Reverse transcription-based loop-mediated isothermal amplification strategy for real-time miRNA detection with phosphorothioated probes
Graphical abstract
A novel reverse transcription-based loop-mediated isothermal amplification (LAMP) strategy for miRNA detection has been developed. This method consists of two stem-loop probes inspired by the dumbbell-shaped amplicons and inner primers used in conventional LAMP reactions. The final signal is monitored by a toehold-mediated strand exchange reaction (one-step strand displacement probe), which is able to minimize background noise. We hope this strategy can provide a highly selective and sensitive platform for miRNA detection.
Introduction
MiRNAs are small non-coding RNAs of approximately 19–24 nucleotides in length, which inhibit the translation of proteins by binding to the three prime untranslated region (3′-UTR) of the target mRNA [1]. Studies have increasingly found a close relationship between aberrant expression levels of miRNAs and the development of diverse diseases [2]; thus, miRNAs have been regarded as significant biomarkers for clinical diagnoses. However, the intrinsic characteristics of miRNA, including short sequence lengths, large variability in per-cell copy number, and high sequence similarity within families of expressed miRNAs, make them challenging analytical targets [3,4]. Therefore, it is necessary to establish highly sensitive and selective miRNA detection assays for biomedical and clinical applications. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the near-unanimous choice for quantifying miRNAs due to its high sensitivity [[5], [6], [7], [8]]; however, its drawbacks include a time-consuming analytical process, and a risk for false-positive results as a result of nonspecific amplification [9,10]. Moreover, PCR requires a precise thermal cycling, which is not suitable for point-of-care (POC) diagnostics in the field. In order to overcome these drawbacks, various miRNA detection methods based on isothermal amplification have been developed, such as the rolling circle amplification (RCA) [[11], [12], [13], [14], [15], [16]], duplex-specific nuclease (DSN)-based amplification [[17], [18], [19], [20], [21]], strand-displacement amplification (SDA) [[22], [23], [24], [25], [26]], and hybridization chain reaction (HCR) [[27], [28], [29], [30]]. Among these protocols, the loop-mediated isothermal amplification (LAMP) has attracted particular attention and offered new opportunities for applications in biological analysis because of its rapidness, high sensitivity of detection, simplified operation, and low cost.
LAMP is a highly efficient nucleic acid amplification technique which is performed under isothermal conditions [[31], [32], [33], [34], [35]]. In a conventional LAMP reaction, four primers are designed from an extremely long single-strand DNA (ssDNA) template, which forms a concatemerized amplicon that ensures exponential amplification from a continuous strand displacement DNA synthesis. The final LAMP product comprises large-molecular-weight concatemers that contain self-priming hairpins and four single-stranded free loops. Meanwhile, some studies have reported detection of miRNAs by combining LAMP with other amplification methods [[36], [37], [38], [39], [40]]. However, some drawbacks of the LAMP-based miRNA assays remain. Some methods are based on miRNA-primed synthesis of a dumbbell-shaped initiator for cycling, which may lead to an excessive background noise due to the high reaction temperature required to facilitate strand displacement with backward primers. Some methods use nonspecific signal transduction schemes such as SYBR Green for real-time detection, which is unable to distinguish the side-products of nonspecific amplification.
Herein, we introduced a novel reverse transcription-based LAMP strategy for miRNA detection with a highly simplified probe design. Termed “terminal hairpin formation and self-priming” (THSP) reaction, this method requires no primers for DNA extension; the self-folding at the terminal of the hairpin structure accomplishes this. The efficiency of self-folding has been improved by incorporating phosphorothioates (PS) into the nucleic acid substrate [41,42]. A stem-loop probe (SL probe; used for reverse transcription of target miRNA into cDNA), a hairpin probe with PS modifications (PS–H probe), and a forward/backward inner primer (FIP/BIP) were designed. Target miRNA is reverse transcribed with the SL probe, which induces the PS-H probe to hybridize with the new SL probe, followed by DNA extension at the 3′end of the two probes, which yields a double-stranded DNA structure. Owing to the activity of the PS-H probe, the THSP reaction entails self-folding at the terminal end to form a double stem-loop structure in the DNA. This is the starting material for subsequent LAMP cycles, which use FIP and BIP. Inspired by the dumbbell-shaped amplicons used in LAMP reactions, we creatively designed two detection probes (SL and H probes), which are activated by reverse transcription in the presence of target miRNA. The final signal is monitored by a toehold-mediated strand exchange reaction (termed the one-step strand displacement [OSD] probe), which is able to minimize background noise through its ability to successfully distinguish side-products from true amplicons due to the inherent sequence specificity of toehold-mediated strand exchange (Scheme S1). Hence, the strategy developed here (RT-THSP-LAMP) can provide a highly selective and sensitive platform for miRNA detection.
Section snippets
Material and apparatus
MiRNA 1st Strand cDNA Synthesis Kit was purchased from Vazyme (Nanjing, China), Bst 2.0 DNA polymerase and the corresponding isothermal buffer were purchased from New England Biolabs (USA), dNTPs and RNase-free water were all obtained from Takara (Dalian, China). All nucleic acid in this work including SL probe, H probe, inner primer (FIP, BIP) were synthesized and purified by Sangon Biotech (Shanghai, China). MiRNAs were synthesized and purified by Genepharma (Shanghai, China). The sequences
Design principle of the RT-THSP LAMP assay
This miRNA RT-THSP-LAMP assay consists of two hairpin DNA probes (SL and PS-H) and two primers, which are used in typical LAMP reactions. The principle underlying the amplification is based on the reverse transcription process that occurs in THSP and LAMP reactions. As shown in Scheme 1, the SL probe was first elongated after a reverse transcription reaction with the addition of miRNA. The elongated sequence at the 3′ end of the SL probe was complementary to part of the 3′ end of PS-H probe,
Conclusion
In summary, we have established a novel and versatile assay for the real-time quantification of miRNA with excellent sensitivity and selectivity, which integrates the advantages of LAMP and THSP. Two stem-loop probes inspired by the dumbbell-shaped amplicons and the inner primers used in conventional LAMP reactions have been introduced. With the use of PS-modified THSP, the LAMP reaction can be initiated by the self-folding and extension of DNA in the presence of target miRNA. The final signal
CRediT authorship contribution statement
Abdu Ahmed Abdullah AL-maskri: Methodology, Software, Investigation, Writing - original draft. Jiawei Ye: Formal analysis, Visualization. Jadera Talap: Data curation, Writing - original draft. Haihong Hu: Validation, Formal analysis, Visualization. Lianli Sun: Visualization, Investigation. Lushan Yu: Supervision. Sheng Cai: Writing - review & editing. Su Zeng: Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
We acknowledge financial support from the National Key Project of China (2017YFC0908600), National Natural Science Foundation of China (Grant 81773817), the Fundamental Research Funds for the Central Universities (2019FZA7017), Leading Talent of “Ten Thousand Plan”-National High-Level Talents Special Support Plan.
References (43)
- et al.
Research advances in the detection of miRNA
J. Pharm. Anal.
(2019) - et al.
Droplet digital PCR improves urinary exosomal miRNA detection compared to real-time PCR
Clin. Biochem.
(2019) - et al.
Ultrasensitive detection of miRNA via one-step rolling circle-quantitative PCR (RC-qPCR)
Anal. Chim. Acta
(2019) - et al.
An ultrasensitive electrochemical biosensor for detection of microRNA-21 based on redox reaction of ascorbic acid/iodine and duplex-specific nuclease assisted target recycling
Biosens. Bioelectron.
(2019) - et al.
Ultrasensitive electrochemical microRNA-21 biosensor coupling with carboxylate-reduced graphene oxide-based signal-enhancing and duplex-specific nuclease-assisted target recycling
Sens. Actuators, B
(2019) - et al.
Near-infrared triggered strand displacement amplification for MicroRNA quantitative detection in single living cells
Chem. Sci.
(2018) - et al.
G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection
Anal. Chim. Acta
(2016) - et al.
Rapid and ultrasensitive detection of microRNA based on strand displacement amplification-mediated entropy-driven circuit reaction
Sens. Actuators, B
(2018) - et al.
Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells
Anal. Chim. Acta
(2016) - et al.
A graphene-based biosensor for detecting microRNA with augmented sensitivity through helicase-assisted signal amplification of hybridization chain reaction
Sens. Actuators, B
(2018)
Rolling circle extension-actuated loop-mediated isothermal amplification (RCA-LAMP) for ultrasensitive detection of microRNAs
Biosens. Bioelectron.
Regulation of microRNA biogenesis
Nat. Rev. Mol. Cell Biol.
Oncomirs: the potential role of non-coding microRNAs in understanding cancer
Bioinformation
The discovery approaches and detection methods of microRNAs
Mol. Biol. Rep.
Comprehensive data analysis for development of custom qRT-PCR miRNA assay for glioblastoma: a prevalidation study
Epigenomics
Identification of early lung cancer miRNA biomarkers using qRT-PCR and novel label-free nanoarray technology
Canc. Res.
A novel diagnostic and prognostic biomarker for colorectal cancers: detection of circulating miRNA by quantitative PCR
Biomarkers Med.
Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer
Clin. Chem.
Ultrasensitive quantification of mature microRNAs by real-time PCR based on ligation of a ribonucleotide-modified DNA probe
Chem. Commun.
In situ electrochemical generation of electrochemiluminescent silver naonoclusters on target-cycling synchronized rolling circle amplification platform for microRNA detection
Anal. Chem.
Off" to "On" surface-enhanced Raman spectroscopy platform with padlock probe-based exponential rolling circle amplification for ultrasensitive detection of microRNA 155
Anal. Chem.
Cited by (31)
Multifunctional dumbbell probes-based logic circuits: microRNAs logic detection and tumor cells identification
2023, Analytica Chimica ActaUltrasensitive detection porcine transmissible gastroenteritis virus based on the dual-photoelectrode photofuel cell with in-situ isothermal amplification
2023, Journal of Electroanalytical ChemistrySensitive detection of cancer biomarker with enzyme-free mediated cascade signal amplification empowered undisturbed dual-mode assay
2023, Sensors and Actuators B: ChemicalHigh specific microRNA detection based on G-quadruplex sensitive fluorescence under isothermal amplification
2023, Chinese Journal of Analytical Chemistry
- 1
These authors equally contributed to this work.