High Specific and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based Exponential Rolling Circle Amplification

Liu, Haiyun; Li, Lü; Duan, Lili; Xu, Wei; Xie, Yanxia; Tong, Lili; Wang, Qian; Tang, Bo

doi:10.1021/ac401715k

Cited by 223 publications

(125 citation statements)

References 51 publications

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“…[19][20][21][22][23][24][25][26] The let-7 family of miRNAs in particular has been shown to play significant roles in ovarian, prostate, liver and pancreatic cancer. [27][28][29][30] Since miRNAs are short, polymerase chain reaction (PCR) cannot be carried out on the unmodified target.…”

Section: Author Manuscript Author Manuscriptmentioning

confidence: 99%

“…[21][22][23][24][25][26] This can in principle be extended yet further with hyperbranched RCA. 11 Isothermal detection of miRNAs via rolling circle templates has been reported previously, using templated fluorogenic chemistry 10,31 or DNA-binding fluorescent dyes [22][23][24][25] to report on the products.Experiments in the presence of ARNs showed that the 22mer let-7a RNA could indeed prime DNA synthesis by the highly processive ɸ29 DNA polymerase, using a 50 nt circular DNA complementary to the miRNA as template. Signal appeared above background for reactions as short as 1 h ( Figure S10), and longer polymerase reactions produced yet greater signals.…”

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confidence: 96%

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ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

Mohsen

Harcourt

et al. 2016

Angewandte Chemie

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We report a new strategy to produce luminescence signals from DNA synthesis by designing chimeric nucleoside tetraphosphate dimers in which ATP, rather than pyrophosphate, is the leaving group. We describe the synthesis of ATP-releasing nucleotides (ARNs) as derivatives of the four canonical nucleotides. We find that the four are good substrates for DNA polymerase, with K m values averaging 13-fold higher than those of natural dNTPs, and k cat values within 1.5-fold of those of native nucleotides. Importantly, ARNs are found to yield very little background signal with luciferase. DNA synthesis experiments show that the ATP byproduct can be harnessed to elicit a chemiluminescence signal in the presence of luciferase. Using a polymerase together with the chimeric nucleotides, target DNAs/RNAs trigger the release of stoichiometrically large quantities of ATP, allowing sensitive isothermal luminescence detection of nucleic acids as diverse as phage DNAs and short miRNAs. TOC imageChimeric dinucleotides were designed to release ATP during polymerase synthesis of DNA. This ATP can be used to generate a signal with luciferase, allowing the sensitive isothermal detection of both large (phage DNA) and small (miRNA) nucleic acids.* kool@stanford.edu.Supporting information for this article is given via a link at the end of the document. The use of luciferase signaling can also provide sensitive reporting of DNA synthesis. This enzyme is employed in the "pyrosequencing" methodology developed for high-throughput DNA sequencing. 8 In this technology, four enzymes are employed, two of them to recycle the pyrophosphate product of the DNA polymerase reaction, generating modified ATP. This method is sensitive, but is also relatively complex, and as a result, the method is not used broadly beyond its application in pyrosequencing instruments. HHS Public AccessFurther improvements in methods for general reporting of polymerase synthesis may be useful in amplified detection of native nucleic acids, in reporting on isothermal amplification methods such as rolling circle amplification (RCA), 9-12 and in future-generation approaches to DNA sequencing. To this end, it would be desirable to take advantage of the high sensitivity and specificity of luciferase in detecting DNA synthesis.Here we describe the design and application of ATP-releasing nucleotides (ARNs, Figure 1) as reporters of DNA synthesis. These tetraphosphate-bridged chimeric RNA-DNA dinucleotides are employed sequentially as substrates for DNA polymerases and for luciferase. In this design, DNA polymerase uses the ARNs to copy a target strand, releasing one equivalent of ATP for every deoxynucleotide incorporated. In a subsequent reaction, luciferase processes the ATP products to generate light signals in the presence of luciferin. In principle, the longer the target nucleic acid molecule, the more signals are generated, thus giving the possibility of high sensitivity.Although dimeric polyphosphate-linked nucleotides are known in the literature, 13-15 the ATP-rele...

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Section: Author Manuscript Author Manuscriptmentioning

confidence: 99%

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confidence: 96%

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

Mohsen

Harcourt

et al. 2016

Angewandte Chemie

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“…Since many important miRNAs are in quite low abundance in biological samples, many nucleic acid amplification techniques have been employed, such as RT-PCR, 6,7 ligase chain reaction, 8,9 ribozyme amplification, 10,11 loop-mediated isothermal amplification (LAMP), 12,13 exponential amplification reaction (EXPAR), 14 and rolling circle amplification (RCA). 15,16 These methods indeed have greatly improved the sensitivity of miRNAs detection; however, they were highly enzyme dependent and label needed. Therefore, the development of simple, label-free, enzyme-free, and nontarget-amplification methods for miRNA analysis is desired.…”

Section: Introductionmentioning

confidence: 99%

Sensitive Colorimetric Detection of MicroRNA Based on Target Catalyzed Double-arm Hairpin DNA Assembling

Tian

Zheng

2016

ANAL. SCI.

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The common drawbacks of the current colorimetric sensing platform using gold nanoparticles (AuNP) as an indictor is its relatively low sensitivity, which restrict their analytical application for low-level analytes, such as the detection of the microRNA (miRNA). In the present work, we developed a novel strategy to construct a colorimetric sensing platform for miRNA based on target catalyzed hairpin DNA assembling. Unlike a single-stranded DNA probe or a single-arm hairpin structure DNA probe, in our strategy the double-arm hairpin structure DNA probe was first designed, and was further demonstrated to work well in catalysis the of hairpin DNA assembly reaction, which significantly enhanced the sensitivity of the AuNP based colorimetric sensing platform. In addition, compared to other miRNA detection schemes reported previously, the proposed strategy is not only enzyme-free, label-free, immobilization-free, but also eliminates the need for any sophisticated instrumentation. The proposed strategy may open a new way to allow miRNAs expression to be profiled in a decentralized setting, such as at point-of-care.

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“…[6] However, it is difficult to analyze the miRNAs because of their unique characteristics, including their small size, sequence homology among family members, and low abundance in total RNA samples. [7] So, the strategies for specific and sensitive quantitative detection of miRNAs are in urgent need. [8][9][10][11] Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is the gold standard for miRNA quantification.…”

Section: Introductionmentioning

confidence: 99%

“…[14][15][16][17] Among these isothermal amplification techniques, rolling circle amplification (RCA) has become increasing popular for the quantitative detection of miRNA. [7,[18][19][20][21][22][23][24][25][26][27] In RCA process, a short DNA (or RNA) is amplified to form a long single-stranded DNA (ssDNA) in the presence of a circular DNA template and special DNA polymerase. The RCA product is a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template.…”

Section: Introductionmentioning

confidence: 99%

Photoinduced Electron Transfer-Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA

et al. 2016

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[a] MicroRNAs (miRNAs) detection is crucial for further understanding the biological functions of miRNAs and early cancer diagnosis and therapeutics, but now remains a great challenge. Here, we present a novel homogenous biosensing strategy for fluorescence detection of miRNA. In this strategy, a preliminary synthesized circular DNA was used as recognition probe for hybridization with miRNA target, and then miRNA target primed a rolling circle amplification (RCA) reaction. The RCA product could be hybridized with thousands of carboxyfluorescein (FAM)-labeled linear DNA probes, which led FAM to be close to -GGG-base of RCA product, accompanying with the significant fluorescence quenching due to photoinduced electron transfer (PET) between FAM and guanine. This is for the first time to integrate RCA and PET into one detection process. With highly efficient amplification of RCA and excellent signal readout of PET, this method exhibited a high sensitivity toward target miRNA with a detection limit of 6 aM. The targetdependent circularization of the padlock probe and the ligation reaction could improve the specificity effectively, leading to discrimination between miRNA family members. This method provides a simple, isothermal, and low-cost approach for sensitive detection of miRNA and holds great potential for early diagnosis in gene-related diseases.

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High Specific and Ultrasensitive Isothermal Detection of MicroRNA by Padlock Probe-Based Exponential Rolling Circle Amplification

Cited by 223 publications

References 51 publications

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

Sensitive Colorimetric Detection of MicroRNA Based on Target Catalyzed Double-arm Hairpin DNA Assembling

Photoinduced Electron Transfer-Based Fluorescence Quenching Combined with Rolling Circle Amplification for Sensitive Detection of MicroRNA

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