A novel sensitive and selective ligation-based ATP assay using a molecular beacon

Abstract

In this paper, a novel, facile fluorometric ATP assay with high sensitivity and excellent selectivity has been reported. This approach utilizes a molecular beacon, T4 DNA ligase and two short oligonucleotides. In the presence of ATP, the T4 DNA ligase catalyzes the ligation reaction and the ligation product restores the fluorescence of the molecular beacon. Owing to the high sensitivity of the molecular beacon and T4 DNA ligase's high substrate dependence, this novel ATP assay demonstrates exceptional selectivity and high sensitivity down to 0.14 nM in homogeneous solution. Cellular ATP concentrations in several cell lines have been determined by measuring the lysate sample containing 8.0 × 10(3) cells.

A label-free fluorescence strategy for sensitive detection of ATP based on the ligation-triggered super-sandwich

“…The correlation equation is ΔF¼59.45þ 65.97 log C with a correlation coefficient of 0.9872. The detection limit was calculated to be 200 pM (3s/k, where s is the relative standard deviation and k is the slope of the calibration graph, n ¼11), providing superior detection sensitivity compared with aptamer-based fluorescence strategies (Sanghavi et al, 2013;Xu et al, 2014;Zhang et al, 2012), and it is comparable with the T4 DNA-based strategies (Ma et al, 2012(Ma et al, , 2013. The high sensitivity can be achieved owing to the following factors: (1) T4 DNA ligase can efficiently help the formation of the super-sandwich in the homogeneous solution and (2) an 800-1000 fold increase of the fluorescence intensity of SG I can be obtained when it is intercalated into the duplex DNA while the formed super-sandwich can provide more binding points for the dye.…”

“…These methods have shown high sensitivity and selectivity but it usually takes several hours to finish the detection. Several novel strategies for ATP detection based on the ATP-dependent ligation reaction have been reported (Ma et al, 2008(Ma et al, , 2012(Ma et al, , 2013. Owing to the high sensitivity of the molecular beacon and the high substrate dependence of T4 DNA ligase, these methods have shown very good analytical performances.…”

“…The aptamer-based ATP methods generally use the conformational change of the aptamers upon ATP binding which needs a long incubation time. Among these methods, nanoparticles , graphene oxide (Wang et al, 2010) and enzymes (Ma et al, 2013) have been widely applied to improve their performance. These methods have shown high sensitivity and selectivity but it usually takes several hours to finish the detection.…”

Real-time monitoring of small biological molecules by ligation-mediated polymerase chain reaction

“…Notably, the sensitivity of the proposed method has improved by more than 6 orders of magnitude as compared with that of aptamer-based fluorescence amplification assay (4×10 -8 M), 17 and by 4 orders of magnitude as compared with that of T4 DNA ligase-based fluorescent method (1×10 -10 M, 1.4×10 -10 M, and 2×10 -10 M, respectively). [21][22][23] The improved sensitivity may be attributed to the exceptional amplification efficiency of PCR and the reduced background signal induced by Exo I and Exo III. To verify the selectivity of the proposed method for ATP assay, we measured its analogues including adenosine-monophosphate (AMP), adenosine-diphosphate (ADP), uridine triphosphate (UTP), cytidine triphosphate (CTP), and guanosine triphosphate (GTP).…”

Simple fluorescence-based detection of protein kinase A activity using a molecular beacon probe

“…20,21 In addition, a novel method for detecting biologic small molecules such as ATP and nicotinamide adenine dinucleotide (NAD) has been developed. 22,23 In this study, we developed a simple, fast, and sensitive assay for detecting protein kinase activity using MBs, based on DNA ligation. …”