Abstract
Most antisense oligonucleotide experiments are performed with molecules containing RNase H-competent backbones. However, RNase H may cleave nontargeted mRNAs bound to only partially complementary oligonucleotides. Decreasing such “irrelevant cleavage” would be of critical importance to the ability of the antisense biotechnology to provide accurate assessment of gene function. RNase P is a ubiquitous endogenous cellular ribozyme whose function is to cleave the 5′ terminus of precursor tRNAs to generate the mature tRNA. To recruit RNase P, complementary oligonucleotides called external guide sequences (EGS), which mimic structural features of precursor tRNA, were incorporated into an antisense 2′-O-methyl oligoribonucleotide targeted to the 3′ region of the PKC-α mRNA. In T24 human bladder carcinoma cells, these EGSs, but not control sequences, were highly effective in downregulating PKC-α protein and mRNA expression. Furthermore, the downregulation is dependent on the presence of, and base sequence in, the T-loop. Similar observations were made with an EGS targeted to the bcl-xL mRNA.
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References
Stein, C.A. & Cheng, Y.-C. Antisense oligonucleotides as therapeutic agents—Is the bullet really magic? Science 261 , 1004–1002 (1993).
Wagner, R. Gene inhibition using antisense oligodeoxynucleotides. Nature 372, 333–335 (1994).
Stein, C.A. & Krieg, A. eds. Applied antisense oligonucleotide technology. (Wiley-Liss, New York; 1998).
Mulamba, G., Hu, A., Azad, R., Anderson, K. & Coen, I. Human cytomegalovirus mutant with sequence-dependent resistance to the phosphorothioate oligonucleotide fomivirsen (Isis 2922) . Antimicrob. Agents Chemother. 42, 971– 973 (1998).
Walder, R.Y. & Walder, J.A. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 85, 5011–5015 ( 1988).
Minshull, J. & Hunt, T. The use of single-stranded DNA and RNase-H to promote quantitative “hybrid arrest of translation” of mRNA–DNA hybrids in reticulocyte lysate cell-free translations. Nucleic Acids Res. 14, 6433–6451 (1986).
Hausen, P. & Stein, H. Ribonuclease H. An enzyme degrading the RNA moiety of DNA–RNA hybrids. Eur. J. Biochem. 14, 278–283 (1970).
Stein, C.A., Subasinghe, C., Shinozuka, K. & Cohen, J. Physicochemical properties of phosphorothioate oligodeoxyribonucleotides. Nucleic Acids Res. 16, 3209–3221 (1988).
Giles, R. & Tidd, D. Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures. Nucleic Acids Res. 20, 763– 770 (1992).
Giles, R., Spiller, D. & Tidd, D. Chimeric oligodeoxynucleotide analogues: enhanced cell uptake of structures that direct ribonuclease H with high specificity. Anti-Cancer Drug Res. 8, 33–51 (1993).
Giles, R., Ruddell, C., Spiller, D., Green, J. & Tidd, D. Single base discrimination for ribonuclease H dependent antisense effects within intact human leukaemia cells. Nucleic Acids Res. 23, 954–961 (1995).
Monia, B. et al. Evaluation of 2′-modified oligonucleotides containing 2′-deoxy gaps as antisense inhibitors of gene expression. J. Biol. Chem. 268, 14154–14522 ( 1993).
Benimetskaya, L. et al. Cationic porphyrins: novel delivery vehicles for antisense oligonucleotides. Nucleic Acids Res. 26, 5310–5317 (1998).
Forster, A. & Altman, S. External guide sequences for an RNA enzyme. Science 249, 783– 786 (1990).
Altman, S. RNA enyme-directed gene therapy. Proc. Natl. Acad. Sci. USA 90, 10898–10900 (1993).
Ma, M. et al. Nuclease-resistant external guide sequence-induced cleavage of target RNA by human ribonuclease P. Antisense Nucleic Acid Drug Devel. 8, 415–426 ( 1998).
Heidenreich, O., Benseler, F., Fahrenholz, A. & Eckstein, F. High activity and stability of hammerhead ribozymes containing 2′-modified pyrimidine nucleosides and phosphorothioates. J. Biol. Chem. 269, 2131–2138 (1994).
Yuan, Y., Hwang, E. & Altman, S. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA 89, 8006– 8010 (1992).
Dean, N., McKay, R., Condon, T. & Bennett, F. Inhibition of protein kinase C-α expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters. J. Biol. Chem. 269, 16416– 16424 (1994).
Dean, N. & McKay, R. Inhition of protein kinase C-α expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 91 , 11762–11766 (1994).
Loria, A. & Pan, T. Recognition of the T stem-loop of a pre-tRNA substrate by the ribozyme from Bacillus subtilis ribonuclease P. Biochemistry 36, 6317–6325 (1997).
Kahle, D., Wehmeyer, U. & Krupp, G. Substrate recognition by RNase P and by the catalytic M1 RNA: identification of possible contact points in pre-tRNAs. EMBO J. 9, 1929–1937 ( 1990).
Werner, M., Rose, E., Al Emran, O., Goldberg, A. & George, S. Targeted cleavage of RNA molecules by human RNase P using minimized external guide sequences. Antisense Nucleic Acid Drug Devel. 9, 81–98 ( 1999).
Werner, M., Rosa, E., Nordstrom, J., Goldberg, A. & George, S. Short oligonucleotides as external guide sequences for site-specific cleavage of RNA molecules with human RNase P. RNA 4, 847–855 (1998).
Ortigao, J. et al. Antisense effect of oligodeoxynucleotides with inverted terminal internucleotidic linkages: a minimal modification protecting against nucleolytic degradation. Antisense Res. Devel. 2, 129 –146 (1992).
Sinha, N. et al. Labile exocyclic amine protection of nucleosides in DNA, RNA and oligonucleotide analog synthesis facilitating N-deacylation, minimizing depurination and chain degradation. Biochimie 75, 13–23 (1993).
Acknowledgements
This work was generously funded (C.A.S.) by Innovir Pharmaceuticals. We thank Andrei Laikhter of Annovis, Inc. (Ashton, PA) for the synthesis of the bcl-xL EGS, and S. Altman for criticism.
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Ma, M., Benimetskaya, L., Lebedeva, I. et al. Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences. Nat Biotechnol 18, 58–61 (2000). https://doi.org/10.1038/71924
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DOI: https://doi.org/10.1038/71924
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