1. Rapid identification of efficient target cleavage sites using a hammerhead ribozyme library in an iterative manner 
Wei-Hua Pan, Ping Xin, Vuong Bui, Gary A Clawson 
Molecular Therapy 
Volume 7, Issue 1, Pages (January 2003)
DOI: /S X(00)
Copyright © 2002 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Schematic representation of the random Rz selection library. (A) Diagram of a hammerhead ribozyme, showing a central domain for a trans-acting Rz (the catalytic core nucleotides are underlined), flanked by two random 9-nt 5′/3′ flanking regions. Arrowhead indicates the site of cleavage, just 3′ to the NUH triplet in the target RNA. (B) Procedure for generating the library of random Rz-RNA transcripts. Primers are annealed together and subjected to PCR amplification to yield a double-stranded DNA library. The Sp6 RNA polymerase promoter (underlined) is then utilized to transcribe the 79-nt random Rz library. (C) The dsDNA library was generated and sequenced using a PCR-based method; the products were then analyzed on a 6% sequencing gel under standard conditions. The results confirm the presence of the catalytic core and the two random 9-nt regions of the library.
Molecular Therapy 2003 7, DOI: ( /S X(00) )
Copyright © 2002 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Schematic representation of the target RNA. (A) Diagram of a hammerhead ribozyme tail, showing a catalytic core with fixed helix I, a “P” part that was the 3′ end of target RNA, and a “Q” part reverse complementary to the P portion of the target RNA. Arrowhead depicts the site of cleavage, just 3′ to the GUC triplet in the target RNA. (B) Procedure for generating the template of target RNA transcripts. Pretemplate, dsDNA generated by RT/PCR was subjected to PCR amplification with 5′/3′-end primers to yield a double-stranded DNA library. The T7 RNA polymerase promoter (underlined) was then utilized to transcribe the target RNA with Rz tail. (C) Target RNA with a precise 3′ end was self-liberated during in vitro transcription; the transcripts were then analyzed on a 6% sequencing gel under standard conditions.
Molecular Therapy 2003 7, DOI: ( /S X(00) )
Copyright © 2002 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Schematic overview of the library selection procedure. The Rz-library RNA and target RNA are annealed to form RNA–RNA complex (A(a)). The complexes are then isolated (A(b)) and regenerated (A(c)) by RT/PCR and in vitro transcription. The reamplified Rz-library RNA and the 5′- or 3′-end 32P-labeled target RNA in the presence of magnesium (A(d)) and then are mixed to initiate Rz-catalytic activity (A(e)). Finally, the cleaved products are separated on a 6% sequencing gel under standard conditions (A(f) and B). In B, lanes 1 and 2 are the target RNA incubated with random and selected Rz-library RNA (respectively), lanes 3 and 4 are G and A hydrolysis ladders generated from target RNA by RNase T1 and U2 digestions (respectively). The sizes of the cleavage products are shown to the left.
Molecular Therapy 2003 7, DOI: ( /S X(00) )
Copyright © 2002 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
In vitro cleavage analyses of HPV16 E6/E7 targeted sRz. (A) Comparative analysis of sRz activity in vitro. 40 nM individual sRz and 10 nM 5′-end 32P-labeled HPV16 target RNA (782 nt) were incubated for 30 min at 37°C in 20 mM Tris–HCl (pH 7.4), also containing 1, 5, or 25 mM MgCl2 (shown). Parallel analyses were performed with Rz427, which was the most active Rz identified using our SELEX selection protocol [38]. Following cleavage, the products were separated by denaturing PAGE, and the gel was dried and subjected to autoradiography. Shown are five sRz that were selected on the basis of preliminary tests. (B) In vitro cleavage analyses of HPV16 E6/E7 targeted sRz59. 1, 2.6, 6.4, 16, 40, and 100 nM sRz59 and 10 nM 5′-end 32P-labeled HPV16 target RNA (782 nt) were incubated for 30 min at 37°C in 20 mM Tris–HCl (pH 7.4), also containing 25 (shown) or 5 mM MgCl2. Following cleavage, the products were separated by denaturing PAGE, and the gel was dried and subjected to autoradiography.
Molecular Therapy 2003 7, DOI: ( /S X(00) )
Copyright © 2002 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
mFold plot of HPV16 E6/E7 target mRNA. Full-length HPV16 E6/E7 RNA was modeled using the RNA folding server found at Positions of the Rz sites identified by library selection are shown as sRz, and those chosen on the basis of the mFold modeling are shown as mRz.
Molecular Therapy 2003 7, DOI: ( /S X(00) )
Copyright © 2002 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Reduction of HPV16 E6/E7 target RNA in cotransfection experiments. 293T cells were cotransfected with plasmids encoding the HPV16 E6/E7 mRNA and the SNIPAAsRz constructs as indicated: SNIPAAsRz constructs are denoted by target cut site, while “AA” denotes the “empty” SNIPAA cassette (i.e., containing no E6/E7-targeted trans-acting Rz). After 3 or 5 days, RNA was isolated. (A) Quantification of E6/E7 transcript using radiolabeled RT/PCR. Primers were chosen to yield a 345-bp product for HPV16 E6/E7 and a 186-bp fragment for 18S rRNA, amplified concurrently as a standard. Following amplifications, samples were analyzed by PAGE and quantified using PhosphorImager analysis. (B) Quantification of HPV16 E6/E7 transcript using quantitative (Q) PCR. Repeat experiments were performed, RNA was isolated, and QPCR was performed as described. Results were analyzed using the REST program using the pair-wise fixed reallocation randomization test, with TBP as reference (results were similar without normalization). Essentially all reductions in E6/E7 transcript levels are statistically significant at P < Statistically significant reductions were also evident on day 1 and day 7, with average decreases of 34 and 47% (respectively; data not shown). (C) Quantification of HPV16 E6/E7 transcript using QPCR. Repeat experiments were performed, RNA was isolated, and QPCR was performed as described. Results were analyzed using the REST program using the pair-wise fixed reallocation randomization test, with TBP as reference (results were similar without normalization). MRz59 and mRz427 denote SNIPAA constructs containing catalytically inactive versions of Rz59 and Rz427. I Rz denotes an irrelevant SNIPAA construct containing a trans-acting Rz targeted to hepatitis B virus [38], and pVAX1 denotes the vector control. In contrast to results with the SNIPAARz constructs, no significant reductions were observed with the catalytically inactive versions.
Molecular Therapy 2003 7, DOI: ( /S X(00) )
Copyright © 2002 The American Society of Gene Therapy Terms and Conditions