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

Abstract

Herein, we propose a novel and universal biosensing platform based on a polymerase-nicking enzyme synergetic isothermal quadratic DNA machine (ESQM). This platform tactfully integrates two signal amplification modules, strand displacement amplification (SDA) and nicking enzyme signal amplification (NESA), into a one-step system. A bifunctional DNA probe with a stem-loop structure was designed to be partly complementary to the SDA product and digested substrate of NESA for bridging SDA and NESA. ESQM can be performed by using only the enzymes and buffer involved in the SDA module. In the presence of a target, this DNA machine is activated to afford a high quadratic amplified signal. Using Pb 2+ and DNA adenine methylation (Dam) methyltransferase (MTase) as analytes, a sensitive biosensing platform is demonstrated. Low detection limits of 30 fM Pb 2+ and 0.05 U ml − 1 Dam MTase were achieved within a short assay time (40 min), which were each superior to those of most previously reported methods. This DNA machine exhibited high selectivity for Pb 2+ . Furthermore, the successful detection of complex environmental water samples demonstrated the applicability of the proposed strategy in real samples, holding great potential for its application in environmental monitoring, biomedical research and clinical diagnosis. INTRODUCTIONSignal amplification technologies have a critical role in molecular biology research, environmental and food monitoring, forensic identification and clinical diagnosis. Since its creation in 1985, polymerase chain reaction has become the most well-known and widely used amplification technique. 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) 7-9 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. Among these methods, EXPAR has been widely used, which is attributed to its rapid amplification kinetics (several minutes) and exceedingly high amplification efficiency (10 6 -to 10 9 -fold). For example, Ye's group 11 and Zhang's group 12,13 have proposed several interesting methods for the highly sensitive detection of microRNA and protein based on EXPAR, respectively. Nevertheless, the EXPAR-based methods involve the weaknesses of early phase non-specific background amplification resulting from the binding of the polymerase to the single-stranded template. 14 Conversely, the linear amplification ...

An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity

“…Notably, the LOD of this biosensor is the lowest to our knowledge. The sensitivity of this biosensor platform has improved by as much as 2 orders of magnitude compared with that of rolling circle amplification (RCA) based chemiluminescence assay,21 by 3 orders of magnitude compared with that of the methylation response DNAzyme colorimetric assay,22 by 3 orders of magnitude compared with that of quantum dots-mediated FRET assay,36 and as well as being an improvement over other methods 37–39. The extremely low LOD could be attributed to (i) the high amplification efficiency because of making full use of the integrated probe-reporter MB substrates in the SDA amplification process; (ii) multiple highly efficient SDA cycles were included to enhance the signal; and (iii) low background due to few oligos being applied in the whole system.…”

Zero‐Background Helicase‐Dependent Amplification and Its Application to Reliable Assay of Telomerase Activity in Cancer Cell by Eliminating Primer–Dimer Artifacts

“…(This is not involved in other amplification strategies,s uch as PCR and strand displacementa mplification.) [28][29][30][31][32][33][34][35][36][37] Thus, we speculated ATPc oncentration might have an effect on HDA performance. To our surprise, as ATPc oncentration was reduced from 3t o 1.8 mm,t he primer-dimer artifacts reduced significantly (Figure 2A), and specific amplification was also reduced.…”

“…Next, zero-background HDA was used for the analysis of telomerase activity.H uman telomerase is ar ibonucleoprotein that maintains telomere length by adding tandemr epeats (TTAGGG) to the ends of chromosomes;i ti sc losely associated with cellular immortality and carcinogenesis. [33][34][35][36][37][38][39][40] Upregulation (or reactivation) of telomerase activity hasb een observed in the vast majority of human tumors (85-90 %). [40,41] Telomerase activity is relatively low (or undetectable) in most normals omatic cells.…”

Growing prospects of DNA nanomaterials in novel biomedical applications

“…195 By integrating strand displacement and nicking enzyme signal amplication into a one-step system, polymerase-nicking enzyme synergetic quadratic DNA machines have been designed to sense Pb 2+ , miRNA and DNA methyltransferase. 196,197 In addition, powered by the hydrolysis of NAD + , a repair ligation-mediated light-producing DNA machine has been universally developed for DNA, thrombin, adenosine, K + and endonuclease detection in human serum. 198 Yuan 199 has constructed an Fc-switched ECL "off-on" sensor for the sensitive detection of cardiac troponin with a DNA walking machine.…”