Reverse transcription-based loop-mediated isothermal amplification strategy for real-time miRNA detection with phosphorothioated probes

Highlights

• A novel method of reverse transcription-based miRNA detection has been introduced.

• It is based on the dumbbell-shaped amplicons and inner primers used in LAMP.

• It has been termed as “terminal hairpin formation and self-priming” (THSP) reaction.

• Self-folding efficiency was improved by phosphorothioated modification.

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. Termed “terminal hairpin formation and self-priming” (THSP), this reaction incorporates phosphorothioated (PS) modifications to achieve DNA folding and extension without primers. The final signal is monitored by a sequence-specific detection probe, which minimizes the background noise. We suggest that our rapid, facile, and reliable LAMP method will be a promising candidate for detecting miRNA in biomedical applications.

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.