Highly sensitive fluorescence assay of DNA methyltransferase activity via methylation-sensitive cleavage coupled with nicking enzyme-assisted signalamplification

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

“…To the best of our knowledge, this is the most sensitive method for DNA MTase assay reported so far. In comparison with the reported DNA MTase assays, 15 , 18 , 21 – 23 , 25 the sensitivity of the proposed method has been improved by 5 orders of magnitude as compared with that of methylation-responsive DNAzyme-based colorimetric assay (0.25 U mL –1 ), 15 2 orders of magnitude as compared with that of rolling circle amplification (RCA)- and G-quadruplex DNAzyme-based chemiluminescence assay (1.29 × 10 –4 U mL –1 ), 25 5 orders of magnitude as compared with AuNP–DNA and Ru( iii )-mediated dual signal amplification-based electrochemical assay (0.3 U mL –1 ), 18 4 orders of magnitude as compared with that of nicking enzyme-assisted signal amplification-based fluorescence assay (0.06 U mL –1 ), 23 4 orders of magnitude as compared with that of exonuclease III-mediated target recycling-based fluorescence assay (0.01 U mL –1 ), 22 and 3 orders of magnitude as compared with that of QD-mediated FRET assay (0.002 U mL –1 ). 21 The enhanced sensitivity can be ascribed to (1) the high specificity of RNase HII-catalyzed single-ribonucleotide excision, (2) the high amplification efficiency of cyclic ligation-dependent SDA, and (3) the low background signal resulting from the inhibition of nonspecific amplification by single-ribonucleotide excision and specific ligation-dependent strand displacement polymerase extension.…”

“…Typical examples include exonuclease III-aided target recycling 22 and nicking endonuclease ( e.g. Nt.BbvCI and Nt.Alwl)-assisted cyclic signal probe cleavage 23 , 24 -based fluorescence assays, and rolling circle amplification (RCA)-based chemiluminescence assay. 25 Despite the improved sensitivity, these assays involve the careful design of molecular beacons 22 – 24 and the complicated preparation of circular templates, 25 and usually suffer from a high background signal resulting from either nonspecific digestion 22 – 24 or nonspecific amplification.…”

Polymerase/nicking enzyme synergetic isothermal quadratic DNA machine and its application for one-step amplified biosensing of lead (II) ions at femtomole level and DNA methyltransferase

“…1 However, this technique suffers from an intrinsic drawback, the requirement of precision thermal cycling between three different temperatures in a costly thermal cycler, limiting its popularization and application in point-of-care testing. On the other hand, various alternative isothermal amplification methods, such as rolling circle amplification (RCA), 2,3 strand displacement amplification (SDA), 4 loop-mediated isothermal amplification, 5,6 nicking enzyme signal amplification (NESA) [7][8][9] and the exponential amplification reaction (EXPAR) 10 have been proposed in the past two decades. These isothermal methods have been developed mainly based on some new findings in molecular biology concerning DNA synthesis and some accessory proteins together with their mimicking in vitro for nucleic acid amplification.…”

DNA Methyltransferase Activity Assays: Advances and Challenges

“…Signal amplification progressed further by tapping on the advances made in DNA research like isothermal DNA amplification techniques. Consequently, rolling circle amplification (RCA) 83 and processes involving nicking enzymes 84 have evolved into DNA MTase activity assays for improvement purposes. On the other hand, the need for label-free DNA MTase activity assays has grown tremendously in recent years.…”

“…Due to their high specificity and efficiency, they are frequently found operating in assays involving the detection of nucleic acids, protein and small biological molecules 93 - 96 . Thereby, Zhao and co-workers in 2013 developed a DNA MTase activity assay based on recurring cycles of the action of a nicking enzyme Nt.BbvCl 84 . In their assay, molecular beacons and Nt.BbvCl are critical participants besides the intricately designed hairpin-shaped DNA probes with 5' overhangs, DNA MTase and methylation-sensitive restriction endonuclease DpnI.…”