Single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification for sensitive and specific detection of DNA methyltransferase

Abstract

DNA methylation is a predominant epigenetic modification that plays crucial roles in various cellular processes. DNA methyltransferase (MTase) is responsible for DNA methylation, and its dysregulation may induce aberrant methylation patterns that are closely related to cancers. Conventional methods for DNA MTase assay are usually cumbersome and laborious with poor sensitivity. Alternatively, some signal amplification strategies are employed to improve the sensitivity, but they suffer from poor specificity and consequently limited sensitivity due to the nonspecific amplification. Herein, we develop for the first time a new fluorescence method to specifically and sensitively detect DNA MTase activity on the basis of single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. In the presence of DNA MTase, the hairpin substrate is methylated and cleaved by endonuclease Dpn I, releasing a 24-nt cleavage product. The 24-nt cleavage product may function as a primer and adjacently hybridize with the ligation probes (LP1 and LP2) to form the template (LP1-LP2) for strand displacement amplification (SDA), initiating the single-ribonucleotide repair-mediated cyclic ligation-dependent SDA to produce a large number of reporter probes. The reporter probe can subsequently hybridize with the signal probe that is modified with FAM and BHQ1 to form a stable double-stranded DNA (dsDNA) duplex with a ribonucleotide mismatch. Ribonuclease HII (RNase HII) can excise the single ribonucleotide, resulting in the cyclic cleavage of signal probes and the generation of an enhanced fluorescence signal. Taking advantage of the high specificity of RNase HII-catalyzed single-ribonucleotide excision and the high amplification efficiency of cyclic ligation-dependent SDA, this assay exhibits the highest sensitivity reported so far with a detection limit of 4.8 × 10^-6 U mL^-1 and a large dynamic range of 5 orders of magnitude. Moreover, this method can be used for the discrimination of Dam MTase from other DNA MTases, the accurate quantification of Dam MTase activity in E. coli cells, and the screening of Dam MTase inhibitors, providing a new paradigm for biomedical research and clinical diagnosis.

Scheme 1. Schematic illustration of DNA MTase assay based on single-ribonucleotide repair-mediated ligation-dependent cycling signal amplification. This assay involves three reaction steps: (1) Dam MTase-directed cleavage of hairpin substrates, (2) single-ribonucleotide repair-mediated cyclic ligation-dependent SDA, and (3) RNase HII-catalyzed cyclic cleavage of signal probes for the generation of a distinct fluorescence signal.

Fig. 1. Mechanism of RNase HII-catalyzed single-ribonucleotide repairing. The single ribonucleotide misincorporated within the 5′-DNA-RNA-DNA-3′/3′-DNA-5′duplexes can be specifically recognized and excised by RNase HII through hydrolyzing the phosphodiester bonds 5′ to the ribonucleotide at the DNA–RNA junction, leaving a single nucleotide gap with 5′ PO₄ and 3′ OH ends.

Fig. 2. (A) Nondenaturing PAGE analysis of the products of the Dam MTase-directed cleavage reaction under different experimental conditions. Lane M, the DNA ladder marker; lane 1, in the absence of Dam MTase and Dpn I; lane 2, in the absence of Dpn I; lane 3, in the absence of Dam MTase; lane 4, in the presence of Dam MTase and Dpn I. SYBR gold is used as the fluorescent indicator. (B) Nondenaturing PAGE analysis of the products of the single-ribonucleotide repair-mediated cyclic ligation-dependent SDA reaction under different experimental conditions. Lane M, the DNA ladder marker; lane 1, in the absence of Dam MTase; lane 2, in the presence of Dam MTase; lane 3, the synthesized LP1; lane 4, the synthesized LP2; lane 5, the synthesized hairpin substrate. A silver staining kit is used to stain the gel. (C) Measurement of the fluorescence signal in the presence of Dam MTase (red curve) and in the absence of Dam MTase (control, black curve). The concentration of Dam MTase is 40 U mL^–1.

Fig. 3. (A) Fluorescence spectra in response to different-concentration Dam MTase. (B) Variance of fluorescence intensity with the concentration of Dam MTase from 1 × 10^–4 to 40 U mL^–1. The inset shows the linear relationship between the fluorescence intensity and the logarithm of Dam MTase concentration from 1 × 10^–4 to 10 U mL^–1. Error bars show the standard deviations of three experiments.

Fig. 4. Measurement of fluorescence intensity in response to the reaction buffer (control, black column), 40 U mL^–1 M.CviPI (green column), 40 U mL^–1 M.SssI (blue column), and 40 U mL^–1 Dam MTase (red column), respectively. Error bars show the standard deviation of three independent experiments.

Fig. 5. Variance of the relative activity of Dam MTase in response to different-concentration gentamycin. The inset shows the chemical structure of gentamicin. The concentration of Dam MTase is 40 U mL^–1. Error bars show the standard deviation of three experiments.

Fig. 6. (A) Measurement of Dam MTase in lysis buffer (control, black column), JM110 (green column) and GW5100 (red column) E. coli cells, respectively. The total protein concentration is 0.015 mg mL^–1 for GW5100 and 0.015 mg mL^–1 for JM110 E. coli cells, respectively. (B) Variance of the fluorescence intensity with different-concentration total protein extracted from GW5100 E. coli cells. The inset shows the linear relationship between the fluorescence intensity and the logarithm of the total protein concentration of GW5100 in the range from 1.5 × 10^–4 to 4.5 × 10^–2 mg mL^–1. Error bars represent the standard deviation of three independent experiments.