The CChMVd-HHRwas synthesized by overnight
in vitro transcription of the PCR products. To avoid cleavage during transcription,
a deoxyribonucleotide (5′-CATGGATCTTCATCAGGACACC
GAC-3′), complementary to a part of the HHR [15]was used in the transcription
mixture at a concentration of 10 μM. The RNAs obtained were
purified by denaturing (7 M urea) 15% polyacrylamide gel electrophoresis
(PAGE) and eluted from the gel overnight in 300 mM sodium
acetate, pH 5.2 at 4 °C. The RNAs were recovered from the solution by
filtration through 0.22 μm diameter micro-filters and precipitation
with ethanol. The purified ribozyme was finally resuspended in water
and stored at −20 °C.