the deoxyribozyme (blue and red nucleotides). Ten of these nucleotides are absolutely essential (red nucleotides). Interest- ingly, these crucial nucleotides are distributed into three sequence elements separated by some important nucleotides (shown in black). The methylation interference experiment shown in Figure 5 supports the notion that these nucleotides are involved in the formation of the active site of the deoxyri- bozyme: several guanine residues in or near these regions cannot tolerate the methylation at N7 positions while some other guanines are prone to methylation. The three special nucleotides in the substrate domain— the ribonucleotide, the ﬂuorophore-modiﬁed dT (F) and the quencher-modiﬁed dT (Q) apparently make important contribution to the structure and/or the function of the deoxyri- bozyme, reﬂected by the observations that (i) neither the ﬂuorophore nor the quencher can be removed and (ii) the adenine located on the ribose at the cleavage site cannot be mutated into other bases. We speculate that the active site of the deoxyribozyme is rather complex and involves the ribonucleotide, the ﬂuorophore and the quencher, and some or all of the 10 absolutely conserved nucleotides in the three highly crucial sequence elements. This active site is very likely further supported by the nucleotides that sur- round the three sequence elements. The precise structure of pH4DZ1 can only be revealed by high-resolution techniques including NMR and X-ray crystallography, which will be the subject of future experimentation. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS This work was supported by research grants from the Canadian Institutes for Health Research, MDS-Sciex, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation and the Ontario Innovation Trust for support of this work. Y.L. is a Canada Research Chair. Funding to pay the Open Access publication charges for this article was provided by the Canadian Institute of Health Research. Conflict of interest statement. None declared. REFERENCES 1. Joyce,G.F. (2004) Directed evolution of nucleic acid enzymes. Annu. Rev. Biochem., 73, 791–836. 2. Silverman,S.K. (2004) Deoxyribozymes: DNA catalysts for bioorganic chemistry. Org. Biomol. Chem., 2, 2701–2706. 3. Achenbach,J.C., Chiuman,W., Cruz,R.P. and Li,Y. (2004) DNAzymes: from creation in vitro to application in vivo. Curr. Pharm. Biotechnol., 5, 321–336. 4. Breaker,R.R. (2000) Making catalytic DNAs. Science, 290, 2095–2096. 5. Lu,Y. (2002) New transition-metal-dependent DNAzymes as efficient endonucleases and as selective metal biosensors. Chemistry, 8, 4589–4596. 6. Carmi,N., Balkhi,S.R. and Breaker,R.R. (1998) Cleaving DNA with DNA. Proc. Natl Acad. Sci. USA, 95, 2233–2237. Nucleic Acids Research, 2005, Vol. 33, No. 22 7175 7. Sreedhara,A., Li,Y. and Breaker,R.R. (2004) Ligating DNA with DNA. J. Am. Chem. Soc., 126, 3454–3460. 8. Santoro,S.W. and Joyce,G.F. (1997) A general purpose RNA-cleaving DNA enzyme. Proc. Natl Acad. Sci. USA, 94, 4262–4266. 9. Flynn-Charlebois,A., Wang,Y., Prior,T.K., Rashid,I., Hoadley,K.A., Coppins,R.L., Wolf,A.C. and Silverman,S.K. (2003) Deoxyribozymes with 20-50 RNA ligase activity. J. Am. Chem. Soc., 125, 2444–2454. 10. Li,Y. and Breaker,R.R. (1999) Phosphorylating DNA with DNA. Proc. Natl Acad. Sci. USA, 96, 2746–2751. 11. Li,Y., Liu,Y. and Breaker,R.R. (2000) Capping DNA with DNA. Biochemistry, 39, 3106–3114. 12. Li,Y. and Sen,D. (1996) A catalytic DNA for porphyrin metallation. Nature Struct. Biol., 3, 743–747. 13. Chinnapen,D.J. and Sen,D. (2004) A deoxyribozyme that harnesses light to repair thymine dimers in DNA. Proc. Natl Acad. Sci. USA, 101, 65–69. 14. Santoro,S.W. and Joyce,G.F. (1998) Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry, 37, 13330–13342. 15. Li,J., Zheng,W., Kwon,A.H. and Lu,Y. (2000) In vitro selection and characterization of a highly efficient Zn(II)-dependent RNA- cleaving deoxyribozyme. Nucleic Acids Res., 28, 481–488. 16. Peracchi,A. (2000) Preferential activation of the 8-17 deoxyribozyme by Ca(II) ions. Evidence for the identity of 8-17 with the catalytic domain of the Mg5 deoxyribozyme. J. Biol. Chem., 275, 11693–11697. 17. Liu,Z., Mei,S.H., Brennan,J.D. and Li,Y. (2003) Assemblage of signaling DNA enzymes with intriguing metal-ion specificities and pH dependences. J. Am. Chem. Soc., 125, 7539–7545. 18. Mei,S.H., Liu,Z., Brennan,J.D. and Li,Y. (2003) An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling. J. Am. Chem. Soc., 125, 412–420. 19. Wang,W., Billen,L.P. and Li,Y. (2002) Sequence diversity, metal specificity, and catalytic proficiency of metal-dependent phosphorylating DNA enzymes. Chem. Biol., 9, 507–517. 20. Brown,A.K., Li,J., Pavot,C.M. and Lu,Y. (2003) A lead-dependent DNAzyme with a two-step mechanism. Biochemistry, 42, 7152–7161. 21. Jayasena,V.K. and Gold,L. (1997) In vitro selection of self-cleaving RNAs with a low pH optimum. Proc. Natl Acad. Sci. USA, 94, 10612–10617. 22. Achenbach,J.C., Jeffries,G.A., McManus,S.A., Billen,L.P. and Li,Y. (2005) Secondary-structure characterization of two proficient kinase deoxyribozymes. Biochemistry, 44, 3765–3774. 23. Shen,Y., Brennan,J.D. and Li,Y. (2005) Characterizing the secondary structure and identifying functionally essential nucleotides of pH6DZ1, a fluorescence-signaling and RNA-cleaving deoxyribozyme. Biochemistry, 44, 12066–12076. 24. Breaker,R.R. and Joyce,G.F. (1994) A DNA enzyme that cleaves RNA. Chem. Biol., 1, 223–229. 25. Breaker,R.R. and Joyce,G.F. (1995) A DNA enzyme with Mg(II)- dependent RNA phosphoesterase activity. Chem. Biol., 2, 655–660. 26. Roth,A. and Breaker,R.R. (1998) An amino acid as a cofactor for a catalytic polynucleotide. Proc. Natl Acad. Sci. USA, 95, 6027–6031. 27. Santoro,S.W., Joyce,G.F., Sakthivel,K., Gramatikova,S. and Barbas,C.F., 3rd. (2000) RNA cleavage by a DNA enzyme with extended chemical functionality. J. Am. Chem. Soc., 122, 2433–2439. 28. Cruz,R.P., Withers,J.B. and Li,Y. (2004) Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme. Chem. Biol., 11, 57–67. 29. Li,Y. and Breaker,R.R. (1999) Kinetics of RNA degradation by specific base catalysis of transesterification involving the 20-hydroxyl group. J. Am. Chem. Soc., 121, 5364–5372. 30. Feldman,A.R. and Sen,D. (2001) A new and efficient DNA enzyme for the sequence-specific cleavage of RNA. J. Mol. Biol., 313, 283–294. 31. Schlosser,K. and Li,Y. (2004) Tracing sequence diversity change of RNA-cleaving deoxyribozymes under increasing selection pressure during in vitro selection. Biochemistry, 43, 9695–9707. 32. Faulhammer,D. and Famulok,M. (1997) Characterization and divalent metal-ion dependence of in vitro selected deoxyribozymes which cleave DNA/RNA chimeric oligonucleotides. J. Mol. Biol., 269, 188–202. 33. Faulhammer,D. and Famulok,M. (1996) The Ca2+ ion as a cofactor for a novel RNA-cleaving deoxyribozyme. Angew. Chem. Int. Ed. Engl., 35, 2837–2841. 34. Zuker,M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res., 31, 3406–3415.