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    Rolling Circle Amplification

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    Rolling circle amplification (RCA) is an isothermal enzymatic process in which a short nucleic acid primer is amplified to form a long single-stranded nucleic acid using a circular template and special nucleic acid polymerases. Furthermore, this approach can be further engineered into a device for point-of-need monitoring of environmental pollutants.

    1. Introduction

    In recent years, the discharge of contaminants from industrial and agricultural activities and urban wastewater has caused serious contamination of the aqueous system, posing a great potential threat to human health and aquatic life. These contaminants can be divided into three categories: (i) inorganic chemical substances, (ii) organic pollutants and (iii) microorganisms. These substances can cause adverse effects on the environment [1][2][3][4], for example, the disruption of hormones and the endocrine system and the induction of cytotoxicity and/or genotoxicity and carcinogenesis [5][6]. The variable composition of pollutants and their location in aqueous environments over time have resulted in increasing focus on new technologies that use cheap and real-time strategies to monitor pollutants. Most of these strategies are based on laboratory platforms, such as inductively coupled plasma mass spectrometry (ICP-MS) for the detection of heavy metal ions, liquid chromatography-tandem mass spectrometry (LC-MS) for the detection of small organic chemicals or their metabolites, and polymerase chain reaction (PCR) for the detection of nucleic acids and genetic information, which require preprocessing and frequent data sampling, which means that they are both expensive and slow. These aspects highlight the need to develop a new strategy that is more sensitive, portable, and efficient for on-site detection of pollutants composed of multiple substances [7][8][9][10].

    Recently, rolling circle amplification (RCA)-based analytical methods have received increasing attention in environmental monitoring. RCA is an uncomplicated and efficient isothermal enzymatic process using unique DNA and RNA polymerases to produce long single-stranded DNA (ssDNA) and RNA [11][12]. In RCA, the polymerase will spontaneously and continuously add nucleotides to the primers that bind to the circular template, generating long ssDNA with tandem repeats of tens to hundreds of orders of magnitude. Unlike PCR, which requires a thermal cycler and thermostable DNA polymerase. RCA can be in solution, on a solid support, or in a complex biological environment at a constant temperature (room temperature to 37 °C). The ability of RCA to grow a long DNA chain on a solid support or inside a cell from one molecular binding event enables the detection of targets at a single molecule level [13][14][15]. In addition, an RCA product comprising repeating cyclic sequences complementary to template DNA can be customized by template design. By designing the template, the customizable DNA product includes functional sequences, including aptamers, DNAzymes, spacer domains, and restriction endonuclease sites. Of course, by hybridizing the RCA product with a complementary nucleic acid linked to a functional part including biotin [16][17], fluorophores [18][19], antibodies [20], and nanoparticles [21][22][23][24], it is easy to synthesize a multifunctional material with a variety of properties, including biorecognition and biosensing. Collectively, the properties of high-efficiency isothermal amplification, single-molecule sensitivity, versatility of structure and composition, and multivalences make RCA a powerful tool in aqueous environments [25][26][27]. Currently, RCA has been extensively studied to develop sensitive methods for detecting DNA, RNA, DNA methylation, single nucleotide polymorphisms, small molecules, proteins, and cells. In addition to diagnosis, RCA has also been proven to be effective for cell-free cloning and sequencing [28][29], in situ genotyping and genome-wide analysis of cells and tissues [30][31][32][33][34]. Recently, RCA has received widespread attention for its use in the production of DNA nanostructures such as origami, nanoribbons, nanotubes, DNA nanoscaffolds, and DNA metamaterials for periodic nanocomponents [11][35][36][37][38]. Importantly, these materials have high prospects in a wide range of applications, including environmental monitoring, drug delivery, and in vivo imaging of manufacturing electronic circuits, including DNA-based materials.

    2. Advantages and Disadvantages of the RCA Assay

    2.1. Fundamentals of RCA

    The RCA reaction typically requires four components: (1) DNA polymerase (e.g., Phi29 DNA polymerase), which includes an appropriate buffer; (2) a short nucleic acid primer; (3) a circular DNA template; and (4) deoxynucleotide triphosphate (dNTP) (monomer or structural unit of RCA product) [11][39][40][41]. In polymerases, Phi29 DNA polymerase is most commonly used because of its excellent capability and continuous strand displacement synthesis capability. Phi29 can handle topological constraints, four-way cross connections, and multiple circular DNA template complexes [39][42][43]. For RCA primers, both RNA and DNA (usually the “target” molecule to be detected) can achieve this goal. Indeed, the target DNA and RNA can be used to connect the first template mediated as a padlock probe (PLP) using RCA reaction circular template cyclizing [44][45]. The circular DNA template (usually 15–200 nucleotides (NT) in length) is a component that can be enzymatically or chemically synthesized through intramolecular phosphate and hydroxyl end groups. Most commonly, the template is a circular DNA template mediated by enzymatic ligation (e.g., T4 DNA ligase) or the use of a special DNA ligase enzyme with a template-free connection to a synthetic CircLigase [15][46]. By designing primers and circular templates, RCA product length, sequence, composition, structure and rigidity may be appropriately adjusted, thereby becoming a highly versatile RCA technique (summarized in Table 1).
    Table 1. Comparison of RCA, PCR and real-time PCR for the detection of DNA.
    Features Conventional PCR Assay Real Time-PCR Assay RCA Assay
    Sensitivity Sensitive Highly sensitive Highly sensitive
    Specificity Specific Specific Specific
    Temperature conditions Thermal cycle Thermal cycle Isothermal
    Inhibition by biological samples Yes Yes No
    Instruments required Thermocycler Thermocycler Not required
    Post-assay analysis Required Required Generally not required
    Amplicon detection methods Gel electrophoresis Real-time detection/amplification graph Gel electrophoresis, Turbidity measurement by visual inspection or using a real-time turbidimeter; dye-based visual detection
    Qualitative detection Yes Yes Yes
    Quantitative detection No Yes Semi-quantitative
    Portability Partially Yes Yes
    Overall assay time 3–5 h 2.5–4 h 1–1.5 h
    Cost effectiveness Less expensive Expensive Less expensive

    2.2. Exponential RCA Amplification

    One of the powerful functions of RCA is the ability to design a circular template so that the signal generated by a single binding event is exponentially amplified [47][48][49]. Using a plurality of primers hybridizing to the same ring can lead to amplification of a plurality of events, thereby producing a plurality of RCA products [50][51] (Figure 1). The number of primers that one circular template can accommodate depends on the length of the primers and the circle. Another method for exponential amplification of RCA uses a so-called hyperbranched RCA (HRCA) (branched or amplification) method, in which the RCA product used as a template for the second and third groups is further expanded using primers [52][53][54][55]. Note that a primer can be integrated into the hyperbranched RCA method to increase the sensitivity, especially when the target is detected at low abundance [56]. Additionally, restriction enzyme digestion followed by enzymatic ligation template-mediated, linear RCA products may be converted to a variety of cyclic products [57][58]. A second set of primers may then be used to incorporate these new cyclic products for further amplification. This “circle to circle amplification” restriction digestion process, cyclization and amplification may be repeated for additional amplification. Finally, after hybridization with a second set of circles, the RCA product may be treated with nicking enzyme to generate a plurality of primers. The hybridized primer/circular template product obtained from the nickase reaction can be directly used for the next cycle of RCA amplification.
    Biosensors 11 00352 g001

    This entry is adapted from 10.3390/bios11100352


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