K+-responsive off-to-on switching of hammerhead ribozyme through dual G-quadruplex formation requiring no heating and cooling treatment

Abstract

Functional RNAs that switch their activities in response to K(+) may sense the intracellular (100 mM) and extracellular (5 mM) K(+) concentrations and regulate their functions accordingly. Previously, we developed a quadruplex hammerhead ribozyme (QHR) whose conformational change, from a duplex to a G-quadruplex, triggered by K(+) results in expression of the activity. However, this QHR required heating and cooling treatment (annealing) to induce the K(+)-responsive conformational change and activity. Here, we developed a new quadruplex hammerhead ribozyme (QHR) system that does not require annealing to induce the K(+)-responsive conformational change and activity. This system is composed of QHR and a G-quadruplex-forming complementary DNA strand (QCS). In the absence of K(+), QCS formed a duplex with QHR, which suppressed the residual activity. Upon elevation of the K(+) concentration, QCS dissociated from QHR was trapped in a G-quadruplex, and then QHR could form a G-quadruplex and exerted the activity. The 11.6-fold higher activity was induced by K(+) with an EC50 value of 23 mM, but not by Na(+), which is desirable when the activity switching between the intra-/extracellular environment is aimed at. This is the first report of the activation of functional RNA through a 'dual G-quadruplex formation system'.

A new class of ribozyme from hepatitis B virus

“…Using circular dichroism analysis, Chakraborty and Ghosh found that an alternative (mutually exclusive) structure, a G‐quadruplex, may be present within in the epsilon region (which is part of the ribozyme). However, the structure formed independently of monovalent K + or Li + ions, which is not typical of a G‐quadruplex . G‐quadruplexes are well studied in many systems and have been suggested to play a role in replication in other viruses .…”

“…The K + ‐dependent structure, possibly a G‐quadruplex, would be consistent with synthetic ribozyme studies. In engineered constructs, G‐quadruplexes and potassium ions have been found to stabilise and promote the activities of small ribozymes including those from HDV .…”