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

Abstract

Long noncoding RNAs (lncRNAs) are valuable biomarkers and therapeutic targets, and they play essential roles in various pathological and biological processes. So far, the reported lncRNA assays usually suffer from unsatisfactory sensitivity and time-consuming procedures. Herein, we develop a mix-and-read assay based on multiple cyclic enzymatic repairing amplification (ERA) for sensitive and rapid detection of mammalian metastasis-associated lung adenocarcinoma transcript 1 (lncRNA MALAT1). In this assay, we design two three-way junction (3WJ) probes including a 3WJ template and a 3WJ primer to specifically recognize lncRNA MALAT1, and the formation of a stable 3WJ structure induces cyclic ERA to generate triggers. The resulting triggers subsequently hybridize with a free 3WJ template and act as primers to initiate new rounds of cyclic ERA, generating abundant triggers. The hybridization of triggers with signal probes forms stable double-stranded DNA duplexes that can be specifically cleaved by apurinic/apyrimidinic endonuclease 1 to produce a high fluorescence signal. This assay can be carried out in a mix-and-read manner within 10 min under an isothermal condition (50 °C), which is the rapidest and simplest method reported so far for the lncRNA MALAT1 assay. This method can sensitively detect lncRNA MALAT1 with a limit of detection of 0.87 aM, and it can accurately measure endogenous lncRNA MALAT1 at the single-cell level. Moreover, this method can distinguish lncRNA MALAT1 expression in breast cancer patient tissues and their corresponding healthy adjacent tissues. Importantly, the extension of this assay to different RNAs detection can be achieved by simply replacing the corresponding target recognition sequences.

Elongation and Ligation-Mediated Differential Coding for Label-Free and Locus-Specific Analysis of 8-Oxo-7,8-dihydroguanine in DNA

“…Recently, a series of enzyme-assisted isothermal amplification strategies (e.g., exponential amplification reaction (EXPAR), 29 rolling circle amplification (RCA), 30 deoxynucleotidyl transferase (TdT)-assisted signal amplification 31,32 and loop-mediated isothermal amplification 33 have been introduced to enhance the sensitivity. Among them, RCA is a high-efficiency and simple amplification method that utilizes a circular template to obtain large amounts of repetitive singlestranded DNAs (ssDNAs).…”

“…Recently, a series of enzyme-assisted isothermal amplification strategies (e.g., exponential amplification reaction (EXPAR), rolling circle amplification (RCA), deoxynucleotidyl transferase (TdT)-assisted signal amplification , and loop-mediated isothermal amplification have been introduced to enhance the sensitivity. Among them, RCA is a high-efficiency and simple amplification method that utilizes a circular template to obtain large amounts of repetitive single-stranded DNAs (ssDNAs). , The specific incorporation of thioflavin T (ThT) into the G-quadruplex chambers can achieve a 2100-fold fluorescence enhancement, and the reasonable introduction of G-quadruplex structures into the amplified product paves the way for label-free and real-time monitoring of RCA reaction. , Despite several ligation-based methods for the detection of modifications in nucleic acids, , the enzymatic synthesis of G-quadruplex for locus-specific analysis of 8-oxoG has not been explored so far.…”

Construction of Genetically Encoded Light-Up RNA Aptamers for Label-free and Ultrasensitive Detection of CircRNAs in Cancer Cells and Tissues

“…Recently, a series of new methods on the basis of isothermal amplification have been adapted for sensitive measurement of circRNAs, including hybridization chain reactions, catalytic hairpin assembly, , rolling circle amplification, , cyclic enzymatic amplification, , and loop-mediated isothermal amplification . However, these strategies typically involve multiple hairpin probes, specific circular templates, and fluorophore-/quencher-labeled signal probes. − Consequently, the construction of new biosensors for rapid and sensitive detection of circRNAs is highly desirable.…”

Target-activated T7 transcription circuit-mediated multiple cycling signal amplification for monitoring of flap endonuclease 1 activity in cancer cells

“…Recently, a variety of isothermal nucleic acid amplification techniques including rolling circle amplification (RCA), 27,28 strand displacement amplification (SDA), 29 exponential isothermal amplification reaction (EXPAR), 30 and loop-mediated isothermal amplification (LAMP), 31 have been introduced into biosensing systems to improve the sensitivity. Superior to DNA amplification, T7 transcription amplification is a novel RNA amplification technique based on isothermally repeated RNA synthesis with a specific T7 promoter, and it can transcribe T7-promoter downstream DNA into abundant ssRNAs within 1 h. 32 T7 transcription has the inherent advantages of excellent specificity, flexible sequence design, and rapid enzyme kinetics, and has been widely applied for the detection of DNAs, RNAs, and proteins.…”