A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection

Zhou, Yuntao; Huang, Qing; Gao, Jimin; Lü, Jianxin; Shen, Xizhong; Fan, Chunhai

doi:10.1093/nar/gkq556

Cited by 172 publications

(138 citation statements)

References 36 publications

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“…RTC is less efficient than ordinary rolling-circle amplification (RCA) (Demidov 2002) and, therefore, cannot serve for direct detection of target sequences without the additional signal amplification provided here by qPCR. Although RCA and some other non-isothermal approaches to signal amplification have been previously applied to detection of miRNAs (Allawi et al 2004;Hartig et al 2004;Jonstrup et al 2006;van Huffel et al 2006;Cheng et al 2009;Siva et al 2009;Yao et al 2009;Zhou et al 2010), these methods were less sensitive and accurate than RT-PCR.…”

Section: Introductionmentioning

confidence: 99%

miR-ID: A novel, circularization-based platform for detection of microRNAs

Kumar¹,

Johnston²,

Kazakov³

2010

RNA

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MicroRNAs (miRNAs) are important regulators of gene expression and have great potential as biomarkers, prognostic indicators, and therapeutic targets. Determining the expression patterns of these molecules is essential for elucidating their biogenesis, regulation, relation to disease, and response to therapy. Although PCR-based assays are commonly used for expression profiling of miRNAs, the small size, sequence heterogeneity, and (in some cases) end modifications of miRNAs constrain the performance of existing PCR methods. Here we introduce miR-ID, a novel method that avoids these constraints while providing superior sensitivity and sequence specificity at a lower cost. It also has the unique ability to differentiate unmodified small RNAs from those carrying 29-OMe groups at their 39-ends while detecting both forms. miR-ID is comprised of the following steps: (1) circularization of the miRNA by a ligase; (2) reverse transcription of the circularized miRNA (RTC), producing tandem repeats of a DNA sequence complementary to the miRNA; and (3) qPCR amplification of segments of this multimeric cDNA using 59-overlapping primers and a nonspecific dye such as SYBR Green. No chemically modified probes (e.g., TaqMan) or primers (e.g., LNA) are required. The circular RNA and multimeric cDNA templates provide unmatched flexibility in the positioning of primers, which may include straddling the boundaries between these repetitive miRNA sequences. miR-ID is based on new findings that are themselves of general interest, including reverse transcription of small RNA circles and the use of 59-overlapping primers for detection of repetitive sequences by qPCR.

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Section: Introductionmentioning

confidence: 99%

miR-ID: A novel, circularization-based platform for detection of microRNAs

Kumar¹,

Johnston²,

Kazakov³

2010

RNA

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“…This type of RCA is called RNA-primed RCA (RPRCA) and, indeed, RPRCA is used for microRNA and small RNA detection without reverse transcription. 21,40,[42][43][44][45][46][47][48] When several circular ssDNA probes are tested in detecting in vitro transcribed GFP messenger RNA (mRNA) as a standard RPRCA procedure, the RPRCA reaction is observed under a certain ssDNA probe (Fig. 4).…”

Section: ·2 Rna Detectionmentioning

confidence: 99%

“…When circular DNA probes for vtx1 and vtx2 detection are prepared, RPRCA initiates in test samples with 10 9 copies/mL or more mRNA. On the other hand, circular DNA probes, designed so as to be a dumbbell in shape, 46 successfully improve the detection levels of verotoxin mRNA up to 10 4 copies/mL (data not shown). This is presumably because the dumbbellshaped circular DNA probe would lower any non-specific binding of RNA to the probes.…”

Section: ·3 Challenges For Enterohemorrhagic E Coli Detectionmentioning

confidence: 99%

Expanding Possibilities of Rolling Circle Amplification as a Biosensing Platform

Kobori

Takahashi

2014

ANAL. SCI.

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“…[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.…”

mentioning

confidence: 96%

“…[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.…”

mentioning

confidence: 99%

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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A dumbbell probe-mediated rolling circle amplification strategy for highly sensitive microRNA detection

Cited by 172 publications

References 36 publications

miR-ID: A novel, circularization-based platform for detection of microRNAs

miR-ID: A novel, circularization-based platform for detection of microRNAs

Expanding Possibilities of Rolling Circle Amplification as a Biosensing Platform

ATP‐Releasing Nucleotides: Linking DNA Synthesis to Luciferase Signaling

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