A Ligation Triggered Label-Free Fluorescent Assay for Adenosine-Triphosphate Based on Nicking Endonuclease Signal Amplification and Ligand Responsive G-Quadruplex Formation

Abstract

A novel label-free fluorescent assay strategy was developed for sensitive and selective detection of adenosine-triphosphate (ATP) based on nicking endonuclease signal amplification (NESA) and N-Methyl mesoporphyrin (NMM)-responsive G-quadruplex formation. In this assay, the nick in the specially designed DNA duplex can be sealed by T4 DNA ligase in the presence of ATP. The following replication and strand displacement yield the duplex that contains the full recognition site for nicking endonuclease. Nicking enzyme's cutting the replicated DNA duplex results in a new replication site for polymerase and the continuous displacement of the nicked G-rich strand. The nicked strand can fold into G-quadruplex structure in the presence of K⁺ and selectively binds with NMM, yielding a significant fluorescence signal. Under the optimal conditions, the fluorescent assay strategy showed a dynamic response to ATP in the concentration range from 10 nM to 1000 nM and a detection limit of 0.8 nM, which compared favorably with most of the previous ATP assay methods. In addition, the proposed strategy exhibited superior selectivity toward ATP over other nucleosides and its analogues. The fluorescent assay was also applied in detecting ATP in serum with satisfactory results.

Keywords:

• ATP detection
• G-quadruplex
• Nicking endonuclease signal amplification (NESA)
• NMM
• T4 DNA ligase

Acknowledgments

This work was supported by the NSFC (21025521, 21035001, 21190041), the National Key Basic Research Program (2011CB911000), the CSIRT Program and the NSF of Hunan Province (10JJ7002).

Notes

LrRET, long-range resonance energy transfer; LOD, detection of limit; CRET, chemiluminescence resonance energy transfer.