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.