7164–7175 Nucleic Acids Research, 2005, Vol. 33, No. 22 doi:10.1093/nar/gki1013 Characterization of a catalytically efficient acidic RNA-cleaving deoxyribozyme Srinivas A. Kandadai and Yingfu Li* Department of Biochemistry and Biomedical Sciences and Department of Chemistry, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5 Received August 23, 2005; Revised November 11, 2005; Accepted November 25, 2005 ABSTRACT We previously demonstrated—through the isolation of RNA-cleaving deoxyribozymes by in vitro selec- tion that are catalytically active in highly acidic solutions—that DNA, despite its chemical simplicity, could perform catalysis under challenging chemical conditions [Liu,Z., Mei,S.H., Brennan,J.D. and Li,Y. (2003) J. Am. Chem. Soc. 125, 7539–7545]. One remarkable DNA molecule therefrom is pH4DZ1, a self-cleaving deoxyribozyme that exhibits a kobs of 1 min 1 at pH 3.8. In this study, we carried out a series of experiments to examine the sequence and catalytic properties of this acidic deoxyribozyme. Extensive nucleotide truncation experiments indic- ated that pH4DZ1 was a considerably large deoxyri- bozyme, requiring 80 out of the original 123 nt for the optimal catalytic activity. A reselection experiment identified ten absolutely conserved nucleotides that are distributed in three catalytically crucial sequence elements. In addition, a trans deoxyri- bozyme was successfully designed. Comparison of the observed rate constant of pH4DZ1 with experi- mentally determined rate constant for the uncatalyzed reaction revealed that pH4DZ1 achieved a rate enhancement of 106-fold. This study provides valu- able information about this low-pH-functional deoxyribozyme and paves way for further structural and mechanistic characterization of this unique catalytic DNA. INTRODUCTION Catalytic DNAs (also known as deoxyribozymes, DNAzymes or DNA enzymes) refer to single-stranded DNA molecules with a catalytic function (1–5). Through in vitro selection, a large number of deoxyribozymes have been isolated from random-sequence DNA libraries for catalyzing many chemical reactions that include cleavage and ligation of DNA or RNA strands (6–9), 50-phosphorylation and 50-adenylylation of DNA (10,11), porphyrin metallation (12) and thymine dimer repair (13). Deoxyribozymes are interesting molecules to study both for their relevance to inherent catalytic capab- ilities of nucleic acids and for their practical values. Indeed, a large number of deoxyribozymes are currently being studied as unique molecular tools for a variety of chemical and biological applications (2,3). Despite very limited chemical functionalities, DNA is cap- able of executing efﬁcient catalysis, reﬂected by the fact that many deoxyribozymes exhibit kcat values at or above 1 min 1 (8,14–20). Most known deoxyribozymes are reported to function at or near physiological conditions. Encouraged by a study conducted by Jayasena and Gold in which self- cleaving ribozymes functional at low pH were isolated from a random-sequence RNA pool (21), we recently carried out an in vitro selection experiment to derive deoxyribozymes from a random-sequence DNA pool that could perform catalysis in a highly acidic solution (17). Several self-cleaving deoxyri- bozymes were indeed obtained for catalyzing RNA cleavage in highly acidic solutions. One of these deoxyribozymes, named pH4DZ1, is a highly effective catalyst, achieving a kobs of 1 min 1 with a pH optimum of 3.8. DNA at this pH has considerably different properties due to the extensive protonation at the N1 atom of adenosine and the N3 atom of cytosine (1). Studying deoxyribozymes such as pH4DZ1 that have unusual catalytic capability is expected not only to expand our general understanding of catalysis by both natural and artiﬁcial polymers, but also to recover useful information that could facilitate the development of deoxyribozymes for applications under unusual chemical conditions. Here we report on a comprehensive characterization of the catalytic and sequence properties of pH4DZ1. We conducted a series of experiments designed to achieve the following four speciﬁc goals: (i) minimizing the size of pH4DZ1, (ii) identifying the functionally essential nucleotides within its sequence, (iii) designing a trans-acting deoxyribozyme system and (iv) assessing the rate enhancement achieved by pH4DZ1. The results from these experiments are described below. *To whom correspondence should be addressed. Tel: +1 905 525 9140; Fax: +1 905 522 9033; Email: liying@mcmaster.ca The Author 2005. Published by Oxford University Press. All rights reserved. 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