2.8. RNase H assay
In a 10 Al volume, 1 nM a-32P-labeled uidA RNA
transcript, 50 nM oligodesoxynucleotide, 40 mM Tris–
HCl pH 7.5, 4 mM MgCl2, 1 mM DTT and 5 units
RNase H were mixed and incubated at 37 8C for 1 min.
The reaction was stopped by addition of 5 Al stopping
buffer. Reaction products were separated by polyacryl-
amide gel electrophoresis (5%) and gels were exposed
to an X-ray film after drying on Whatman filter paper.
Reaction products for each oligodesoxynucleotide were
determined twice in separate assays.
3. Results
3.1. In vitro analyses of ribozymes targeting the uidA
mRNA
The secondary structure of the uidA mRNA (1811 bp)
was analysed in silico using the program RNAdraw
(Matzura and Wennborg, 1996) and searched for sin-
gle-stranded regions containing a GUC sequence, which
follows the NHH rule and represents an efficient ribo-
zyme cleavage site (Ohkawa et al., 1995; Kore et al.,
1998). Seven such sites were identified within the uidA
mRNA. Four target sites are located in the 5V-region (nt
10, 67, 219, 297), two target sites are located in the
middle region (nt 476, 762) and one site within the 3V-
region of the substrate mRNA (nt 1332; data not shown).
Ribozymes targeting these cleavage sites (rz4–rz10)
were developed (Table 1), transcribed in vitro and ex-
amined qualitatively for cleavage activity of in vitro
transcribed uidA mRNAs. Three uidA substrate lengths
were tested, namely a short version buidAshortQ (nt 1–
217), a medium size version buidAmedQ (nt 1–1090) and
a full-length version buidAlongQ (nt 1–1811). We chose
three different substrate lengths as RNA transcripts
often show reduced stability with increased transcript
length.
Hendry and McCall (1996) reported that a relatively
short arm I (5 bp) together with a longer arm III (10 bp)
improves the cleavage activity of hammerhead ribo-
zymes. To analyse the influence of binding arm length
on cleavage activity, we constructed the ribozymes rz4
and rz5 in two versions, i.e. a symmetric version with
10 binding nucleotides on both arms I and III (rz4sym
and rz5sym), and an asymmetric version with five
binding nucleotides in arm I and 10 binding nucleotides
in arm III (rz4asym and rz5asym). Fig. 3 shows exem-
plarily the result of an in vitro ribozyme assay with
uidAshort as substrate and the symmetric and asym-
metric versions of ribozymes rz4 and rz5. All four