Label-Free miRNA-21 Analysis Based on Strand Displacement and Terminal Deoxynucleotidyl Transferase-Assisted Amplification Strategy

Abstract

MicroRNAs (miRNAs) are regarded as a rising star in the biomedical industry. By monitoring slight increases in miRNA-21 levels, the possibilities of multi-type malignancy can be evaluated more precisely and earlier. However, the inconvenience and insensitivity of traditional methods for detecting miRNA-21 levels remains challenging. In this study, a partially complementary cDNA probe was designed to detect miRNA-21 with target-triggered dual amplification based on strand displacement amplification (SDA) and terminal deoxynucleotidyl transferase (TdT)-assisted amplification. In this system, the presence of miRNA-21 can hybridize with template DNA to initiate SDA, generating a large number of trigger molecules. With the assistance of TdT and dGTP, the released trigger DNA with 3′-OH terminal can be elongated to a superlong poly(guanine) sequence, and a notable fluorescence signal was observed in the presence of thioflavin T. By means of dual amplification strategy, the sensing platform showed a good response tomiRNA-21 with a detection limit of 1.7 pM (S/N = 3). Moreover, the specificity of this method was verified using a set of miRNA with sequence homologous to miRNA-21. In order to further explore its practical application capabilities, the expression of miRNA in different cell lines was quantitatively analyzed and compared with the qRT-PCR. The considerable results of this study suggest great potential for the application of the proposed approach in clinical diagnosis.

Applications of Terminal Deoxynucleotidyl Transferase Enzyme in Biotechnology

“…New approaches for sensitive detection of miRNA have been developed using systems coupled with TdT‐dependent amplification of target signals [62] . A label‐free strategy incorporating strand displacement and TdT‐assisted amplification was used to optimize the detection of miRNA‐21, a key biomarker for several malignancies [63] . Another approach incorporating TdT with copper nanoclusters was used for quantitative detection of miRNA‐21 [64] .…”

“…[62] A label-free strategy incorporating strand displacement and TdT-assisted amplification was used to optimize the detection of miRNA-21, a key biomarker for several malignancies. [63] Another approach incorporating TdT with copper nanoclusters was used for quantitative detection of miRNA-21. [64] A multiple amplification strategy that utilizes TdT target amplification was used for the detection of plant microRNA-162a.…”

Simple Isothermal and Label-Free Strategy for Colorectal Cancer Potential Biomarker miR-625-5p Detection

“…Although typical realtime quantitative PCR (RT-qPCR) can also quantitatively analyze it, the detection results may be false positive for specific miRNA levels, and the process is time-consuming and depends on specific instruments [15,16]. In order to improve the effect of detecting specific miRNA, more and more new detection principles have been proposed, such as rolling circle amplification (RCA) [17,18], strand displacement amplification (SDA) [19,20] and catalyzed hairpin assembly amplification (CHA) [21]. Although these methods can improve efficiency and do not depend on specific instruments, RCA might generate non-specific amplification [22] and CHA is limited by an uncatalyzed reaction called circuit 'leakage' [23].…”