In Vitro Selection of External Guide Sequences for Directing RNase P-mediated Inhibition of Viral Gene Expression
2002; Elsevier BV; Volume: 277; Issue: 33 Linguagem: Inglês
10.1074/jbc.m200183200
ISSN1083-351X
AutoresTianhong Zhou, Joseph Kim, Ahmed F. Kilani, Kihoon Kim, Walter Dunn, Solomon Jo, Edward Nepomuceno, Fenyong Liu,
Tópico(s)Viral Infections and Immunology Research
ResumoExternal guide sequences (EGSs) are small RNA molecules that bind to a target mRNA, form a complex resembling the structure of a tRNA, and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. An in vitro selection procedure was used to select EGSs that direct human RNase P to cleave the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1. One of the selected EGSs, TK17, was at least 35 times more active in directing RNase P in cleaving TK mRNAin vitro than the EGS derived from a natural tRNA sequence. TK17, when in complex with the TK mRNA sequence, resembles a portion of tRNA structure and exhibits an enhanced binding affinity to the target mRNA. Moreover, a reduction of 95 and 50% in the TK expression was found in herpes simplex virus 1-infected cells that expressed the selected EGS and the EGS derived from the natural tRNA sequence, respectively. Our study provides direct evidence that EGS molecules isolated by the selection procedure are effective in tissue culture. These results also demonstrate the potential for using the selection procedure as a general approach for the generation of highly effective EGSs for gene-targeting application. External guide sequences (EGSs) are small RNA molecules that bind to a target mRNA, form a complex resembling the structure of a tRNA, and render the mRNA susceptible to hydrolysis by RNase P, a tRNA processing enzyme. An in vitro selection procedure was used to select EGSs that direct human RNase P to cleave the mRNA encoding thymidine kinase (TK) of herpes simplex virus 1. One of the selected EGSs, TK17, was at least 35 times more active in directing RNase P in cleaving TK mRNAin vitro than the EGS derived from a natural tRNA sequence. TK17, when in complex with the TK mRNA sequence, resembles a portion of tRNA structure and exhibits an enhanced binding affinity to the target mRNA. Moreover, a reduction of 95 and 50% in the TK expression was found in herpes simplex virus 1-infected cells that expressed the selected EGS and the EGS derived from the natural tRNA sequence, respectively. Our study provides direct evidence that EGS molecules isolated by the selection procedure are effective in tissue culture. These results also demonstrate the potential for using the selection procedure as a general approach for the generation of highly effective EGSs for gene-targeting application. external guide sequence precursor tRNA thymidine kinase herpes simplex virus 1 multiplicity of infection Antisense technology has been shown to be a promising gene-targeting approach for use in basic research and clinical therapeutic applications. The gene-targeting agents used can be a conventional antisense oligonucleotide, an antisense catalytic molecule (ribozyme or DNA enzyme), or an antisense molecule with an additional (guide) sequence that targets the mRNA for degradation by endogenous RNases such as RNase L and RNase P (1Rossi J.J. Chem. Biol. 1999; 6: R33-R37Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 2Kruger M. Beger C. Wong-Staal F. Methods Enzymol. 1999; 306: 207-225Crossref PubMed Scopus (16) Google Scholar, 3Maran A. Maitra R.K. Kumar A. Dong B. Xiao W., Li, G. Williams B.R. Torrence P.F. Silverman R.H. Science. 1994; 265: 789-792Crossref PubMed Scopus (211) Google Scholar, 4Stein C.A. Cheng Y.C. Science. 1993; 261: 1004-1012Crossref PubMed Scopus (1265) Google Scholar, 5Santoro S.W. Joyce G.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4262-4266Crossref PubMed Scopus (1285) Google Scholar, 6Yuan Y. Altman S. Science. 1994; 263: 1269-1273Crossref PubMed Scopus (83) Google Scholar). Antisense molecules with guide sequences have several unique features as gene-targeting agents. Targeting with these molecules results in irreversible cleavage and the cleavage can be in a catalytic fashion. Moreover, this targeting approach uses the cellular endogenous RNases (e.g. RNase P) for degradation of the target mRNA and, therefore, assures the stability and efficiency of the targeting enzymes in the cellular environment. Ribonuclease P (RNase P) is a ribonucleoprotein complex found in all organisms examined. It is one of the highly active enzymes in cells and is responsible for the maturation of 5′ termini of all tRNAs, which account for approximately 2% of total cellular RNA (7Altman S. Kirsebom L.A. Gesteland R.F. Cech T.R. Atkins J.F. The RNA World. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 351-380Google Scholar, 8Frank D.N. Pace N.R. Annu. Rev. Biochem. 1998; 67: 153-180Crossref PubMed Scopus (399) Google Scholar). This enzyme catalyzes a hydrolysis reaction to remove the leader sequence of precursor tRNA (9Guerrier-Takada C. Gardiner K. Marsh T. Pace N. Altman S. Cell. 1983; 35: 849-857Abstract Full Text PDF PubMed Scopus (2067) Google Scholar). Human RNase P has at least nine polypeptides and a RNA subunit (H1 RNA) (7Altman S. Kirsebom L.A. Gesteland R.F. Cech T.R. Atkins J.F. The RNA World. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 351-380Google Scholar, 10Bartkiewicz M. Gold H. Altman S. Genes Dev. 1989; 3: 488-499Crossref PubMed Scopus (139) Google Scholar). One of the unique features of RNase P is its ability to recognize the structures, rather than the sequences, of the substrates, which allows the enzyme to hydrolyze different natural substrates in vivo or in vitro. Accordingly, any complex of two RNA molecules that resembles a tRNA molecule can be recognized and cleaved by RNase P (Fig. 1, A and B) (11Forster A.C. Altman S. Science. 1990; 249: 783-786Crossref PubMed Scopus (259) Google Scholar, 12Yuan Y. Hwang E. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8006-8010Crossref PubMed Scopus (128) Google Scholar). One of the RNA molecules is called the external guide sequence (EGS).1 In principle, an mRNA sequence can be targeted for RNase P cleavage by using EGSs to hybridize with the target RNA and direct RNase P to the site of cleavage. The EGSs used to direct human RNase P for targeted cleavage resemble three-quarters of a tRNA molecule and consist of two sequence elements: a targeting sequence complementary to the mRNA sequence and a guide sequence, which is a portion of the natural tRNA sequence and is required for RNase P recognition (Fig. 1B) (11Forster A.C. Altman S. Science. 1990; 249: 783-786Crossref PubMed Scopus (259) Google Scholar, 12Yuan Y. Hwang E. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8006-8010Crossref PubMed Scopus (128) Google Scholar). Subsequent studies have shown that expression of EGSs in human cells can reduce the expression of both cellular and viral genes (11Forster A.C. Altman S. Science. 1990; 249: 783-786Crossref PubMed Scopus (259) Google Scholar, 12Yuan Y. Hwang E. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8006-8010Crossref PubMed Scopus (128) Google Scholar, 13Dunn W. Trang P. Liu F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14831-14836Crossref PubMed Scopus (31) Google Scholar, 14Ma M. Benimetskaya L. Lebedeva I. Dignam J. Takle G. Stein C.A. Nat. Biotechnol. 2000; 18: 58-61Crossref PubMed Scopus (27) Google Scholar, 15Plehn-Dujowich D. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7327-7332Crossref PubMed Scopus (89) Google Scholar). For example, we have previously shown that EGSs efficiently direct human RNase P to cleave the mRNA sequence encoding the thymidine kinase (TK) of herpes simplex virus 1 (HSV-1) in vitro (16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar). A reduction of 50–70% in the TK mRNA and protein expression was observed in HSV-1-infected cells expressing the EGSs. Targeted cleavage of mRNA by RNase P using EGSs provides a unique approach to inactivate any RNA of known sequence expressed in vivo. Further studies aimed at increasing the targeting activity of the EGSs are needed to develop the EGS-based technology as a general tool for use in gene-targeting applications. Although little is known about the rate-limiting step of EGS-targeting reactions in cultured cells, we believe that binding of the EGSs to the target mRNA as well as the efficiency of cleavage are important for the efficacy of the EGSs. Indeed, recent studies on ribozymes and antisense phosphothioate molecules suggest that binding of these molecules to their target RNAs appears to be rate-limiting in vivo(17Lee N.S. Bertrand E. Rossi J. RNA. 1999; 5: 1200-1209Crossref PubMed Scopus (26) Google Scholar, 18Pal B.K. Scherer L. Zelby L. Bertrand E. Rossi J.J. J. Virol. 1998; 72: 8349-8353Crossref PubMed Google Scholar, 19Sullenger B.A. Cech T.R. Science. 1993; 262: 1566-1569Crossref PubMed Scopus (186) Google Scholar, 20zu Putlitz J., Yu, Q. Burke J.M. Wands J.R. J. Virol. 1999; 73: 5381-5387Crossref PubMed Google Scholar, 21Yu Q. Pecchia D.B. Kingsley S.L. Heckman J.E. Burke J.M. J. Biol. Chem. 1998; 273: 23524-23533Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). In our previous studies, EGS RNAs were used to target a region of TK mRNA that is accessible to modification by dimethyl sulfate in cell culture and is also accessible to EGS binding (16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar). Moreover, the EGSs were expressed primarily in the nuclei by using the promoter of small nuclear U6 RNA (12Yuan Y. Hwang E. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8006-8010Crossref PubMed Scopus (128) Google Scholar, 16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar, 22Bertrand E. Castanotto D. Zhou C. Carbonnelle C. Lee N.S. Good P. Chatterjee S. Grange T. Pictet R. Kohn D. Engelke D. Rossi J.J. RNA. 1997; 3: 75-88PubMed Google Scholar). This design would increase the probability for the constructed EGSs to bind to its target mRNA sequence and co-localize with RNase P, which is localized exclusively in the nuclei. Under such conditions, it is possible that the EGS efficacy in culture cells is dictated by the overall efficiency (Vmax/Km) of the EGS-induced RNase P cleavage. If this is the case, increasing the targeting activity of the EGS may lead to a more effective inhibition of the target mRNA expression in cultured cells. In the present study, we employed an in vitro selection procedure (23Burke J.M. Berzal-Herranz A. FASEB J. 1993; 7: 106-112Crossref PubMed Scopus (51) Google Scholar, 24Gold L. Allen P. Binkley J. Brown D. Schneider D. Eddy S.R. Tuerk C. Green L. MacDougal S. Tasset D. Gesteland R.R. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1993: 497-509Google Scholar, 25Joyce G.F. Sci. Am. 1992; 267: 90-97Crossref PubMed Scopus (92) Google Scholar, 26Szostak J.W. Ellington A.D. Gesteland R.R. Atkins J.F. The RNA World. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1993: 511-533Google Scholar) to isolate highly active EGSs from an EGS pool that contained random mutations. One of the selected EGSs exhibited a targeting activity at least 35 times higher than that of the EGS derived from the natural tRNA sequence. When the selected EGS was expressed in cells infected with HSV-1, a reduction of 95% in TK expression was observed. These studies demonstrate the feasibility of developing effective EGSs for gene-targeting applications. DNA template coding for substrate tk46 was constructed by annealing oligonucleotide OliT7 (5′-TAATACGACTCACTATAG-3′) with OliTK46 (5′-ACCGCGCAGCCTGGTCGAACGCAGACGCGTGTTGATGGCAGGGGTCTATAGTGAGTCGTATTA-3′). The DNA sequence coding for the EGS TK-Ser was synthesized by the polymerase chain reaction (PCR), using DNA that encodes yeast tRNASer as the template (27Drainas D. Zimmerly S. Willis I. Soll D. FEBS Lett. 1989; 251: 84-88Crossref PubMed Scopus (26) Google Scholar), and was cloned under the control of the T7 RNA polymerase promoter. The 5′ and 3′ primer were oligo1 (5′-GTTAACGTCGGACAGACTC-3′) and oligo2 (5′-AAGCTTTAAACGTCTGCGGCAGGATTTG-3′), respectively. DNA sequence coding for TK17 was generated by PCR using the selected substrate construct C17 as the template, and oligonucleotides TK24EGS (5′-GGAATTCTAATACGACTCACTATAGGTTAACGTC-3′) and TK23 (5′-AAACGTCTGCG-3′) as the 5′ and 3′ primers, respectively. C-TK-Ser and C-TK17 were derived from TK-Ser and TK17, respectively, and contained point mutations (5′-UUC-3′ → AAG) at the three highly conserved positions in the T loop of these EGSs (see Fig. 4, A and C). The DNA sequences coding for EGS C-TK-Ser and C-TK17 were generated by introducing point mutations into the TK-Ser and TK17 sequences, as described previously (16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar). Human RNase P was prepared from HeLa cellular extracts as described previously (10Bartkiewicz M. Gold H. Altman S. Genes Dev. 1989; 3: 488-499Crossref PubMed Scopus (139) Google Scholar, 12Yuan Y. Hwang E. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8006-8010Crossref PubMed Scopus (128) Google Scholar). EGS RNAs and substrate tk46 were synthesizedin vitro by T7 RNA polymerase and further purified on 8% urea-polyacrylamide gels. Subsequently, the EGS RNAs and32P-labeled tk46 were incubated with human RNase P. The cleavage reactions were carried out at 37°C in a volume of 10 μl in buffer A (50 mm Tris, pH 7.4, 100 mm NH4Cl, and 20 mmMgCl2) (12Yuan Y. Hwang E. Altman S. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8006-8010Crossref PubMed Scopus (128) Google Scholar). Cleavage products were separated in denaturing gels and analyzed with a STORM840 PhosphorImager (AmershamBiosciences). Double-stranded DNA templates were synthesized by mouse Moloney leukemia virus reverse transcriptase (Roche Molecular Biochemicals), from two DNA oligonucleotides, TK21 (5′-GGAATTCTAATACGACTCACTATAGACCCCTGCCATCAACACGCGTCTGCGTTCGACCAGGCTGCGCGGTTAACGTCG-3′) and TK22 (5′-AAACGTCTGCGNNNNNNNNNNNNNNNNNNNNNNNNNCCGACGTTAA-3′). TK21 contains the leader sequence for the substrate (Fig.1E) and the promoter sequence for T7 RNA polymerase. TK22 contains a randomized sequence indicated as (N)25 and underlined (Fig. 1E). The general strategy for the design of these oligonucleotides followed that described by Yuan and Altman (6Yuan Y. Altman S. Science. 1994; 263: 1269-1273Crossref PubMed Scopus (83) Google Scholar). The RNA substrate was incubated at 37 °C with human RNase P in buffer A. The 3′ proximal cleavage product was separated in and isolated from a denaturing 8% polyacrylamide gel, and was then used as template for reverse transcription to generate cDNA, in the presence of 20 mm primer oligodeoxynucleotide, TK23 (5′-AAACGTCTGCG-3′), and avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals). The mixture was incubated for 2 h at 42 °C. The cDNA was subsequently amplified by PCR with oligodeoxynucleotide primers TK21 and TK23 and was used to generate RNA substrates for the next round of selection. TK21, the 5′ primer for PCR, allows restoration of the T7 promoter sequence and the leader sequence of the RNA substrates. Initially, 20 nmol of the pool of RNA substrates, which contained the randomized sequence and were synthesized in vitro, were digested by human RNase P in a volume of 2 ml. In subsequent cycles of selection, 5 pmol of substrate were used and digested with the appropriate enzyme in a volume of 20 μl. During the first four cycles of selection, substrates were incubated for 120 min at 37 °C with 100 units of RNase P. During the final five cycles of selection, incubation time was shortened to 10 min. The amount of human RNase P used was reduced by 200-fold. This strategy allowed enrichment of all sequences that exhibited similar susceptibility to cleavage. After nine cycles of selection, cDNA that contained substrate sequences was cloned into pUC19 and sequence analysis was performed. RNase T1, nuclease S1, and RNase V were purchased fromAmersham Biosciences or Invitrogen. The procedures for mapping the structure of tk46-EGS complexes were carried out as described previously (6Yuan Y. Altman S. Science. 1994; 263: 1269-1273Crossref PubMed Scopus (83) Google Scholar, 28Trang P. Hsu A.W. Liu F. Nucleic Acids Res. 1999; 27: 4590-4597Crossref PubMed Scopus (22) Google Scholar). RNases were diluted and incubated at 37 °C for 10 min with the tk46-EGS complexes in buffer A that had been supplemented with 10 μg of bulk tRNA from Escherichia coliin a final volume of 10 μl. Cleavage products were separated in 10% polyacrylamide gels that contained 7 m urea. RNA substrates were incubated in buffer A (50 mm Tris, pH 7.4, 100 mm NH4Cl, and 20 mmMgCl2) at 37 °C with human RNase P and cleavage products were separated on 8% denaturing gels and quantitated with a STORM840 PhosphorImager. Assays to determine kinetic parameters were performed under multiple turnover conditions, as described previously (29Herschlag D. Cech T.R. Biochemistry. 1990; 29: 10159-10171Crossref PubMed Scopus (307) Google Scholar, 30Kilani A.F. Trang P., Jo, S. Hsu A. Kim J. Nepomuceno E. Liu F. J. Biol. Chem. 2000; 275: 10611-10622Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 31Liu F. Altman S. Cell. 1994; 77: 1083-1100Abstract Full Text PDF PubMed Scopus (131) Google Scholar) (see supplemental data available on-line). In brief, the cleavage of substrates was assayed in buffer at various concentrations of substrates, both above and below the Km for each respective substrate. The amount of substrates are in large excess to that of the enzymes to assure that saturation with the substrate was achieved in the multiple-turnover conditions (see supplemental data). Aliquots were withdrawn from reaction mixtures at regular intervals and analyzed in polyacrylamide-urea gels. Values ofKm(apparent) and Vmax(apparent) were obtained from Lineweaver-Burk double-reciprocal plots. The procedures to measure the equilibrium dissociation constants (Kd) of complexes of the EGSs and the substrates were modified from Pyle et al. (32Pyle A.M. McSwiggen J.A. Cech T.R. Proc. Natl. Acad. Sci. 1990; 87: 8187-8191Crossref PubMed Scopus (137) Google Scholar). In brief, various concentrations of EGSs were preincubated in buffer B (50 mm Tris, pH 7.5, 100 mm NH4Cl, 20 mm MgCl2, 3% glycerol, 0.1% xylene cyanol, 0.1% bromphenol blue) for 10 min before mixing with an equal volume of different concentrations of substrate RNA preheated under identical conditions. The samples were incubated for 10–120 min to allow binding, loaded on a 5% polyacrylamide gel, and run at 10 watts. The electrophoresis running buffer contained 100 mm Tris-Hepes, pH 7.5, and 10 mm MgCl2 (32Pyle A.M. McSwiggen J.A. Cech T.R. Proc. Natl. Acad. Sci. 1990; 87: 8187-8191Crossref PubMed Scopus (137) Google Scholar). The value ofKd was then extrapolated from a graph plotting percentage of product bound versus EGS concentration. The values were the average of three experiments. Constructs pTK-Ser, pC-TK-Ser, pTK17, and pC-TK17 were generated by placing the DNA sequences that coded for EGS TK-Ser, C-TK-Ser, TK17, and C-TK17 under the control of the U6 promoter, respectively (16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar). Human 143tk− cells were cotransfected with pTK116 (containing the neomycin-resistant gene) and the EGS plasmids, with the aid of a mammalian transfection kit (Stratagene Inc., La Jolla, CA). At 48 h after transfection, neomycin (Invitrogen) was added to the culture medium in a final concentration of 400 μg/ml. Cells were subsequently selected under neomycin for 2 weeks and were eventually cloned. The level of EGS expression in individual cell clone was determined by Northern analyses with probes complementary to EGSs. Only those cell clones that expressed similar levels of EGSs were used for subsequent experiments. Approximately 106 cells in a T25 flask were either mock-infected or infected with HSV-1 in 1.5 ml of Medium 199 at a multiplicity of infection (m.o.i.) as specified under "Results." At 8–16 h after infection, cells were harvested and RNA and protein extracts were prepared as described previously (33Liu F. Roizman B. J. Virol. 1991; 65: 5149-5156Crossref PubMed Google Scholar). The RNA probes used to detect TK mRNA and the transcripts of the α47, Us10. Us11 genes were synthesized from pTK129 and pTK141, and RNase protection assays were performed as described previously (34Liu F. Altman S. Genes Dev. 1995; 9: 471-480Crossref PubMed Scopus (109) Google Scholar). The protected RNA products were separated in 8% urea-polyacrylamide denaturing gels and quantitated with a STORM840 PhosphorImager. The denatured, solubilized polypeptides from cell lysates were separated on 9% (v/v) SDS-polyacrylamide gels cross-linked withN, N″-methylenebisacrylamide (16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar). The separated polypeptides were transferred electrically to nitrocellulose membranes and reacted with the antibodies against HSV-1 TK (16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar) or ICP27 (purchased from Goodwin Institute of Cancer Research, Plantation, FL). The membranes were subsequently stained with a chemiluminescent substrate with the aid of a Western chemiluminescent substrate kit (Amersham Biosciences) and quantitated with a STORM840 PhosphorImager. Quantitation was performed in the linear range of RNA and protein detection (e.g. 2-fold changes in RNA and protein samples result in a 2-fold change in signal bracketing the range of experimental values; see supplemental data). The RNA substrate tk46 used in the selection experiment contains a 5′ TK mRNA sequence of 46 nucleotides (Figs. 1E and4A). This sequence has been shown to be accessible to modification by dimethyl sulfate and, presumably, to EGS binding in mammalian cell culture (16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar). We have previously showed that EGSs derived from ptRNASer can direct human RNase P to cleave tk46 in vitro and inhibit HSV-1 TK expression in cultured cells (16Kawa D. Wang J. Yuan Y. Liu F. RNA. 1998; 4: 1397-1406Crossref PubMed Scopus (55) Google Scholar). However, the cleavage reaction is inefficient compared with the cleavage of a natural tRNA substrate (i.e.ptRNASer). It is believed that the acceptor stem and D stem and loop sequences within a natural tRNA molecule are involved in tertiary interactions with different parts of the tRNA (e.g.variable and T stem and loop regions) and are important for folding of the tRNA and interactions with RNase P (7Altman S. Kirsebom L.A. Gesteland R.F. Cech T.R. Atkins J.F. The RNA World. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1999: 351-380Google Scholar, 8Frank D.N. Pace N.R. Annu. Rev. Biochem. 1998; 67: 153-180Crossref PubMed Scopus (399) Google Scholar). The reduced susceptibility of the tk46-EGS complex to be cleaved by RNase P, compared with a natural tRNA, is probably a result of replacing the natural tRNA sequence at the acceptor and D stem regions with the tk46 and its complementary sequence in the complex of tk46 and the EGS. These substitutions may disrupt some of the tertiary interactions that are potentially important in maintaining the proper tRNA-like conformation and in recognition by human RNase P. Restoration of these interactions or introducing additional interactions by changing other parts of the EGS sequence, such as those resembling the variable region, and T stem and loop, may increase the susceptibility of the mRNA-EGS complex to be cleaved by RNase P. To generate EGSs that are highly active in directing RNase P to cleave tk46, a pool of chimeric, covalently linked tk46-EGS substrates that contain partially randomized sequences was constructed (Fig.1E) and selected for the ability to be cleaved by human RNase P (Fig. 1D). The chimeric RNA, with its 5′ region consisting of the tk46 sequence, contains the sequences that base pair with tk46 at the regions resembling the acceptor and D stem of a tRNA and, in addition, a randomized sequence of 25 nucleotides at the positions corresponding to the regions resembling the variable and T stem and loop regions (Fig. 1E). The anticodon region, which is dispensable for EGS activity (6Yuan Y. Altman S. Science. 1994; 263: 1269-1273Crossref PubMed Scopus (83) Google Scholar), was not included in the chimeric substrate (Fig. 1, C and E). The pool of tk46-EGS chimeric substrates was synthesized in vitro by T7 RNA polymerase. In each round of selection, the pool of RNAs was digested with human RNase P in buffer A (50 mm Tris, pH 7.4, 100 mm NH4Cl, and 20 mmMgCl2) and the 3′ cleavage products were isolated in denaturing gels. cDNA molecules were then synthesized and amplified from these RNA molecules by reverse transcription followed by PCR and used as the templates for synthesis of EGS RNA molecules for the next round of selection. The 5′ primer for the PCR reaction contains the tk46 sequence as well as the T7 promoter sequence and, therefore, allows the restoration of these sequences for the next cycle of selection. The stringency of the selection was increased at each cycle by reducing the amount of human RNase P and the time allowed for the cleavage reaction, such that only those substrates that were rapidly cleaved by the enzyme were selected. The cleavage efficiency of the substrate population of each generation was monitored (Fig.2A). Moreover, EGSs were also constructed from the selected population of each generation and tested for their activity to direct human RNase P-mediated cleavage (Fig.2B). These results indicate that the susceptibility of the chimeric substrate as well as the targeting activity of the EGSs increase with each selection cycle (Fig. 2). The selection procedure was repeated nine times, until no apparent enhancement of the cleavage efficiency of the substrate population was observed after a short period of incubation (10 min) (Fig. 2A). Twenty-four sequences coding for the EGSs isolated after nine cycles of selection were cloned and determined (Table I). These EGSs were divided into two sets based on their primary nucleotide sequences. Each sequence in set 1 either had the same sequence or extensive homology to other sequences of the same set. In contrast, the three sequences in set 2 did not exhibit significant sequence homology to each other or to those in set 1.Table IPartial sequences of 24 substrates obtained after selection with human RNase P In our selection procedure, the tk46-EGS chimeric substrates were selected for their susceptibility to be cleaved by human RNase P. To determine whether the selected substrates can be cleaved by RNase P, one of the selected substrates, C17, which is the most abundant selected sequence (Table I), was assayed for cleavage by RNase P. Kinetic analyses indicate that human RNase P cleaved substrate C17 as efficiently as ptRNASer (Table II), suggesting that C17 may possess proper tertiary interactions and conformations, which are found in natural tRNA substrate and are required for optimal recognition by human RNase P.Table IIMeasurement of the kinetic parameters [Vmax(apparent), Km(apparent), and Vmax(apparent)/Km(apparent)] in the RNase P cleavage of tRNASer or tk46 in the presence of different EGSsSubstrateKm(apparent)Vmax(apparent)Vmax(apparent)/Km(apparent)Kdμmpmol/minpmol μm−1 min−1nmptRNASer0.015 ± 0.0030.04 ± 0.0072.7 ± 0.4C170.020 ± 0.0030.06 ± 0.0083.0 ± 0.5TK mRNA (tk46)+ TK-Ser0.5 ± 0.090.03 ± 0.0050.06 ± 0.011960 ± 110+ TK170.3 ± 0.040.65 ± 0.012.2 ± 0.417 ± 2+ C-TK-SerNDND<0.0001950 ± 120+ C-TK17NDND<0.000115 ± 3+ TK-G0NDND<0.0001ND Open table in a new tab To analyze the relationship between the EGS sequences from the selected tk46-EGS substrates and the capabilities of these EGSs to direct human RNase P for targeting cleavage, an EGS sequence, TK17, was constructed from the sequence of C17. Substrate tk46 was incubated with TK17 in the presence of human RNase P, and the cleavage products were analyzed in denaturing gels (Fig. 3). Kinetic analyses of the cleavage reactions directed by the selected EGS TK17 as well as by EGS TK-Ser, which was derived from the ptRNASer sequence, were performed under multiple-turnover conditions. Under these conditions, the amounts of substrates were in large excess to that of the enzyme to assure that saturation with the substrate was achieved (see supplemental data). The values ofKm(apparent) and Vmax(apparent) as well as the overall cleavage efficiency [Vmax(apparent)/Km(apparent)] were determined (Table II). Under the selection conditions (i.e. buffer A), TK17 was extremely efficient in directing human RNase P to cleave tk46 (Fig. 3, lane 3) and was at least 2 × 104-fold more active than the EGS molecules (i.e. TK-G0) that were derived from the initial randomized tk46-EGS chimeric substrate pool (G0) (Table II). Moreover, in the presence of TK17, RNase P-mediated cleavage of tk46 was at least 35-fold more efficient than that in the presence of TK-Ser (compare lanes 3and 2) (Table II). These observations indicate that highly active EGS RNAs were successfully selected using the in vitro selection procedure. Because TK17 is among the most abundant sequence found after nine cycles of selection and exhibits highly active targeting activity among the selected EGSs tested (Fig. 3, Table II, and data not shown), this EGS was further characterized and expressed in tissue culture to determine its efficacy in inhibiting TK expression (see below). In our selection, the mRNA-EGS chimeric molecules were subjected to digestion by RNase P and selected for their susceptibility to cleavage by RNase P. As the selection proceeded, the EGS sequence in the chimeric molecule was selected for
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