7168 Nucleic Acids Research, 2005, Vol. 33, No. 22 in Figure 1C. Truncating as many as 10 nt from the 30 terminus had no apparent effect on the catalytic activity as the mutant deoxyribozymes EC5-7 (having 5, 8 and 10 nucleotides deleted from the 30 end of the deoxyribozyme) all retained full activity. Removing ﬁve additional nucleotides from the same end reduced the catalytic activity by 100-fold (EC8, +++) while truncating ﬁve more nucleotides nearly abolished the RNA-cleaving activity (EC9, +). These results indicate even some of the nucleotides located within the 30-primer- binding site are required for the full catalytic activity of the deoxyribozyme. Deletion of internal nucleotides The terminal nucleotide truncation experiments demonstrated that all functionally important nucleotides (required to support full catalytic activity of pH4DZ1) within the catalyst domain are located between G31 and G90. To determine whether some of the internal nucleotides could also be removed, we carried out a ‘deletion walking’ experiment and the data are shown in Figure 2. More speciﬁcally, we conducted a series of sequential, 6 nt deletions, starting from G16 and ending at A91. Each new deletion had a 3 nt overlap with the previous deletion. EC6 was used as the reference deoxyribozyme for this experiment. Surprisingly, the deletion walking experiment clearly indic- ated that pH4DZ1 could not tolerate well a continuous 6 nt deletion anywhere between G31 and A91. Most of the 6 nt deletion mutants caused at least 1000-fold activity reduction (++ or lower), and only EC21 (++++), EC29 (+++) and EC33 (+++) had the activity above ++. These results suggest that a large number of nucleotides in this region participate in inter- actions critical to the folding and/or catalysis of the deoxyri- bozyme. More importantly, the deletion walking experiment has identiﬁed three separate sequence elements (shaded in grey in Figure 2) containing the nucleotides that appeared to be most crucial to the function of the deoxyribozyme as each 6 nt deletion within these elements caused either com- plete loss of activity ( ) or near-complete loss of activity (+). Identification of functionally essential nucleotides by in vitro selection We turned our attention next to the identiﬁcation of individual nucleotides within the three crucial sequence elements high- lighted in grey in Figure 2. Since there are a total of 30 nt in these regions, it would be extremely time-consuming to assess the functionality of individual nucleotides using single-nucleotide mutation strategy. For this consideration, we chose to conduct an in vitro selection experiment. A chem- ically synthesized, partially mutagenized pH4DZ1 DNA lib- rary was constructed in which (i) the nonessential nucleotides between 1 and 25 were removed and (ii) every position in G32– T43, C56–G67 and G80–T86 (upper-case letters in large font, Figure 3B) was mutagenized at a mutation rate of 24% (76% of the wild-type nucleotide and 8% each of the other 3 nt) fol- lowing a previous protocol (10). The in vitro selection scheme was similar to that used for the isolation of pH4DZ1 (17) (re-shown in Figure 3A) except for the use of different DNA molecules. Brieﬂy, the DNA library L1 was ligated to substrate S3, in the presence of the template T1 and T4 DNA ligase, to yield 105 nt S3-L1 constructs Figure 3. In vitro selection of functional pH4DZ1 mutants. (A) Selection scheme. Each selection cycle consists of steps I–VIII. (I) 88 nt DNA L1 is ligated to 17 nt substrate S3. (II) Ligated 105 nt DNA is isolated by PAGE. (III) Purified 105 nt DNA is incubated with divalent metal ions for RNA cleavage. (IV) The 90 nt 30-cleavage fragment is isolated by PAGE. (V) The recovered 90 nt DNA is amplified by PCR using primers P1 and P2. (VI) 105 bp PCR product in (V) is further amplified by PCR using primers P2 and P3 to introduce a ribonucleotide linkage embedded in front of the L1 sequence. (VII) The result- ing double-stranded DNAs are treated with NaOH to cleave the ribonucleotide linkage. (VIII) The cleavage fragments are phosphorylated at 50 end, and used to initiate the next round. (B) All the DNA sequences used for in vitro selection. The lower-case letters in L1 are primer-binding sites; the letters in larger fonts in L1 are partially randomized nucleotides. Other nucleotides in L1 are not ran- domized. The underlined letters are ribonucleotides. (C) Selection progress. The white bars are the selection with a 1 min RNA cleavage time while the black bars are for 5 h selection. The x-axis represents the selection round; the y-axis plots the percentage of detected RNA cleavage in each selection round. (step I). After puriﬁcation by 10% denaturing PAGE (step II), the S3-L1 chimeras were allowed to cleave in the selection buffer (step III). The 30 cleavage fragment (90 nt) was ﬁrst puriﬁed by 10% denaturing PAGE (step IV) and then ampli- ﬁed by two successive rounds of PCR (steps V and VI). The purpose of the second PCR was to introduce a ribonucleotide right before the start of L1 sequence so that single-stranded L1 could be regenerated by alkaline digestion (VII). After 50-phosphorylation by PNK, the single-stranded L1 molecules were used to initiate a new round of selective ampliﬁcation. The sequences of all the DNA molecules used for the in vitro