Ribozymes and sirna Protocols

METHODS IN MOLECULAR BIOLOGY TM TM Volume 252 Ribozymes and sirna Protocols SECOND EDITION Edited by Mouldy Sioud pred GFP pred GFP sirna

strsv-derived Hairpin Ribozymes 339 25 Design, Targeting, and Initial Screening of strsv- Derived Hairpin Ribozymes for Optimum Helix 1 Length and Catalytic Efficiency In Vitro Max W. Richardson, Linda Hostalek, Michelle Dobson, Jason Hu, Richard Shippy, Andrew Siwkowski, Jonathan D. Marmur, Kamel Khalili, Paul E. Klotman, Arnold Hampel, and Jay Rappaport Summary Hairpin ribozymes derived from the negative strand of satellite RNAs from the tobacco ringspot virus (strsv) can be engineered to target and cleave a variety of heterologous RNAs from both cellular and viral transcripts. Attention to design and targeting rules and optimization of helix 1 length and catalytic efficiency in vitro may increase the efficacy of hairpin ribozymes in reducing the expression of targeted transcripts. Here, principles for the design and targeting of strsv-derived hairpin ribozymes are described, as well as methods and materials for optimizing helix 1 length, and for conducting an initial screen of catalytic efficiency to identify promising candidates for further evaluation. Examples are provided for hairpin ribozymes that target human and mouse transforming growth-factor beta (TGF-β), as well as human polycystic kidney disease gene 1 (PKD1) and JC virus large T-antigen. The tetraloop modification of the strsv hairpin ribozyme is considered superior to designs based on the native strsv hairpin ribozyme, given its potential to yeild considerable improvements in stability and catalytic efficiency. Key Words: Tetraloop; hairpin; ribozyme; strsv; TGF-β; HIV-1; PKD1; JCV; T-antigen. 1. Introduction The development of recombinant hairpin ribozymes that are capable of targeting and cleaving heterologous RNA sequences in trans as tools for gene therapy for a variety of diseases has generated considerable excitement (1,2). In particular, two catalytically efficient ribozymes were developed that targeted the 5' long terminal repeat (LTR) U5 leader sequence and the protease From: Methods in Molecular Biology, vol. 252: Ribozymes and sirna Protocols, Second Edition Edited by: M. Sioud Humana Press Inc., Totowa, NJ 339

340 Richardson et al. gene of HIV-1, and initially showed very encouraging results in reducing or blocking viral replication in vitro in both transformed T-cell lines and primary cultures (3 6). Unfortunately, despite preliminary evidence of the safety and stability in vivo of autologous CD8-depleted cells transduced ex vivo with a murine retroviral vector expressing an anti-hiv-1 ribozyme (7), no clinical study to date has shown evidence of the utility of the hairpin ribozyme approach in vivo. This may reflect difficulty in maintaining long-term expression of ribozyme constructs, and the short half-life of ribozyme transcripts in vivo. Despite the lack of immediate success, hairpin ribozymes are still being studied as potential tools for gene therapy, and may show efficacy in the future as technology improves in terms of delivery and stability of expression, and the stability of the ribozymes themselves. It is also worth noting that although ribozymes are catalytically ineffective in vitro compared to conventional enzymes, they may be more efficient in vivo because of the presence of protein co-factors (8), particularly if directed to the right subcellular environment. The rules that govern the targeting and design of native and tetraloop hairpin ribozymes derived from the negative strand of the satellite RNA from tobacco ringspot virus (strsv) are well-defined, and were previously described in detail (9). Caveats for the design of ribozymes derived from the satellite RNA of chicory yellow mottle virus (scymv1) have also been welldescribed (10). Briefly, for strsv-derived hairpin ribozymes, targeting a heterologous RNA requires identification of the sequence BN*GUC at one or more positions in its transcript, in which B is any base but A, N is any base, * denotes the site of cleavage, and GUC are required. Particular attention should be given to identifying target sites in the 5' and 3' regions of transcripts, and splice donor sites and regions with considerable secondary structure in general should be avoided. In terms of substrate complementarity, helix 1 may be of variable length generally from 6 10 bases helix 2 is fixed at four bases. The tetraloop modification of the strsv ribozyme appears to have few drawbacks. It may lead to both increased stability and a considerable increase in catalytic efficiency relative to the native hairpin ribozyme, and is therefore recommended (see Note 1). This chapter provides specific examples from the development of strsv-derived native and tetraloop hairpin ribozymes that target both human transforming growth factor-β (htgf-β) and murine TGF-β (mtgf-β), in the context of their overexpression in HIV-1-associated nephropathy (HIVAN) and in a transgenic mouse model with some characteristics of HIVAN (11). TGF-β also has been implicated in HIV-induced central nervous system (CNS) disorders (12,13). This chapter also describes the development of ribozymes targeting transcripts of the polycystic kidney disease gene 1 protein (PKD1) (14), and the tumorigenic JC virus (JCV) large T-antigen (15). The results are intended to provide an example of the application of targeting

strsv-derived Hairpin Ribozymes 341 and design rules in an initial screen of ribozymes for catalytic efficiency to identify the most promising candidates for subsequent testing in vitro (see Note 2). 2. Materials 2.1. Identification of Potential Ribozyme Cleavage Sites in Targeted Transcripts Sequences that are potentially amenable to targeting with hairpin ribozymes have been identified in the transcripts of targeted genes using the commercially available DNA sequence manipulation program MacVector (Accelrys Inc., San Diego, CA). Similarly, sequences of transcripts from targeted genes were obtained from publicly available sequence information (http:// www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=nucleotide). Native hairpin and tetraloop ribozymes have been designed, as previously described (9). A tetraloop design template is provided here (Fig. 1). T7 promoter regions must be added to the negative-strand template for in vitro transcription; restriction sites may be added and the plus strand must be synthesized for subsequent subcloning into appropriate expression vectors. 2.2. Oligonucleotide Templates for In Vitro Transcription of Ribozyme and Substrate RNA Oligonucleotide templates for in vitro transcription using T7 RNA polymerase were ordered from commercial manufacturers (Midland Certified Reagent Company, Midland, Texas; Invitrogen, La Jolla, CA), or synthesized in house using an ABI 392 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). Commercial oligonucleotides were gel-purified. Oligonucleotides that were synthesized in-house were high-performance liquid chromatography (HPLC)-purified as previously described using an RP-300 Brownlee reverse-phase column and an acetonitrile-triethylammonium acetate, ph 7.0 gradient (16,17). HPLC-purified oligonucleotides performed better in subsequent transcription reactions compared to those that were gel-purified. Oligonucleotide templates used for in vitro transcription of ribozymes and substrates, as well as the corresponding T7 promoter primers, are listed here; target regions of ribozymes are indicated in lower-case lettering. The mrnas for human and murine TGF-β, PKD1, and JCV large T-antigen were targeted. The wild-type strsv ribozyme and substrate templates are also included, and were used as a control for kinetic analysis. 1. Human transforming growth factor beta (htgf-β) ribozymes and substrates: 960R tetraloop (156 160) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT cca gtt ctt gag acg acc CTA TAG TGA GTC GTA TTA 3'

342 Fig. 1. strsv-derived tetraloop hairpin ribozyme design template. 342 Richardson et al.

strsv-derived Hairpin Ribozymes 343 760S substrate (156 160) 5' TCG TCT CAG ACT CTG GCG CTA TAG TGA GTC GTA TTA 3' 961R tetraloop (230 234) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT agc ttt ctg gga gaa gcc CTA TAG TGA GTC GTA TTA 3' 761S substrate (230 234) 5' CTT CTC CCG ACC AGC TCG CTA TAG TGA GTC GTA TTA 3' 962R tetraloop (400 404) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT acg gtt ctc ctc agg ccc CTA TAG TGA GTC GTA TTA 3' 762S substrate (400 404) 5' GCC TGA GGG ACG CCG TCG CTA TAG TGA GTC GTA TTA 3' 963R tetraloop (438 442) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT cgg gtt ctg ccg acc ccc CTA TAG TGA GTC GTA TTA 3' 763S substrate (438 442) 5' GGG TCG GCG ACT CCC GCG CTA TAG TGA GTC GTA TTA 3' 964R tetraloop (517 521) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT ggc ctt ctt cct gag ccc CTA TAG TGA GTC GTA TTA 3' 764S substrate (517 521) 5' GCT CAG GAG ACA GGC CCG CTA TAG TGA GTC GTA TTA 3' 966R tetraloop (2188 2192) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT ctg gtt ctt cca tcc ccc CTA TAG TGA GTC GTA TTA 3' 766S substrate (2188 2192) 5' GGG ATG GAG ACC CCA GCG CTA TAG TGA GTC GTA TTA 3' 967R tetraloop (2252 2256) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT tgc ctt ctt gca cta tcc CTA TAG TGA GTC GTA TTA 3' 767S substrate (2252 2256) 5' ATA GTG CAG ACA GGC ACG CTA TAG TGA GTC GTA TTA 3' 968R tetraloop (2377 2381) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT att ctt cta cca tag ccc CTA TAG TGA GTC GTA TTA 3' 768S substrate (2377 2381) 5' GCT ATG GTG ACT GAA TCG CTA TAG TGA GTC GTA TTA 3' 2. Murine transforming growth factor beta (mtgf-β) ribozymes and substrates: 835R native (23 27) 5' TAC CAG GTA ATA TAC CAC AAC GTG TGT TTC TCT GGT ctt gtt ctc ctc gca tcc CCC TAT AGT GAG TCG TAT TA 3' 838R tetraloop (23 27) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT ctt gtt ctc ctc gca tcc CCC TAT AGT GAG TCG TAT TA 3' 836S substrate (23 27) 5' GGA TGC GAG GGA CTC AAG CGC TAT AGT GAG TCG TAT TA 3'

344 Richardson et al. 842R native (532 536) 5' TAC CAG GTA ATA TAC CAC AAC GTG TGT TTC TCT GGT cgc ctt ctc ccc aag cca CCC TAT AGT GAG TCG TAT TA 3' 843R tetraloop (532 536) 5' TAC CAG GTA ATA TAC CAC GGA CCG AAG TCC GTG TGT TTC TCT GGT cgc ctt ctc ccc aag cca CCC TAT AGT GAG TCG TAT TA 3' 837S substrate (532 536) 5' TGG CTT GGG GGA CTG GCG CGC TAT AGT GAG TCG TAT TA 3 3. Human polycystic kidney disease gene 1 protein (PKD1) ribozyme and substrate: PKDR native (13255 13259) 5' TAC CAG GTA ATA TAC CAC AAC GTG TGT TTC TCT GGT cat ctt ctt gtc tgt ggg CCC TAT AGT GAG TCG TAT TA 3' PKDS substrate (13255 13259) 5' CCC ACA GAC AGA CAG ATG CGC TAT AGT GAG TCG TAT TA 3' 4. JCV virus large T-antigen ribozyme and substrate. JCVR native (4931 4927) 5' TAC CAG GTA ATA TAC CAC AAC GTG TGT TTC TCT GT ttc ctt cta tga gaa aag ctc CCT ATA GTG AGT CGT ATT A 3' JCVS substrate (4931 4927) 5' AGC TTT TCT CAT GAC AGG AAC GCT ATA GTG AGT CGT ATT A 3' 5. strsv control ribozyme and substrate. strsvr native (control) 5' TAC CAG GTA ATA TAC CAC AAC GTG TGT TTC TCT GGT tga ctt ctc tgt ttc CCT ATA GTG AGT CGT ATT A 3' strsv substrate (control) 5' AAA CAG GAC TGT CAC GCT ATA GTG AGT CGT ATT A 3' 6. T7 promoter ribozyme and substrate oligonucleotides. 773R (T7comp-R) 5' TAA TAC GAC TCA CTA TAG GG 3' 772S (T7comp-S) 5' TAA TAC GAC TCA CTA TAG CG 3' 2.3. End-Labeling of DNA Mol-Wt Markers Five micrograms each of oligonucleotides of 93, 71, 50, 47, and 22 nucleotides (nt) were pooled, lyophilized, and resuspended in 25 µl of dh 2 O. The sequences of these oligonucleotides are as follows: 93 nt 5' CCC TGC AGA ATT CTT GGG AGA AGC GAA CCA GAG CGT CAC ACG GAC TTC GGT CCG TGG TAT ATT ACC TGG TAT TTT TTT AAG CTT ATC GAT CCC 3' 71 nt 5' AAT TCA CAC AAC AAG AAG GCA ACC AGA GAA ACA CAC GGA CTT CGG TCC GTG GTA TAT TAC CTG GTA CCC GG 3' 50 nt 5' GCT CGA GAC GTT GCA ACG TTG CAA CGT GGA TCC TCG ACG TGA GAG CTC GG 3' 47 nt 5' AAT TCT TTT TCT TCT AGA ATG TCT TGA TTG TTG AGG TAA GTG CTG CA 3' 22 nt 5' GGT CGA CTA CCA GGT AAT ATA C 3' The following reagents are also necessary for end-labeling the oligonucleotide pool:

strsv-derived Hairpin Ribozymes 345 1. T4 polynucleotide kinase (PNK) and 10X kinase buffer (Composition), 10 mm spermidine. 2. γp 32 -adenosine 5' triphosphate (ATP) (4500 Ci/mmol); 660 µm ATP. 2.4. In Vitro Transcription of Ribozyme and Substrate RNA Reagents required for annealing of ribozyme and substrate templates to their respective T7-promoter primers, and for subsequent transcription and purification of transcription products, have been described previously in detail (16). A brief summary of required reagents is provided here. The following reagents are required for annealing ribozyme and substrate templates with their respective T7 promoter primers, and for subsequent in vitro transcription of ribozyme and substrate RNAs. 1. Annealed ribozyme and substrate templates. Equimolar amounts of ribozyme or substrate template and the appropriate T7 promoter primer are required approx 10.0 µg of ribozyme template with 2.5 µg T7 promoter 773, and 10.0 µg of substrate template with 5.5 µg of T7 promoter primer 772. The following formula may be used in a spreadsheet program such as Excel (Microsoft Inc., Bellevue, WA) to calculate equimolar amounts of T7 promoter primers. Amounts of T7 primers are calculated based on the use of 10 µg of ribozyme or substrate template, and equimolar amounts are calculated by adjusting for size in basepairs (bp) of primers: T7 µl = [10 (bp of T7 primer)/(bp of ribo or sub primer)]/(t7 primer conc.) 2. 1 M Tris-HCl, ph 7.5. 3. A source of fresh, RNase-free deionized water. DEPC treatment of deionized water is not necessary (16). 4. An accurate, clean set of pipets such as Pipetman, preferably including a P10. 5. Latex or nitrile gloves to prevent RNase contamination. 6. Sterile pipet tips, preferably RNase- and DNase-free; 1.5-mL Eppendorf tubes. 7. A glass beaker or similar device to be heated to 90 C using either a microwave or ring-stand and Bunsen burner, a floating rack, and aluminum foil. 8. 2X transcription buffer mix: 80 mm Tris-HCl, ph 8.0 (1.6 ml 1 M Tris, ph 8.0), 12 mm MgCl 2 (240 µl 1 M MgCl 2 ), 10 mm dithiothreitol (DTT) (200 µl 1 M DTT), 2 mm Spermidine (400 µl 100 mm Spermidine), 8% polyethylene glycol (PEG) 3000 (4 ml 40% PEG 3000), 0.2% Triton X-100 (400 µl 10% Triton X-100), (13.16 ml dh 2 O to 20 ml final). Aliquot and store frozen at 20 C or lower; resuspend completely before use to avoid precipitation. 9. 40 mm final nucleotide triphosphate (NTP) stock (10 mm each NTP). Aliquot and store frozen at 20 C or lower; avoid repeated freeze/thaw cycles. 10. 3000 Ci/mmol αp 32 -CTP (10 µci/µl). 11. T7 RNA polymerase (20 U/µL). 12. RNasin or RNase inhibitor (40 U/µL).

346 Richardson et al. 13. RNase-free DNase I (2 U/µL). 14. Glycogen (20 mg/ml) from Roche Molecular Biochemicals (Indianapolis, IN). 15. 3 M sodium acetate, ph 5.4 (Sigma). 16. 100% reagent alcohol, HPLC-grade (Fisher Scientific, Pittsburgh, PA). 17. 70% reagent alcohol. 18. 2 mm ethylenediaminetetraacetic acid (EDTA). 19. 98% formamide dye mix 98% deionized formamide (49 ml) with 0.025% xylene cyanol (0.0125 g) and bromophenol blue (0.0125 g) as markers, and dh 2 O (to 50 ml final). Aliquot and store at 20 C or lower. 20. 10% and 15% polyacrylamide, 7 M urea, 1X TBE gels. A 50l-mL solution for pouring a 10% gel consists of 12.5 ml of a 40% stock solution of 19:1 acrylamide:bis-acrylamide (0.22 µm filtered), 5 ml of 10X TBE, ph 8.0, solution, 21 g of urea, and dh 2 O to 50 ml final; stir to mix into solution and filter through 0.22-µm filter unit prior to use. Immediately before pouring, add 350 µl of a 10% ammonium-persulfate solution, mix gently, and then add 75 µl of N,N,N',N'-tetamethyl-ethylenediamine (TEMED), mix again and pour. For a 15% gel, use 18.75 ml of the 40% stock solution. 21. Sterile razor blades and mini grinding pestles (Research Products International, Mt. Prospect, IL) designed for use in 1.5-mL Eppendorf tubes. Also, a standard Eppendorf microcentrifuge and a shaker/mixer (Fisher Scientific, Pittsburgh, PA). 22. Gel slice extraction buffer: 0.5 M ammonium acetate, 0.5 mg/ml sodium dodecyl sulfate (SDS), 2 mm EDTA. 23. Plastic wrap for covering gels; autoradiography cassettes, film, and development materials; scintillation vials capable of holding 1.5-mL Eppendorf tubes; a calibrated scintillation counter of known efficiency. 24. The following formulas for determining the concentration of gel purified ribozyme and substrate transcripts. These are easily incorporated in a spreadsheet format: a. dpm = Cerencov cpm/counting efficieny b. µci in transcript = dpm/2,200,000; 1 µci is 2,200,000 dpm c. µci CTP label remaining = µci added/decay factor; lose 3.5% per d past ref. date d. pmol C in transcript = (pmol cold CTP in rxn) (B/C); 1 mm cold CTP is 50,000 pmol e. pmol RNA in transcript = D/(the number of C residues in the transcript) f. dh 2 O µl for resuspension = E/(desired final concentration in µm) 2.5. Helix 1 Length Optimization and Preliminary Analysis of Catalytic Efficiency 1. Gel-purified ribozyme and substrate RNA of known concentration; 0.25-mL Eppendorf tubes and a thermocycler with a heated lid may be preferable for cleavage reactions. 2. 4X cleavage buffer: 8 mm spermidine, 48 mm MgCl 2, 160 mm Tris-HCl, ph 7.5. 3. Scintillation fluid for quantifying radioactivity in gel slices containing uncleaved and cleaved substrate bands. Alternatively, cleavage may be quantified using a

strsv-derived Hairpin Ribozymes 347 phosphorimager, provided the signal is maintained within the linear range of the screen i.e., the screen is not overexposed. 4. Software for calculating catalytic efficiency, preferably employing a nonlinear algorithm for example, Tablecurve 2D (Jandel Scientific, San Rafael, CA) or more recent releases. 3. Methods Methods for annealing ribozyme and substrate oligonucleotides with their respective T7 promoter primers and for subsequent in vitro transcription, gel purification, quantification, helix 1 length optimization, and kinetic analysis have been described in detail previously (16). The following information is intended as a brief overview of relevant protocols. 3.1. Identification of Potential Ribozyme Cleavage Sites in Targeted Transcripts Nucleotide sequences of transcripts encoding htgf-β, mtgf-β, PKD1, and JCV large T-antigen were obtained from GenBank (see Subheading 2.), and imported into MacVector. Transcripts were then screened using MacVector for sites that could be potentially amenable to cleavage e.g., the BN*GUC sequence, where B is any base but A, N is any base, and GUC are required. Particular attention was given to the 5' and 3' regions of transcripts, although rigorous analysis to identify highly conserved domains and regions of minimal secondary structure was not performed (9,18,19). Once suitable sites were identified, hairpin and/or tetraloop ribozymes were designed to correspond to the targeted region using a convenient design template (Fig. 1), with a 9 10 basepair-complementary region in Helix 1. Substrate templates were designed accordingly, again with 9 10 complementary bases in Helix 1. The following sequences should be added to the 3' end of the minus strand of the ribozyme design template (Fig. 1), and to the 3' end of the complement of the substrate sequence e.g., the substrate template for in vitro transcription: Ribozyme: 5' C CCT ATA GTG AGT CGT ATT A 3' Substrate: 5' C GCT ATA GTG AGT CGT ATT A 3' Once ribozyme and substrate templates were designed, they were either synthesized commercially and gel-purified, or made in-house and HPLC-purified, which appeared to be preferable for subsequent in vitro transcriptions. 3.2. Annealing of Ribozyme and Substrate Templates With T7 Promoter Primers 1. In a 1.5-mL Eppendorf tube, add 10 µg of ribozyme or substrate oligonucleotide template, and an equimolar amount of appropriate T7 promoter primer as calculated using the formula from Subheading 2. Approximately 2.5 µg of T7

348 Richardson et al. promoter primer 773 is used with ribozyme templates, and 5.0 µg of T7 promoter primer 772 is used with substrate templates. 2. Add 1 µl of 1 M Tris, ph 7.5, and dh 2 O to 100 µl final volume. 3. Heat to 95 C in a beaker of water, cover in aluminum foil, and cool slowly to room temperature. Store annealed templates at 20 C prior to use, or use immediately. 3.3. End-Labeling of DNA Mol-Wt Markers 1. Use 5 µl of the 25 µl oligo pool (1 mg/ml) described in Subheading 2. for the following reaction: a. 5 µl DNA pool b. 1 µl 10X kinase buffer c,. 1 µl γp 32 -ATP (4500 Ci/mmol) d. 1 µl 10 mm spermidine e. 1 µl 660 µm ATP f. 1 µl T4 PNK 2. Incubate at 37 C for 1 h. 3. Add 50 µl of formamide dye mix, and use 5 µl of the resulting solution per transcription gel. Alternatively, remove free nucleotide using an Eppendorf format G-25 spin column (5 Prime 3 Prime, Inc., Boulder, CO), and use a similar volume of the flowthrough per transcription gel. For overnight exposures, approx 200,000 500,000 cpm is sufficient; it may be best to determine the exact amount empirically. 3.4. In Vitro Transcription of Ribozyme and Substrate RNA 1. Set up the transcription reaction (50 µl) as follows: a. 25 µl 2X transcription mix b. 5 µl 40 mm NTP mix (10 mm each NTP) c. 6 µl annealed template d. 2 4 µl αp 32 -CTP 10 µci/µl (Ribo = 2 µl; Sub = 4 µl) e. 1 µl RNasin (RNase inhibitor) f. 5 7 µl dh 2 O (Sub = 5 µl; Ribo = 7 µl) g. 1 µl T7 RNA polymerase 2. Incubate at 37 C for 3 h; transcription yields may be improved by adding an additional 2.5 µl of 10 mm guanosine 5' triphosphate (GTP) to the reaction after 1 h (16). 3. After 3 h, add 1 µl of DNase I and incubate for an additional 30 min at 37 C. 4. Stop the reaction by adding 5 µl of 3 M sodium acetate, ph 5.4, 1 µl of glycogen, and 150 µl of 100% reagent alcohol. Freeze on dry ice or at 80 C for 30 min, and precipitate RNA by centrifugation at full speed in a microcentrifuge (>10,000g) for 15 min. After removing the supernatant and air-drying briefly, resuspend the pellet in 8 µl of dh 2 O, add 8 µl of formamide dye mix, heat to 90 C for 3 min and cool rapidly on ice. The sample is now ready to be loaded on a denaturing polyacrylamide gel. Alternatively, reactions may be stopped by the addition of 50 µl of formamide dye mix, heated at 90 C for 3 min, and cooled

strsv-derived Hairpin Ribozymes 349 Fig. 2. Gel purification of ribozyme and substrate transcription products. (A) Ribozyme trancription products; the native strsv-derived ribozyme (lane 1) is somewhat smaller, and therefore migrates more quickly than the tetraloop strsv-derived ribozymes (lanes 2 and 3). (B) Substrate transcription products migrate somewhat variably, based on GC content. The transcript in lane 1 is shorter than it should be, and did not cleave efficiently; the other transcripts are of appropriate size and performed well in subsequent cleavage reactions. Full-length n transcripts are indicated with an arrow; one n-1 transcript is marked with a circle. rapidly on ice, and loaded directly on a gel if it is of appropriate thickness and the wells are large enough to handle a sample volume of 100 µl. 5. Load ribozyme transcripts on 10% polyacrylamide, 7 M urea gels; use 15% gels for purification of substrate transcripts. Run gels until the upper xylene cyanol dye front has migrated approx two-thirds of the way down, or approx 32 W, for 3 h. 6. Disassemble gels, and transfer gel to some type of backing such as an older piece of exposed autoradiography film, wrap in plastic wrap, and expose for 2 10 min at room temperature, which should be sufficient if the transcription reaction has worked well. Place pieces of tape on the edges of the film, and mark them with a pen to orient the film properly after development for excision of transcript bands. Full-length ribozyme transcripts are relatively easy to identify. The lower 22-nt DNA marker migrates at approx 19 bp (20), very close to many full-length substrates. Progressively smaller substrate bands (e.g., n-1) should also be excised for use in Helix 1 length optimization. Identification of the full-length substrate transcript can be somewhat difficult, and confirmation by direct RNA sequencing of purified substrate transcripts is an option (20). An example of gel-purified ribozyme and substrate transcripts is provided (Fig. 2).

350 Richardson et al. 7. Excise bands with sterile razor blades, and grind in a 1.5-mL Eppendorf tube using 0.5 ml of gel-extraction buffer and a mini-grinding pestle. Shake homogenized gels slices vigorously in an Eppendorf mixer/shaker for 60 min, and then centrifuge full-speed (>10,000g) at room temperature in a microcentrifuge for 20 min. Transfer the upper phase to new 1.5-mL Eppendorf tubes, add 1 µl of glycogen and 1 ml of 100% reagent alcohol, and precipitate by freezing on dry ice or at 80 C for 30 min and then centrifuging at full speed in a microcentrifuge for 15 min at 4 C. Wash the pellet twice with 500 µl of ice-cold 70% reagent alcohol, and then dry pellets in a speed-vac, or simply by air-drying. 8. Count radioactivity in RNA pellets by Cerenkov counting e.g., place the entire 1.5-mL Eppendorf containing the pellet in a scintillation vial (without scintillation fluid) and read it in a scintillation counter. 9. Calculate the pmoles of RNA of each ribozyme or substrate pellet using the formulas provided in Subheading 2. It is important to note that the number of Cs in a transcript can be determined by counting the number of Gs in the template oligonucleotide excluding the T7 promoter region but in the case of substrate templates including the additional G residue in the CGC sequence acted to enhance transcription initiation. Resuspend ribozymes at 80 nm, and substrates at 400 nm in dh 2 O initially. Using the formula given in Subheading 2., the volume of dh 2 O to add is calculated by dividing the pmols of RNA by the desired final concentration in µm e.g., 0.08 µm for ribozymes and 0.4 µm for substrates. If time allows and there is enough product, take wet counts in scintillation fluid using 1 µl of resuspended ribozyme or substrate, and adjust concentrations accordingly. Use ribozymes and substrates immediately or store at 80 C prior to use. 3.5. Helix 1 Length Optimization and Preliminary Analysis of Catalytic Efficiency Formal kinetic analysis of individual hairpin ribozymes and their optimized substrates requires considerable effort, including repetition of analysis several times using material produced from various transcription reactions to demonstrate reproducibility. Using multiple-turnover conditions albeit with less than 20% substrate cleavage at least two time-points should be tested for each of several substrate concentrations around the apparent K M. As an initial screen of several potential ribozymes that target the same gene, less rigorous methods may be used to narrow the field to a few promising candidates. The following protocols (16) are provided with the goal of a preliminary screen in mind. 1. Perform an initial cleavage reaction using a 1:5 ratio of ribozyme (0.08 µm stock) to substrate (0.4 µm stock) for 1 h at 37 C to determine whether turnover will occur. Run a native strmv ribozyme and substrate control reaction in parallel. Set up the reactions in a 0.25-mL Eppendorf tube suitable for use in a thermocycler with a heated lid as follows:

strsv-derived Hairpin Ribozymes 351 a. 1 µl 4X cleavage buffer b. 1 µl 0.4 µm substrate stock c. 1 µl dh 2 O d. 1 µl 0.08 µm ribozyme stock 2. Stop the reaction by adding 4 µl formamide dye, heat to 90 C, snap-cool on ice, and separate ribozyme, uncleaved substrate, and 3' (larger) and 5' substrate cleavage products on a 15% polyacrylamide, 7 M urea gel; run gel for approx 1 h at 12 W. Expose to autoradiography film overnight at 80 C, or use a phosphorimager screen for an appropriate amount of time, as determined empirically. 3. Quantitate the percentage of cleavage by excising bands and counting in a scintillation counter, or by phosphorimager analysis. In order for turnover to occur, more than 20% of the substrate must be cleaved, considering the molar ratio of ribozyme to substrate. Percentage of cleavage and nm of substrate cleaved are calculated with the following formulas: % cleavage = 100 [(5' product + 3' product)/(uncleaved + 5' + 3')] nm cleaved substrate = (% cleavage) (nm substrate used in reaction) 4. If turnover did not occur in the initial cleavage reaction, it may be necessary to redesign the ribozyme with a shorter complementary region in helix 1. In either case, it is advisable to make use of the progressively shorter transcripts generated during substrate transcription to generate some information regarding the optimum length of helix 1. Smaller substrate transcripts are progressively shorter on the 3' end, and thus represent n-1, n-2, lengths of helix 1; an n + 1 substrate transcript is also typically present. 5. Set up reactions using equal volumes of 0.08 µm ribozyme and 0.4 µm substrate transcript stock solutions, including n + 1, n and n 1, as previously with the initial cleavage reaction to determine the optimum helix 1 length. It may be necessary to incubate the reaction for a longer period 2 h instead of 1 h. Calculate the percentage of cleavage as in step 3. The optimum helix 1 length corresponds to the substrate transcript with which the highest percent cleavage was observed. An example of a helix optimization experiment is provided (Fig. 3). The ribozyme and substrate templates may be redesigned accordingly, or if there is enough of the appropriate substrate transcript available, further kinetic analysis may be performed immediately. 6. A time-course reaction should be performed next to confirm multiple turnover of the ribozyme, generate an estimate of the turnover rate, and refine the incubation time for subsequent kinetic analysis. Use a 1:5 molar ratio of ribozyme (0.08 µm stock) to substrate (0.4 µm stock). Lower concentrations of ribozyme should be used if >80% cleavage was observed in the initial reaction; lower concentrations of substrate may also be used. For reference, final ribozyme concentrations of 1 5 nm are typically used later in formal kinetic analysis. Set up the time-course reaction as follows:

352 Richardson et al. Fig. 3. Helix 1 length optimization. For this tetraloop ribozyme, helix 1 length was determined by phophorimager analysis to be optimized at n-5, corresponding to a helix 1 length of 5 nucleotides. No cleavage was observed past n-8, presumably because of the inability of the ribozyme to bind substrate efficiently with a helix 1 length of less than two bases. a. 8 µl 4X cleavage buffer b. 8 µl 0.4 µm substrate stock (or lower concentration) c. 8 µl dh 2 O d. 8 µl 0.08 µm ribozyme stock (or lower concentration) 7. Withdraw 3-µL aliquots from the reaction at time 0, and every 15 min subsequently for approx 2 h. Stop reactions by adding an equal volume of formamide dye. Separate products by gel electrophoresis and determine the percentage of cleavage as in step 3. An example of a time-course experiment is provided (Fig. 4). The turnover rate is calculated with the following equation: turnover rate = (nm cleaved substrate/nm ribozyme in reaction)/(time in min) 8. Set up multiple-turnover reactions with constant, limiting amounts of ribozyme and varying amounts of substrate to determine the rate constants k cat and K M. Incubate reactions for the appropriate length of time based on the turnover rate. Keep the total percentage of cleavage of substrate below 20% to maintain linear conditions with regard to product vs time, yet maintain multiple-turnover conditions. Typical final ribozyme concentrations are from 1 5 nm. A range of substrate concentrations should be used, working down from the maximum volume

strsv-derived Hairpin Ribozymes 353 Fig. 4. Time-course experiment to verify multiple-turnover conditions, and obtain an estimate of the rate constant for subsequent kinetics experiments. The ribozyme, uncleaved substrate, and 3' and 5' substrate cleavage products are indicated. The minutes of incubation for each time-point are indicated. Although the amount of ribozyme appears lower at 30 and 60 min, all time-points were drawn from the same reaction, and variability was not observed with the substrate. of concentrated substrate stock capable of being added to the reaction in 0.5-µL increments. High substrate concentrations are essential to approach V max experimentally, as evidenced by a plateau in a graph of velocity vs substrate concentration; accurate estimates of k cat and K M are dependent upon approximating V max. The native strsv ribozyme and substrate should be run in parallel as a control. Separate products by gel electrophoresis and quantitate cleavage products as previously via scintillation counting or phosphorimager analysis. A typical gel from a successful kinetics reaction is provided for reference (Fig. 5). An example set of reactions follows:

354 Richardson et al. Fig. 5. Kinetics experiment with a constant and limiting amount of ribozyme, multiple-turnover conditions and varying amounts of substrate. substrate (400 nm) ribozyme (5 nm) dh 2 O 4X reaction buffer 4.0 µl 2 µl 0 µl 2 µl 3.5 µl 2 µl 0.5 µl 2 µl 3.0 µl 2 µl 1.0 µl 2 µl 2.5 µl 2 µl 1.5 µl 2 µl 2.0 µl 2 µl 2.0 µl 2 µl 1.5 µl 2 µl 2.5 µl 2 µl 1.0 µl 2 µl 3.0 µl 2 µl 0.5 µl 2 µl 3.5 µl 2 µl 9. Calculate preliminary estimates of k cat and K M using Tablecurve 2D (Jandel Scientific, San Rafael, CA). Curves should be fit using nonlinear regression methods. The velocity of the reaction is determined first using the following equation: velocity = nm cleaved substrate/min

Table 1 Summary of Results From the Initial Screen of Ribozymes Targeting htgf-β, mtgf-β, PKD1, and JCV T- Antigen Ribozyme/substrate Gene Site Helix 1 Turnover k cat (min 1 ) K M (nm) k cat /K M (min 1 nm 1 ) 960R tetraloop/760s htgf-β 157 na Yes ND ND ND 961R tetraloop/761s htgf-β 231 na Yes ND ND ND 962R tetraloop/762s htgf-β 401 na Yes 0.093 298 3.12 10 4 963R tetraloop/763sb htgf-β 439 na Yes ND ND ND 964R tetraloop/764s htgf-β 518 na No ND ND ND 966R tetraloop/766s htgf-β 2189 na ND ND ND ND 967R tetraloop/767s htgf-β 2253 na Yes 0.028 216 1.30 10 4 968R tetraloop/768s htgf-β 2378 na Yes 1.86 10 13c 8.36 10 16c 2.22 10 4c 835R native/836s mtgf-β 24 n-2 Yes 0.013 d 43 d 3.02 10 4d 838R tetraloop/836s mtgf-β 24 n-2 Yes 0.05 d 88 d 5.68 10 4d 842R native/837s mtgf-β 533 n-4 Yes 0.038 192 1.98 10 4 842R native /837S mtgf-β 533 n Yes 0.025 168 1.49 10 4 843R tetraloop/837s mtgf-β 533 n-5 Yes 0.039 111 3.51 10 4 PKDR native/pkds PKD1 13256 n-4 Yes 0.084 247 3.40 10 4 PKDR native/pkds PKD1 13256 n Yes 0.021 87 2.41 10 4 JCVR native/jcvs JCV-T ag 4932 n-1 No ND ND ND strsvr/s control Control Control n Yes 0.40 91 4.50 10 4 ND, not determined. a Helix 1 length optimization was not performed; preliminary kinetic analysis was performed with full-length substrate (n). b Of the htgf-β ribozymes for which preliminary kinetic analysis was not performed because of relatively inefficient substrate transcription, the 963R tetraloop ribozyme appeared to be most promising based on the initial time-course reaction using full-length substrate (n). c Although the k cat /K M value for this ribozyme is reasonable, the individual rate constants are unreliable because of the failure of this reaction to plateau in terms of a graph of velocity vs substrate concentration; higher substrate concentrations must be tested with this ribozyme. d Although helix 1 was optimized at n-2 for these ribozymes, n was cleaved comparably, and was used in kinetic analysis because of the availability of the transcript in quantity. strsv-derived Hairpin Ribozymes 355

356 Richardson et al. The following equation is then used to calculate the K M and V max : y = A x/(b + x) where y = velocity; x = initial nm substrate; A = V max ; B = K M. The following equation is then used to determine k cat : k cat = V max /(nm ribozyme) The overall catalytic efficiency of the ribozyme is expressed as k cat /K M. 10. Select the ribozymes with the highest catalytic efficiency for subsequent subcloning into the appropriate expression vectors for in vitro analysis of efficacy in reducing expression of the targeted transcripts (see Notes 3 and 4). If formal rate constants are desired, repeat the kinetic analysis with material from different transcriptions, using multiple time-points for each of several substrate concentrations around the apparent K M. 4. Notes 1. Results of the initial screen of several ribozymes that target htgf-β and mtgf-β, as well as PKD1 and the JCV large T-antigen are provided (Table 1). Several promising ribozymes directed against htgf-β and mtgf-β have been identified. The ribozyme targeting PKD1 also appeared to be worthy of further consideration. In contrast, the ribozyme directed against JCV large T-antigen failed to turn-over, and thus must be redesigned with a shorter substrate or a different target region. 2. Generally, tetraloop ribozymes performed better than native varities. Therefore, it appears to be generally preferable to incorporate the tetraloop modification in the first place, and it may be of little value to also synthesize a native ribozyme for each target site in order to compare the two. 3. The studies described here illustrate methods to identify and optimize ribozyme targets in vitro. The extent to which catalytic efficiency (k cat /K M ) is critical for ribozyme activity is unclear, since antisense effects may also be important in overall function within cells. 4. Additional considerations for the successful use of ribozymes for gene therapy should involve the utilization of genbank data on the human genome to identify potential RNA cleavage sites outside a particular target gene. As therapeutics, major barriers that must be addressed in the future include RNA trafficking and RNA-protein assembly, in which such processes may limit the diffusion, accessibility, and association of ribozymes with target RNAs within cells. Acknowledgments This work was supported by NIH grants to J. R., P. E. K., and A. H. References 1. Hampel, A. (1998) The hairpin ribozyme: discovery, two-dimensional model, and development for gene therapy. Prog. Nucleic Acid Res. Mol. Biol. 58, 1 39.

strsv-derived Hairpin Ribozymes 357 2. Shippy, R., Lockner, R., Farnsworth, M., and Hampel, A. (1999) The hairpin ribozyme. Discovery, mechanism, and development for gene therapy. Mol. Biotechnol. 12, 117 129. 3. Ojwang, J., Hampel, A., Looney, D., Wong-Staal, F., and Rappaport, J. (1992). Inhibition of human immunodeficiency virus type-1 (HIV-1) expression by a hairpin ribozyme. Proc. Natl. Acad. Sci. USA 89, 10,802 10,806. 4. Yu, M., Ojwang, J., Yamada, O., Hampel, A., Rappaport, J., Looney, D., and Wong-Staal, F. (1993) A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90, 6340 6344. 5. Leavitt, M.C., Yu, M., Yamada, O., Kraus, G., Looney, D., Poeschla, E., and Wong-Staal, F. (1994) Transfer of an anti-hiv-1 ribozyme gene into primary human lymphocytes. Hum. Gene Ther. 5, 1115 1120. 6. Yu, M., Poeschla, E., Yamada, O., Degrandis, P., Leavitt, M. C., Heusch, M., et al. (1995) In vitro and in vivo characterization of a second functional hairpin ribozyme against HIV-1. Virology 206, 381 386. 7. Wong-Staal, F., Poeschla, E. M., and Looney, D. J. (1998) A controlled, Phase 1 clinical trial to evaluate the safety and effects in HIV-1 infected human of autologous lymphocytes transduced with a ribozyme that cleaves HIV-1 RNA. Hum. Gene Ther. 9, 2407 2425. 8. Kruger, K., Grabowski, P. J., Zaug, A. J., Sands, J., Gottschling, D. E., and Cech, T. R. (1982) Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31, 147 157. 9. Hampel, A., DeYoung, M. B., Galasinski, S., and Siwkowski, A. (1997) Design of the hairpin ribozyme for targeting specific RNA sequences, in Methods in Molecular Biology, vol. 74, (Turner, P., ed.), Humana Press, Totowa, NJ, pp. 171 177. 10. Lian, Y., DeYoung, M. B., Siwkowski, A., Hampel, A., and Rappaport, J. (1999) The scymv1 hairpin ribozyme: targeting rules and cleavage of heterologous RNA. Gene Ther. 6, 1114 1119. 11. Rappaport J., Kopp, J. B., and Klotman, P. E. (1994) Host virus interactions and the molecular regulation of HIV-1: role in the pathogenesis of HIV-associated nephropathy. Kidney Int. 46, 16 27. 12. Rappaport, J., Josesph, J., Croul, S., Alexander, G., Del Valle, L., Amini, S., and Khalili, K. (1999) Molecular pathway involved in HIV-1-induced CNS pathology: role of viral regulatory protein, Tat. J. Leukoc. Biol. 65, 458 465. 13. Bonwetsch, R., Croul, S., Richardson, M.W., Lorenzana, C., Valle, L.D., Sverstiuk, A.E., et al. (1999) Role of HIV-1 Tat and CC chemokine MIP-1 alpha in the pathogenesis of HIV associated central nervous system disorders. J. Neurovirol. 5, 685 694. 14. Arnaout, M. A. (2001) Molecular genetics and pathogenesis of autosomal dominant polycystic kidney disease. Annu. Rev. Med. 52, 93 123. 15. Safak, M. and Khalili, K. (2001) Physical and functional interaction between viral and cellular proteins modulate JCV gene transcription. J Neurovirol 7, 288 292.

358 Richardson et al. 16. DeYoung, M. B., Siwkowski, A., and Hampel, A. (1997) Determination of catalytic parameters for hairpin ribozymes, in Methods in Molecular Biology, vol. 74, (Turner, P., ed.), Humana Press, Totowa, NJ, pp. 209 220. 17. Siwkowski, A., Humphrey, M., DeYoung, M. B., and Hampel, A. (1997) Screening for important base identities in the hairpin ribozyme by in vitro selection for cleavage. Biotechniques 24, 278 284. 18. DeYoung, M. B. and Hampel, A. (1997) Computer analysis of the conservation and uniqueness of ribozyme-targeted HIV sequences, in Methods in Molecular Biology, vol. 74, (Turner, P., ed.), Humana Press, Totowa, NJ, pp. 27 36. 19. Sczakiel, G. and Tabler, M. (1997) Computer-aided calculation of the local folding potential of target RNA and its use for ribozyme design, in Methods in Molecular Biology, vol. 74, (Turner, P., ed.), Humana Press, Totowa, NJ, pp. 11 15. 20. Siwkowski, A. (1997) T7 transcript length determination using enzymatic RNA sequencing, in Methods in Molecular Biology, vol. 74, (Turner, P., ed.), Humana Press, Totowa, NJ, pp. 91 97. 21. Cavusoglu, E., Chen, I., Rappaport, J., and Marmur, M. D. (2002) Inhibition of tissue factor gene induction and activity using a hairpin ribozyme. Circulation 105, 2282 2287.