was first copied using a synthetic 5-biotinylated mixed DNA/RNA primer. The primer contained an
embedded 12 nucleotide RNA sequence and a 3-DNA tail complementary in sequence to the 3-terminus
of the DNA library. Single-stranded DNA containing both enzyme (within the 50 nucleotide random
sequence) and target RNA sequence was then obtained following capture by streptavidin-coated beads,
denaturation and removal of the non-biotinylated template. This allows folding and interaction of the
enzyme and substrate portions of the immobilised sequence. In the presence of magnesium ion co-factor,
the active sequences undergo self-cleavage within the RNA target section. The released nucleic acid
sequences are then amplified by PCR where one primer is biotinylated. Single-stranded templates are pro-
duced by streptavidin capture of the biotinylated strand ready for the next round of SELEX.
The DNAzyme sequences identified in this work can be simplified and shortened such that cleavage of a
separate substrate strand (transcleavage) can occur. The ‘10–23’enzyme derived from this work comprises
of a catalytic core of 15 nucleotides flanked by two substrate recognition domains in which Watson–Crick
base pairing occurs (Figure 5.28). The structure of the ‘10 –23’ enzyme can be modified to recognise and
Nucleic Acids in Biotechnology 201
Figure 5.27 The generation of a DNAzyme to catalyse the sequence-specific cleavage of RNA
Figure 5.28 Composition of a 10–23 catalytic motif. The catalytic domain of 15 nucleotides is flanked by two
substrate recognition domains that can be varied providing Watson–Crick base-pairing is maintained.
The arrow denotes the site of cleavage