Figure 1. The nucleotide sequence of pH4DZ1 and the activity of various truncated deoxyribozymes. (A) The primary sequence of pH4DZ1. The self- cleaving deoxyribozyme is divided into the ‘substrate’ domain, S1 (italicized letters; numbered 1 to 23 in the 30–50 direction), and the ‘catalyst’ domain (normal letters only; numbered 1 to 100 in the 50–30 direction). The lower-case letters are the primer-binding sequences for the original in vitro selection experiment. (B) Truncating nucleotides from the 50 end of the catalyst domain. (C) Removing nucleotides from the 30 end of the catalyst domain. The reference sequence is pH4DZ1. Underlined nucleotides are the sequence region under investigation, with the vertical arrow pointing to the mutated sequences. The identities of the nucleotides represented by black bars are omitted for clarity. The name of each mutant deoxyribozyme is listed next to its altered sequence. The activity of each construct in this figure (as well as in all remaining figures) is scored using the activity map shown as the insert in (B). All individual deoxyribozymes were constructed by ligating substrate S1 with a relevant 50-32P-labeled synthetic oligonucleotide. F ¼ Fluorescein-dT, Q ¼ DABCYL-dT and A ¼ Adenine ribonucleotide Nucleic Acids Research, 2005, Vol. 33, No. 22 7167 regarded as an efﬁcient catalyst (kobs >0.01 min 1) while a sequence scored +++ or lower is treated as a poor catalyst. It should be noted that the activity-scoring scheme is adopted to simplify the task of comparing relative activities of all deoxyribozyme mutants tested in this report. More precise catalytic rate constants of selective mutant deoxyribozymes were determined and will be discussed later. Truncating nucleotides in the catalyst domain In the original in vitro selection experiment, we utilized a library of 123 nt single-stranded DNA molecules. The use of a relatively long DNA sequence to construct the DNA library was based on the assumption that a large DNA sequence may result in a better catalyst. Since all known RNA-cleaving deoxyribozymes isolated under neutral pH con- ditions (such as 8–17 and 10–23) are fairly small in size, we wondered whether the low-pH-functional pH4DZ1 also used relatively limited nucleotides to construct its catalytic core. We therefore carried out comprehensive sequence truncation experiments (including systematically removing terminal or internal nucleotides from the catalyst domain) and the results are shown in Figures 1 and 2. The effects of deleting nucleotides from the 50 end of the catalyst domain were assessed ﬁrst and the data are presented in Figure 1B. The original primer-binding sequence was com- pletely dispensable (EC1, +++++; E: enzyme; C: cis-acting). In fact, deleting the next 16 nt also produced no signiﬁcant effect because EC2 (28 nt deletion, +++++) and EC3 (31 nt deletion, also +++++) exhibited the activity that was similar to pH4DZ1. Removing the next 3 nt (EC4, +++) resulted in a deoxyribozyme that suffered 100-fold activity reduction. These results indicate that the 31 nt from G1 to G31 have little relevance to the active structure and catalysis of pH4DZ1 while G32, A33, G34, though not catalytically essential, make important contributions to the full activity of the deoxyribozyme. Progressive removal of nucleotides from the 30 end of the catalyst domain was carried out next and the data are presented Figure 2. Activity of deoxyribozyme mutants with sequential internal nucleotide truncation. EC6 was used as the reference deoxyribozyme. Each short dash represents a deleted nucleotide in comparison to the sequence of EC6. Each mutant deoxyribozyme contained continuous 6 nt deletion. The name of each mutant deoxyribozyme is listed next to its sequence. The activity of each mutant deoxyribozyme is scored using the activity map given in Figure 1B. All individual deoxyribozymes were constructed by ligating substrate S1 with a relevant 50-32P-labeled synthetic oligonucleotide. The three regions shaded in grey are identified as the sequence elements that contain functionally most crucial nucleotides based on the observation that each 6 nt removal resulted in either complete loss of activity ( ) or near-complete loss of activity (104 activity reduction, i.e. from +++++ to +).