Target Discovery and Validation Reviews and Protocols

Hammerhead Ribozymes 145
Libraries of randomized hammerhead ribozymes provide a novel tool for the
identification of functional genes (6–10). While the binding arms can be designed
to include sequences complementary to the target RNA, randomization of binding
arms generates a large pool of ribozymes that are capable of cleaving many
mRNA substrates. Such a pool of randomized ribozymes is referred to as a
library.
In a ribozyme-based gene-discovery system, a library of randomized ribozymes
is introduced into cells, where it causes phenotypic changes as a result of
cleaving transcripts that are critically involved in a particular phenotype. The
ribozymes can be recovered from the cells and subsequent sequence analysis,
which involves searching for sequences of a target site in databases with, for
example, the BLAST search program, leads to the identification of the targets of
the ribozymes (6–16). Alternatively, a partial cDNA fragment that subsequently
facilitates the cloning of full-length cDNA can be obtained by 5′ and 3′ rapid
amplification of cDNA ends (RACE), a PCR-based cloning technique (11–15).
Once target genes have been identified, they can be characterized individually
with respect to the specific involvement of each in the phenotype of interest.
The process can then be refined and repeated.
The system is simple and direct, as compared to other “high-throughput”
gene-screening methods, and no prior sequence information is required. Moreover,
because the system allows the identification of direct effectors of a change in
phenotype, it reduces the level of background and false-positive results and produces,
for the most part, interpretable results. Thus, this gene discovery system
based on the ribozyme library is indeed useful for the rapid identification of
functional genes in the post-genome era.
2. Materials
2.1. Construction of the Randomized Hammerhead
Ribozyme-Expression Vector
1. tRNAVal-expression vector: pPUR-KE (a plasmid that contains the promoter of a
human gene for tRNAVal between the EcoRI and BamHI sites of a plasmid pPUR
[Clontech, Palo Alto, CA]; Fig. 1B).
2. Custom-synthesized oligonucleotides.
a. Template DNA: 5′-TCC CCG GTT CGA AAC CGG GCA CTA CAA AAA
CCA ACT TTN NNN NNN CTG ATG AGG CCG AAA GGC CGA AAN
NNN NNG GTA CCC CGG ATA TCT TTT TTT-3′.
b. Primer A: 5′-TCC CCG GTT CGA AAC CGG GCA-3′.
c. Primer B: 5′-GCT TGC ATG CCT GCA GGT CGA CGC GAT AGA AAA
AAA GAT ATC CGG GGT-3′.
3. 10X Taq polymerase buffer: 100 mM Tris-HCl, pH 8.3, 15 mM MgCl2 , 500 mM KCl.
4. dNTP mixture: dATP, dCTP, dGTP, and dTTP, each at 2.5 mM.