Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1995 Mar 28;92(7):2465–2469. doi: 10.1073/pnas.92.7.2465

Total chemical synthesis of a ribozyme derived from a group I intron.

S K Whoriskey 1, N Usman 1, J W Szostak 1
PMCID: PMC42238  PMID: 7708666

Abstract

We describe the complete chemical synthesis of a ribozyme that catalyzes template-directed oligonucleotide ligation. The specific activity of the synthetic ribozyme is nearly identical to that of the same enzyme generated by in vitro transcription with T7 RNA polymerase. The ribozyme is derived from a group I intron and consists of three RNA fragments of 36, 43, and 59 nt that self-assemble to form a catalytically active complex. We have site-specifically substituted ribonucleotide analogs into this enzyme and have identified two 2'-hydroxyl groups that are required for full catalytic activity. In contrast, neither the 2'-hydroxyl nor the exocyclic amino group of the conserved guanosine in the guanosine binding site is necessary for catalysis. By allowing the ribozyme to be modified as easily as its substrates, this synthetic ribozyme system should be useful for testing specific hypotheses concerning ribozyme-substrate interactions and tertiary interactions within the ribozyme.

Full text

PDF
2465

Images in this article

2467Fig. 2
on p.2467
2467Fig. 3
on p.2467

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Caprara M. G., Waring R. B. Important 2'-hydroxyl groups within the core of a group I intron. Biochemistry. 1993 Apr 13;32(14):3604–3610. doi: 10.1021/bi00065a011. [DOI] [PubMed] [Google Scholar]
  2. Doudna J. A., Couture S., Szostak J. W. A multisubunit ribozyme that is a catalyst of and template for complementary strand RNA synthesis. Science. 1991 Mar 29;251(5001):1605–1608. doi: 10.1126/science.1707185. [DOI] [PubMed] [Google Scholar]
  3. Doudna J. A., Szostak J. W., Rich A., Usman N. Chemical synthesis of oligoribonucleotides containing 2-aminopurine: substrates for the investigation of ribozyme function. J Org Chem. 1990 Oct 12;55(21):5547–5549. doi: 10.1021/jo00308a003. [DOI] [PubMed] [Google Scholar]
  4. Fu D. J., McLaughlin L. W. Importance of specific adenosine N7-nitrogens for efficient cleavage by a hammerhead ribozyme. A model for magnesium binding. Biochemistry. 1992 Nov 17;31(45):10941–10949. doi: 10.1021/bi00160a001. [DOI] [PubMed] [Google Scholar]
  5. Green R., Szostak J. W., Benner S. A., Rich A., Usman N. Synthesis of RNA containing inosine: analysis of the sequence requirements for the 5' splice site of the Tetrahymena group I intron. Nucleic Acids Res. 1991 Aug 11;19(15):4161–4166. doi: 10.1093/nar/19.15.4161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Green R., Szostak J. W. Selection of a ribozyme that functions as a superior template in a self-copying reaction. Science. 1992 Dec 18;258(5090):1910–1915. doi: 10.1126/science.1470913. [DOI] [PubMed] [Google Scholar]
  7. Guerrier-Takada C., Altman S. Reconstitution of enzymatic activity from fragments of M1 RNA. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1266–1270. doi: 10.1073/pnas.89.4.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Jarrell K. A., Dietrich R. C., Perlman P. S. Group II intron domain 5 facilitates a trans-splicing reaction. Mol Cell Biol. 1988 Jun;8(6):2361–2366. doi: 10.1128/mcb.8.6.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Koizumi M., Hayase Y., Iwai S., Kamiya H., Inoue H., Ohtsuka E. Design of RNA enzymes distinguishing a single base mutation in RNA. Nucleic Acids Res. 1989 Sep 12;17(17):7059–7071. doi: 10.1093/nar/17.17.7059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Koizumi M., Ohtsuka E. Effects of phosphorothioate and 2-amino groups in hammerhead ribozymes on cleavage rates and Mg2+ binding. Biochemistry. 1991 May 28;30(21):5145–5150. doi: 10.1021/bi00235a005. [DOI] [PubMed] [Google Scholar]
  11. Michel F., Ellington A. D., Couture S., Szostak J. W. Phylogenetic and genetic evidence for base-triples in the catalytic domain of group I introns. Nature. 1990 Oct 11;347(6293):578–580. doi: 10.1038/347578a0. [DOI] [PubMed] [Google Scholar]
  12. Michel F., Westhof E. Modelling of the three-dimensional architecture of group I catalytic introns based on comparative sequence analysis. J Mol Biol. 1990 Dec 5;216(3):585–610. doi: 10.1016/0022-2836(90)90386-Z. [DOI] [PubMed] [Google Scholar]
  13. Milligan J. F., Groebe D. R., Witherell G. W., Uhlenbeck O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 1987 Nov 11;15(21):8783–8798. doi: 10.1093/nar/15.21.8783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Moore M. J., Sharp P. A. Site-specific modification of pre-mRNA: the 2'-hydroxyl groups at the splice sites. Science. 1992 May 15;256(5059):992–997. doi: 10.1126/science.1589782. [DOI] [PubMed] [Google Scholar]
  15. Odai O., Hiroaki H., Sakata T., Tanaka T., Uesugi S. The role of a conserved guanosine residue in the hammerhead-type RNA enzyme. FEBS Lett. 1990 Jul 2;267(1):150–152. doi: 10.1016/0014-5793(90)80311-6. [DOI] [PubMed] [Google Scholar]
  16. Ogilvie K. K., Usman N., Nicoghosian K., Cedergren R. J. Total chemical synthesis of a 77-nucleotide-long RNA sequence having methionine-acceptance activity. Proc Natl Acad Sci U S A. 1988 Aug;85(16):5764–5768. doi: 10.1073/pnas.85.16.5764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Olsen D. B., Benseler F., Aurup H., Pieken W. A., Eckstein F. Study of a hammerhead ribozyme containing 2'-modified adenosine residues. Biochemistry. 1991 Oct 8;30(40):9735–9741. doi: 10.1021/bi00104a024. [DOI] [PubMed] [Google Scholar]
  18. Perreault J. P., Wu T. F., Cousineau B., Ogilvie K. K., Cedergren R. Mixed deoxyribo- and ribo-oligonucleotides with catalytic activity. Nature. 1990 Apr 5;344(6266):565–567. doi: 10.1038/344565a0. [DOI] [PubMed] [Google Scholar]
  19. Pieken W. A., Olsen D. B., Benseler F., Aurup H., Eckstein F. Kinetic characterization of ribonuclease-resistant 2'-modified hammerhead ribozymes. Science. 1991 Jul 19;253(5017):314–317. doi: 10.1126/science.1857967. [DOI] [PubMed] [Google Scholar]
  20. Pyle A. M., McSwiggen J. A., Cech T. R. Direct measurement of oligonucleotide substrate binding to wild-type and mutant ribozymes from Tetrahymena. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8187–8191. doi: 10.1073/pnas.87.21.8187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Scaringe S. A., Francklyn C., Usman N. Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites. Nucleic Acids Res. 1990 Sep 25;18(18):5433–5441. doi: 10.1093/nar/18.18.5433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Vinayak R., Anderson P., McCollum C., Hampel A. Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry. Nucleic Acids Res. 1992 Mar 25;20(6):1265–1269. doi: 10.1093/nar/20.6.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Williams D. M., Pieken W. A., Eckstein F. Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):918–921. doi: 10.1073/pnas.89.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Williams D. M., Pieken W. A., Eckstein F. Function of specific 2'-hydroxyl groups of guanosines in a hammerhead ribozyme probed by 2' modifications. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):918–921. doi: 10.1073/pnas.89.3.918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Yang J. H., Usman N., Chartrand P., Cedergren R. Minimum ribonucleotide requirement for catalysis by the RNA hammerhead domain. Biochemistry. 1992 Jun 2;31(21):5005–5009. doi: 10.1021/bi00136a013. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES