Target Discovery and Validation Reviews and Protocols

Hammerhead Ribozymes 147
3.1.1. Preparation of the Insert DNA
1. Synthesize or purchase the DNA template described here;
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′
Bold letters correspond to the sequence of the ribozyme’s binding arms, and N
stands for any nucleotides (see Note 1).
2. Amplify the DNA template by PCR. For PCR, the reaction mixture contains (final
concentration): 0.02 µM template DNA, 0.5 µM primer A, 0.5 µM primer B, 1 X
Taq buffer, dNTP mix (0.25 mM each), and 10 U Taq polymerase. Place the reaction
mixture in a thermal cycler. Program the cycler to excute 6–8 cycles with the
following program: 95°C, 30 s; 55°C, 30 s; 74°C, 30 s.
3. Purify the PCR fragments by using a column (e.g., QIAquick PCR purification
kit) or phenol-chloroform extraction, and ethanol-precipitation (see Note 2).
4. Digest the PCR fragments with the restriction enzymes, Csp45I and KpnI, in 1X
L (low salt) buffer for >2 h at 37°C. After the reaction, purify the fragments as
described in step 3.
3.1.2. Preparation of Plasmids for Expressing Randomized Ribozymes
1. Digest the vector plasmid “pPUR-KE” with Csp45I and KpnI. After digestion,
load the reaction mixture onto a 1.0% agarose gel and electrophorese the sample
in 1X TAE buffer.
2. Excise gel pieces that contain the fragment of the vector and purify them using a
column (e.g. QIAquick Gel Extraction Kit).
3. Mix 20 µg of the linerized vector and 550 ng of the insert DNA. The ligation reaction
is performed by using DNA Ligation kit Ver.2 at 16°C for >4 h (see Note 3).
4. For transformation, mix 20 mL of E. coli host cells and 1 mL of the ligation mixture
and incubate it on ice for at least 30 min. After heating the mixture at 42°C in
a hot water, add preheated 180 mL of SOC medium at 37°C.
5. Incubate the E. coli mixture at 37°C for 1 h and then plate a part of the mixtures
(10 µL and 100 µL) on LB agar plates that contain ampicillin at 100 µg/mL (see
Note 4). Residual solution is subjected to the plasmid preparation.
6. After colonies have become apparent (12–16 h after plating), count the number of
colonies to statistically check the diversity of the library. For example, if 1000
colonies are found on a plate to which 10 µL of E. coli solution in 200 mL has
been plated, the diversity is estimated as 2 × 10 7 =1000 (colonies) × 200 (mL)/
10 (µL). To confirm the diversity, about 50 independent clones should be picked
up and sequenced.
3.2. Transfection of the Library Expressing Randomized
Hammerhead Ribozymes and Induction of the Selection Pressure
In this system, genes responsible for a specific phenotype of interest can be
isolated using a ribozyme library. When the library is introduced into cells,