2448 Nucleic Acids Research, 2006, Vol. 34, No. 8 Figure 2. (A) Graphical summary of the 8–17 secondary structure and sequence requirements. The 8–17 motif is characterized by a G-T wobble base pair (black dot) at the cleavage site, a 3 base pair stem, a tri-loop and a 4–5 nt single- stranded turn region. Two general binding arms on either side of the 8–17 motif (thick black lines) engage the substrate strand (thick grey lines) through Watson–Crick base pairs (indicated by thin black lines). Underlined nucleotides are strictly conserved, while mutations at other positions are acceptable but associated with different catalytic activity. The approximate relative activity (in Mn2+ buffer) at different positions is indicated. The magnitude and relative order of activities may vary depending on the type of metal ion cofactors used. Cleavage occurs at the G-G junction indicated by the arrow, although it has been previously shown that 8–17 variants can collectively cleave 14 of 16 possible dinucleotide junctions. (B) Proposed secondary structure interaction between the deoxyribozyme domain (black letters) and the RNA substrate domain (grey line/letters) of each sequence class. The outlined letters correspond to P1 and the outlined box corresponds to A1. For simplicity, only the 8–17 containing sec- tion of the deoxyribozyme domain is shown. (C) Evidence to support a catalytic role by 8–17. The autoradiogram depicts the results of self-cleavage assays for truncated versions of each sequence class. The positive control (+) refers to the truncated sequence shown in (B) with a truncated RNA substrate lacking 24 nt from the 30 end (30 min reaction time). Mutants having either a G7 to T or C13 to T mutation are denoted by G and C, respectively (12 h reaction time). Cleavage products (Clv) were separated from the uncleaved precursor substrate (Pre) by 10% denaturing PAGE. omitted since the assay conditions in reference (34) are not directly comparable to our own. The suspected secondary structure interaction between the 8–17 containing DNA domain and the RNA substrate are illustrated in Figure 2B for each class. Approximately 91% of all the observed 8–17 motifs were located at the 50 end of the DNA domain, which allowed them to exploit the 50 primer- binding site (P1) as one binding arm. This unintentional sequence complementarity likely favoured the selection of catalysts that cleave the adjacent G-G dinucleotide junction. Although the observed 8–17 motifs satisfy the sequence requirements established by previous studies, formation of the 8–17 motif is not predicted to be the most energetically favourable according to secondary structure prediction soft- ware, such as mfold (37). This discrepancy is maintained even when full-length molecules are truncated to remove excess sequence elements. Therefore, we synthesized 30 truncated enzymes corresponding to the nucleotide sequences shown in Figure 2B (containing only the suspected 8–17 motif and 8–10 nt binding arms), and ligated them to a truncated RNA substrate lacking 24 consecutive nucleotides from the 30 end (corresponding to the loop region that is not expected to inter- act with the deoxyribozyme domain). Figure 2C indicates that these truncation derivatives are still catalytically active, but the same constructs containing either a G7 to T substitution, or a C13 to T substitution are completely inactive. These experi- mental results are consistent with theoretical expectations (both G7 and C13 are known to be invariant nucleotides), and strongly support a catalytic role for the embedded 8–17 motif. To assess the effect of excess sequence elements on the activity of the 8–17 motif, four different constructs were made for each sequence class, which are depicted in Figure 3. These constructs include: (i) the full-length enzyme, denoted by ‘L’, (ii) a 30 truncated enzyme containing only the 8–17 motif and an 8–10 nt 30 substrate binding arm (corres- ponding to the sequences shown in Figure 2B), denoted by ‘S’ (iii) the same truncated enzyme from part ii with a truncated Figure 3. Schematic diagram of different truncation constructs that were assayed. Dotted arrows define truncation regions. L corresponds to the original full-length molecule. S corresponds to a 30 truncated construct containing only the 8–17 motif and an 8–10 nt 30 binding arm. SS corresponds to the same construct as S, but attached to a truncated RNA substrate lacking 24 contiguous nucleotides from the 30 end. PBA corresponds to the same construct as S, but with an optimized 30 substrate binding arm in which mismatches and bulges were replaced with Watson–Crick base pairs.