Triplex helical formation has been the focus of considerable interest because of possible applications in developing new molecular biology tools as well as therapeutic agents and the possible relevance of H-DNA structures in biology system. We report here that a small-molecule anticancer agent, coralyne, has binding preference to the less stable protonated triplex d(C+-T)(6):d(A-G)(6).d(C-T)(6) over duplex d(A-G)(6).d(C-T)(6) and shows different spectral and electrochemical characteristics when binding to triplex and duplex DNA, indicating that electrochemical technique can detect the less stable protonated triplex formation. (c) 2009 Elsevier B.V. All rights reserved.