) DNA molecules by copying the sequence of a small ss circular DNA template (ssCDT) in a process known as rolling circle amplification (RCA). The use of a ssCDT in RCA, however, comes with a key drawback: the rate of DNA synthesis is significantly reduced. We hypothesize that this issue can be overcome using a very long linear ssDNA template with a repeating sequence. To test this idea, we engineered a DNA assembly, which we denote “micrometer-sized DNA track” (μDT). This μDT, with an average length of ≈13.5 μm, is made of a long chain DNA with a primer-binding domain at its 3′ end and ≈1000 repeating sequence units at its 5′ end, each carrying a DNA anchor. We find that Polφ29 copies μDT at a speed ≈5-time faster than it does a related ssCDT. We use this to design a simple all-in-one printed paper device for rapid and sensitive detection of microRNA let-7. This paper sensor is capable of detecting 1 pM let-7a in 10 minutes.     

Simple and Sensitive Electrochemical DNA Detection of Primer Generation‐Rolling Circle Amplification

A new electrochemical sequence‐specific DNA detection platform based on primer generation‐rolling circle amplification (PG‐RCA), methylene blue (MB) redox indicator, and indium tin oxide (ITO) electrode is reported. In the presence of a specific target sequence, PG‐RCA, an isothermal DNA amplification technique, produced large amounts of amplicons in an exponential manner. In addition to the standard components, the reaction mixture contained MB, which bound with the PG‐RCA amplicons. End‐point electrochemical measurement by differential pulse voltammetry (DPV) was performed using ITO electrode. The amplicon‐bound MB resulted in a lower DPV signal than free MB due to a smaller diffusion coefficient as well as electrostatic repulsion between the negatively charged amplicon‐bound MB and ITO electrode. With simple assay design (recognition probe) and instrumentation (operating temperature at 37 °C and ITO electrode without the need for probe immobilization), this detection platform is well suited for point‐of‐care and on‐site testing. Real‐time measurement was also achieved by pretreating the ITO electrode with bovine serum albumin.     

滚环扩增技术在病原微生物检测中的应用

滚环扩增（RCA）技术是一种简单的恒温DNA扩增技术,在DNA聚合酶的催化下通过扩增闭合环状模板产生成千上万的重复序列。相较于变温核酸扩增技术如聚合酶链式反应（PCR）,RCA无需昂贵的变温仪器,更适合现场检测。该文介绍了RCA技术的原理和分类,综述了其在细菌、病毒以及其它病原微生物检测方面的应用现状,并展望了RCA检测病原微生物的应用前景。RCA在检测病原微生物领域有着巨大潜力,同时可为新型冠状病毒（SARS－CoV－2）的快速检测提供思路和补充。     

Highly Sensitive Determination of microRNA Using Target‐Primed and Branched Rolling‐Circle Amplification

One‐nucleotide differences in microRNAs (miRNAs) can be discriminated in an assay based on a branched rolling‐circle amplification (BRCA) reaction and fluorescence quantification. With the proposed method miRNA can be detected at concentrations as low as 10 fM , and the miRNA in a total RNA sample of a few nanograms can be determined.

     

Self‐Assembled Functional DNA Superstructures as High‐Density and Versatile Recognition Elements for Printed Paper Sensors

Micrometer‐sized functional nucleic acid (FNA) superstructures (denoted as 3D DNA) were examined as a unique class of biorecognition elements to produce highly functional bioactive paper surfaces. 3D DNA containing repeating sequences of either a DNA aptamer or DNAzyme was created from long‐chain products of rolling circle amplification followed by salt aging. The resulting 3D DNA retained its original spherical shape upon inkjet printing and adhered strongly to the paper surface via physisorption. 3D DNA paper sensors showed resistance to degradation by nucleases, suppressed nonspecific protein adsorption, and provided a much higher surface density of functional DNA relative to monomeric FNAs, making such species ideally suited for development of paper‐based biosensors.     

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.     

Patterned Paper Sensors Printed with Long‐Chain DNA Aptamers

There is growing interest in developing printable paper sensors to enable rapid testing of analytes for environmental, food safety, and clinical applications. A major challenge is to find suitable bioinks that are amenable to high‐speed printing and remain functional after printing. We report on a simple and effective approach wherein an aqueous ink composed of megadalton‐sized tandem repeating structure‐switching DNA aptamers (concatemeric aptamers) is used to rapidly create patterned paper sensors on filter paper by inkjet printing. These concatemeric aptamer reporters remain immobilized at the point of printing through strong adsorption but retain sufficient segmental mobility to undergo structure switching and fluorescence signaling to provide both qualitative and quantitative detection of small molecules and protein targets. The convenience of inkjet printing allows for the patterning of internally referenced sensors with multiplexed detection, and provides a generic platform for on‐demand printing of sensors even in remote locations.     

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.