Programmable Ligation-Transcription Circuit-Driven Cascade Amplification Machinery for Multiple Long Noncoding RNAs Detection in Lung Tissues

Abstract

The measurement of long noncoding RNAs (lncRNAs) is essential to diagnosis and treatment of various diseases such as cancers. Herein, we develop a simple method to simultaneously detect multiple lncRNAs using programmable ligation-transcription circuit-driven cascade amplification and single-molecule counting. The presence of targets lncRNA HOTAIR and lncRNA MALAT1 activates the ligation-transcription circuits to produce two corresponding functional RNAs. The functional RNAs then cyclically initiate the digestion of signal probes by duplex-specific nuclease to liberate Cy5 and Cy3 molecules. After magnetic separation, the liberated Cy5 and Cy3 molecules are measured by single-molecule counting. In this assay, a single lncRNA can activate ligation-transcription circuit to generate abundant functional RNAs, endowing this assay with high sensitivity. Integration of single-molecule counting ensures the high sensitivity. This method shows extremely high sensitivity with a limit of detection (LOD) of 0.043 aM for HOX gene antisense intergenic RNA (lncRNA HOTAIR) and 0.126 aM for mammalian metastasis-related lung adenocarcinoma transcript 1 (lncRNA MALAT1). Importantly, this method enables simultaneous measurement of multiple endogenous lncRNAs at the single-cell level, and it may discriminate the expressions of various lncRNA in lung tumor tissues of nonsmall cell lung cancer (NSCLC) patients and their corresponding healthy adjacent tissues, offering a promising platform for clinical diagnosis and biomedical research.

Mix-and-Detection Assay with Multiple Cyclic Enzymatic Repairing Amplification for Rapid and Ultrasensitive Detection of Long Noncoding RNAs in Breast Tissues

“…MALAT1 specifically localizes in nuclear speckles, and its expression level increases in most human cancers. 26 In this assay, we designed a 3WJ primer, a 3WJ template, and a signal probe (Scheme 1). The 3WJ template with a NH 2 modification at the 3′ terminus can avoid the nonspecific polymerization.…”

“…LncRNA MALAT1 is used as a model target. MALAT1 specifically localizes in nuclear speckles, and its expression level increases in most human cancers . In this assay, we designed a 3WJ primer, a 3WJ template, and a signal probe (Scheme ).…”

“…But the involvement of precise temperature cycling control, complicated sample pretreatment, and inevitably false positive limits their clinical applications. Alternatively, a variety of new strategies based on nucleic acid isothermal amplification have been developed to enhance sensitivity, including reverse transcription recombinase polymerase amplification (RT-RPA), reverse transcription loop-mediated isothermal amplification (RT-LAMP), hybridization chain reactions (HCR), , DSN-mediated signal amplification, − rolling circle amplification (RCA), and T7 RNA polymerase transcription-mediated amplification, ,− but these methods usually involve reverse transcription steps, the design of multiple complex probes, time-consuming separation/washing operations, and multiple reaction steps. Hence, the development of rapid and simple methods for lncRNA detection is highly demanded.…”

Long Non-Coding RNA Detection Based on Multi-Probe-Induced Rolling Circle Amplification for Hepatocellular Carcinoma Early Diagnosis

“…Nucleic acid isothermal amplification has also been widely used in RNA detection, such as RCA, LAMP, and SDA, to detect miRNA . At present, the applications of isothermal nucleic acid amplification methods in lncRNA detection are very little, because the large size of lncRNA cannot seve as primers, which is the key reason why isothermal nucleic acid amplification is not often used in lncRNA detection. , Therefore, most of the current schemes used for lncRNA detection are electrochemical methods such as the electrochemical biosensor based on PtPd nanodendrite/nano-flowerlike@GO signal amplification for the detection of lncRNA in serum and an electrochemical sensor based on a composite interface of graphene-like tungsten disulfide/dendritic gold nanostructures (WS2/DGN) to detect lncRNA by using the RNase a-assisted target cycle and the DNA hypertrimer-induced signal enhancement . The electrochemical methods mentioned above involve the use of synthetic materials to improve the detection sensitivity, and the operation steps are complex and time-consuming.…”

Corn-Based Fluorescent Light-Up Biosensors with Improved Signal-to-Background Ratio for Label-Free Detection of Long Noncoding RNAs

“…Up to now, sensitive monitoring of lncRNAs remains a challenge because of their extensive secondary structures, large sizes, and low concentrations in biofluids . In addition to classical microarray and quantitative polymerase chain reaction (qRT-PCR), , various lncRNA assays based on colorimetry, fluorescence, , and electrochemistry have been developed . Among them, fluorescence-based biosensors are the most popular owing to their distinct benefits of simple operation, high sensitivity, and easy readout .…”