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'. (C) 2015 Elsevier Inc. All rights reserved.