Target Discovery and Validation Reviews and Protocols

Hammerhead Ribozymes 151
3.3. Recovery of Active Ribozymes
To isolate plasmids from cells, Hirt’s DNA isolation method is often used.
This method enables the recovery of plasmids without nicking, but it is time
consuming and requires many steps. Thus, instead of it, commercially available
Miniprep kit also can be use, such as QIAGEN’s Miniprep kit, to isolate plasmids
from mammalian cells.
When we introduce the ribozyme library into cells, we must consider that one
transfected cell may have more than one type of plasmid. In such case, if one of
the ribozymes should influence the phenotype, other plasmids are also isolated
from the positive cells. Such unrelated plasmids may be regarded as the falsepositives.
To reduce the false-positives, in many cases, we need to repeat the
same screening at least three to four times. To do this, the isolated plasmids from
first-cycle selection are used to transform competent E. coli cells. The resultant
library should be introduced into cells as second-cycle selection. To determine
the nature of plasmids, we should sequence individual colonies in each cycle.
3.4. Identification of the Candidate Genes Responsible
for Phenotypic Changes
With the sequence information derived from substrate-binding arms of the
ribozyme, we can identify candidate genes responsible for the phenotypic
changes either by 5′ and 3′ rapid amplification of cDNA ends (RACE) or simple
bioinformatics, searching for homology using the entire sequence as query. The
latter method easily leads to the identification of the candidates. In our laboratory,
we screen targets by mining the publicly accessible gene database with the
BLAST program at the NCBI website (http://www.ncbi.nlm.nih.gov/blast/).
1. Access the NCBI website; Click “search for short nearly exact matches.”
2. Fill in the sequence in the search box. The obtained sequence probably is 15 nt
with one gap such as NNNNNNTHNNNNNNN, where H can be filled in this
format (see Notes 5 and 6).
3. Click the [BLAST!] button.
4. Click the [Format!] button. The result will be ready within 5 min.
5. Sequences and the names of genes with score (bits) and E value are listed.
A higher score and lower E value indicate higher homology.
If candidates of interest are found, they must be validated by using other
ribozymes that are capable of cleaving a specific-target mRNA at other sites.
Inactive ribozymes with point mutations within the catalytic site are convenient
controls in such analysis. Alternatively, inactivation by small interfering RNAs
(siRNAs) can be also used to validate candidate genes. Once target genes have
been identified, they can be characterized individually with respect to the specific
involvement of each in the phenotype of interest.