Rolling circle extension-assisted loop-mediated isothermal amplification (Rol-LAMP) method for locus-specific and visible detection of RNA N6-methyladenosine

Abstract

N6-methyladenosine (m6A) is the most prevalent RNA modification in eukaryotic mRNAs. Currently available detection methods for locus-specific m6A marks rely on RT-qPCR, radioactive methods, or high-throughput sequencing. Here, we develop a non-qPCR, ultrasensitive, isothermal, and naked-eye visible method for m6A detection based on rolling circle amplification (RCA) and loop-mediated isothermal amplification (LAMP), named m6A-Rol-LAMP, to verify putative m6A sites in transcripts obtained from the high-throughput data. When padlock probes hybridize to the potential m6A sites on targets, they are converted to circular form by DNA ligase in the absence of m6A modification, while m6A modification hinders the sealing of padlock probes. Subsequently, Bst DNA polymerase-mediated RCA and LAMP allow the amplification of the circular padlock probe to achieve the locus-specific detection of m6A. Following optimization and validation, m6A-Rol-LAMP can ultra-sensitively and quantitatively determine the existence of m6A modification on a specific target site as low as 100 amol under isothermal conditions. Detections of m6A can be performed on rRNA, mRNA, lincRNA, lncRNA and pre-miRNA from biological samples with naked-eye observations after dye incubation. Together, we provide a powerful tool for locus-specific detection of m6A, which can simply, quickly, sensitively, specifically, and visually determine putative m6A modification on RNA.

Enhancing 3D DNA Walker-Induced CRISPR/Cas12a Technology for Highly Sensitive Detection of ExomicroRNA Associated with Osteoporosis

“…Fortunately, in order to address these challenges, a wide range of isothermal amplification strategies have been extensively developed, including rolling circle amplification (RCA), 1 4 , 1 5 exponential isothermal amplification (EXPAR), 16,17 hybridization chain reaction (HCR), 18,19 and catalytic hairpin assembly (CHA). 20−23 However, the DNA walker, as an intriguing type of nanodevices inspired by the concept of natural walker, represents a dynamic molecular system comprised of DNA strands that autonomously move along a predetermined track, leading to improve the local concentration and signal amplification.…”

“…Fortunately, in order to address these challenges, a wide range of isothermal amplification strategies have been extensively developed, including rolling circle amplification (RCA), , exponential isothermal amplification (EXPAR), , hybridization chain reaction (HCR), , and catalytic hairpin assembly (CHA). − However, the DNA walker, as an intriguing type of nanodevices inspired by the concept of natural walker, represents a dynamic molecular system comprised of DNA strands that autonomously move along a predetermined track, leading to improve the local concentration and signal amplification. − The ability of DNA walkers to perform programmable and controllable motion has significant implications in various fields, including nanoscale assembly, molecular biosensor, programmed synthesis, and so on. − Furthermore, compared to one-dimensional (1D) and two-dimensional (2D) track DNA walkers, the three-dimensional (3D) track DNA walker has exhibited remarkable advancements in nucleic acid amplification, featuring a higher walking efficiency, improving the probe loading capacity, and synchronously enhancing sensitivity. , Therefore, by employing a well-designed track, precise identification and multicycle amplification of target exomiRNAs can be achieved for the efficacious detection of exomiRNAs.…”

Isothermal Amplification-Based Detection of Single-Base RNA N6-Methyladenosine

“…When increasing the oligo mixture from 1.25 to 20 pmol, m 6 A-MazF-SAT can distinguish as low as 10% m 6 A (125 fmol; Figure C) to 1% m 6 A (2 fmol; Figure S7) in 200 min. Next, Oligo-1-m 6 A ranging from 100 fmol to 1.5 pmol was tested, which covers most of the RNA targets in biological samples. , All tested oligo amounts can provide m 6 A-MazF-SAT amplification signals, and the fluorescent results exhibited a good linear relationship against the oligo amounts (Figures D and S6B), the equation of which was the relative fluorescence units (RFU) = 0.0083 × m – 0.1688 with R 2 = 0.9857.…”

“…Subsequently, Li et al developed a locusspecific and visible m 6 A detection method, m 6 A-Rol-LAMP, which can qualify and quantify single-base m 6 A targets. 19 Benefiting from the isothermal amplification, m 6 A-Rol-LAMP is sensitive and easy to operate; however, a certain degree of false positive signal remained to be addressed to achieve more accurate m 6 A detection.…”

“…The most commonly used m 6 A detection methods are mass spectrometry as well as high-throughput sequencing based on m 6 A antibody enrichment, such as MeRIP-seq and miCLIP-seq, which can provide the approximate localization of m 6 A or single-base recognition of m 6 A but with complex procedure, respectively. − Recently, locus-specific m 6 A detection methods (e.g., SCARLET, SELECT, and AD-seq) have been developed. − However, SCARLET requires radioactive labeling, which is challenging to operate and time-consuming; SELECT based on the features of m 6 A selectively hinders single-base elongation and nick ligation, which is prone to false-positive results. Subsequently, Li et al developed a locus-specific and visible m 6 A detection method, m 6 A-Rol-LAMP, which can qualify and quantify single-base m 6 A targets . Benefiting from the isothermal amplification, m 6 A-Rol-LAMP is sensitive and easy to operate; however, a certain degree of false positive signal remained to be addressed to achieve more accurate m 6 A detection.…”