A fluorescence biosensor for detecting LncRNA MALAT1 based on isothermal amplification by cyclic extension

Background

Long non-coding RNA (lncRNA) Metastasis-Associated Lung Adenocarcinoma Transcript 1 (MALAT1), a crucial regulator of gene expression, has emerged as a highly promising biomarker in the progression of various cancers. The clinical detection of lncRNA MALAT1 primarily relies on Reverse Transcription-Polymerase Chain Reaction (RT-PCR), which requires skilled operators and large, expensive thermal cycling equipment. These limitations have restricted the application of RT-PCR, particularly in resource-constrained settings.

Results

In this study, we developed a novel signal amplification method, termed Isothermal Amplification by Cyclic Extension (IACE), based on the linear extension of a single-stranded DNA probe. IACE operates through the continuous extension of Probe 1 (a) into long single-stranded DNA with multiple repetitive sequences, facilitated by Probe 2 (a∗a∗) and Bst DNA polymerase. We found that the single-stranded DNA product of IACE could directly activate the CRISPR-Cas12a system without requiring a protospacer adjacent motif (PAM). By integrating IACE with a three-way junction structure and a nicking enzyme, we established a one-step signal amplification strategy for the detection of lncRNA MALAT1, achieving a detection limit as low as 37.5 fM using the CRISPR-Cas system.

Significance

The biosensor developed in the present study simplifies workflows, minimizes contamination risks, and demonstrates exceptional detection performance in tumor patient samples, highlighting its potential to advance clinical tumor diagnostic approaches.