G‑quadruplex-Based Fluorescent Assay of S1 Nuclease Activity and K<sup>+</sup>
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Abstract
Endonuclease plays an important role in many biological
processes,
and an assay of endonuclease activity is of great significance. However,
traditional methods for the assay of endonuclease activity have undesirable
limitations, such as high cost, DNA-consuming and laboriousness. In
the present work, a G-quadruplex-based, fluorescent assay of endonuclease
activity has been developed with protoporphyrin IX (PPIX) as a signal
reporter. S1 nuclease, a single strand DNA (ssDNA)-specific endonuclease,
is employed as model system. In the “on” state, G-quadruplex
DNA can greatly enhance the fluorescence of PPIX. However, if S1 nuclease
could cleave G-quadruplex DNA into small fragments, there would be
no formation of G-quadruplexes, accompanied by low emission response
of PPIX. This fluorescent discrimination before or after digestion
by nuclease can be used to monitor the activity of S1 nuclease. This
assay is simple in design and offers a convenient protocol for homogeneous,
rapid and high-throughput detection. In addition, the proposed strategy
avoids complicated covalent modifications or chemical labeling, and
thus offers advantages of simplicity and cost efficiency. More importantly,
K<sup>+</sup> is found to well inhibit the activity of S1 nuclease
when using certain G-quadruplex DNA as substrate, and thus this system
is further used for turn-on detection of K<sup>+</sup>. S1 nuclease
is critical in the detection of K<sup>+</sup> since it helps to reduce
the background signal