Skip to main content
NCBI home page
RNA logoLink to RNA
. 2001 Apr;7(4):513–523. doi: 10.1017/s1355838201002199

Optimization and optimality of a short ribozyme ligase that joins non-Watson-Crick base pairings.

M P Robertson 1, J R Hesselberth 1, A D Ellington 1
PMCID: PMC1370105  PMID: 11345430

Abstract

A small ribozyme ligase (L1) selected from a random sequence population appears to utilize non-Watson-Crick base pairs at its ligation junction. Mutational and selection analyses confirmed the presence of these base pairings. Randomization of the L1 core and selection of active ligases yielded highly active variants whose rates were on the order of 1 min(-1). Base-pairing covariations confirmed the general secondary structure of the ligase, and the most active ligases contained a novel pentuple sequence covariation. The optimized L1 ligases may be optimal within their sequence spaces, and minimal ligases that span less than 60 nt in length have been engineered based on these results.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

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

  1. Bartel D. P., Szostak J. W. Isolation of new ribozymes from a large pool of random sequences [see comment]. Science. 1993 Sep 10;261(5127):1411–1418. doi: 10.1126/science.7690155. [DOI] [PubMed] [Google Scholar]
  2. Cate J. H., Doudna J. A. A sparse matrix approach to crystallizing ribozymes and RNA motifs. Methods Mol Biol. 1997;74:379–386. doi: 10.1385/0-89603-389-9:379. [DOI] [PubMed] [Google Scholar]
  3. Chapman K. B., Szostak J. W. Isolation of a ribozyme with 5'-5' ligase activity. Chem Biol. 1995 May;2(5):325–333. doi: 10.1016/1074-5521(95)90051-9. [DOI] [PubMed] [Google Scholar]
  4. Conrad R. C., Giver L., Tian Y., Ellington A. D. In vitro selection of nucleic acid aptamers that bind proteins. Methods Enzymol. 1996;267:336–367. doi: 10.1016/s0076-6879(96)67022-0. [DOI] [PubMed] [Google Scholar]
  5. Cuenoud B., Szostak J. W. A DNA metalloenzyme with DNA ligase activity. Nature. 1995 Jun 15;375(6532):611–614. doi: 10.1038/375611a0. [DOI] [PubMed] [Google Scholar]
  6. Ekland E. H., Bartel D. P. The secondary structure and sequence optimization of an RNA ligase ribozyme. Nucleic Acids Res. 1995 Aug 25;23(16):3231–3238. doi: 10.1093/nar/23.16.3231. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ekland E. H., Szostak J. W., Bartel D. P. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science. 1995 Jul 21;269(5222):364–370. doi: 10.1126/science.7618102. [DOI] [PubMed] [Google Scholar]
  8. Ellington A. D., Robertson M. P., Bull J. Ribozymes in wonderland. Science. 1997 Apr 25;276(5312):546–547. doi: 10.1126/science.276.5312.546. [DOI] [PubMed] [Google Scholar]
  9. Ferré-D'Amaré A. R., Zhou K., Doudna J. A. A general module for RNA crystallization. J Mol Biol. 1998 Jun 12;279(3):621–631. doi: 10.1006/jmbi.1998.1789. [DOI] [PubMed] [Google Scholar]
  10. Golden B. L., Podell E. R., Gooding A. R., Cech T. R. Crystals by design: a strategy for crystallization of a ribozyme derived from the Tetrahymena group I intron. J Mol Biol. 1997 Aug 1;270(5):711–723. doi: 10.1006/jmbi.1997.1155. [DOI] [PubMed] [Google Scholar]
  11. Hager A. J., Szostak J. W. Isolation of novel ribozymes that ligate AMP-activated RNA substrates. Chem Biol. 1997 Aug;4(8):607–617. doi: 10.1016/s1074-5521(97)90246-5. [DOI] [PubMed] [Google Scholar]
  12. Hesselberth J., Robertson M. P., Jhaveri S., Ellington A. D. In vitro selection of nucleic acids for diagnostic applications. J Biotechnol. 2000 Mar;74(1):15–25. doi: 10.1016/s1389-0352(99)00005-7. [DOI] [PubMed] [Google Scholar]
  13. Heus H. A., Pardi A. Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops. Science. 1991 Jul 12;253(5016):191–194. doi: 10.1126/science.1712983. [DOI] [PubMed] [Google Scholar]
  14. Jaeger L. The New World of ribozymes. Curr Opin Struct Biol. 1997 Jun;7(3):324–335. doi: 10.1016/s0959-440x(97)80047-4. [DOI] [PubMed] [Google Scholar]
  15. Koizumi M., Soukup G. A., Kerr J. N., Breaker R. R. Allosteric selection of ribozymes that respond to the second messengers cGMP and cAMP. Nat Struct Biol. 1999 Nov;6(11):1062–1071. doi: 10.1038/14947. [DOI] [PubMed] [Google Scholar]
  16. Kumar P. K., Ellington A. D. Artificial evolution and natural ribozymes. FASEB J. 1995 Sep;9(12):1183–1195. doi: 10.1096/fasebj.9.12.7672511. [DOI] [PubMed] [Google Scholar]
  17. Landweber L. F., Pokrovskaya I. D. Emergence of a dual-catalytic RNA with metal-specific cleavage and ligase activities: the spandrels of RNA evolution. Proc Natl Acad Sci U S A. 1999 Jan 5;96(1):173–178. doi: 10.1073/pnas.96.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Li Y., Liu Y., Breaker R. R. Capping DNA with DNA. Biochemistry. 2000 Mar 21;39(11):3106–3114. doi: 10.1021/bi992710r. [DOI] [PubMed] [Google Scholar]
  19. Robertson M. P., Ellington A. D. Design and optimization of effector-activated ribozyme ligases. Nucleic Acids Res. 2000 Apr 15;28(8):1751–1759. doi: 10.1093/nar/28.8.1751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Robertson M. P., Ellington A. D. In vitro selection of an allosteric ribozyme that transduces analytes to amplicons. Nat Biotechnol. 1999 Jan;17(1):62–66. doi: 10.1038/5236. [DOI] [PubMed] [Google Scholar]
  21. Robertson M. P., Ellington A. D. Ribozymes: red in tooth and claw. Curr Biol. 1997 Jun 1;7(6):R376–R379. doi: 10.1016/s0960-9822(06)00179-5. [DOI] [PubMed] [Google Scholar]
  22. Rogers J., Joyce G. F. A ribozyme that lacks cytidine. Nature. 1999 Nov 18;402(6759):323–325. doi: 10.1038/46335. [DOI] [PubMed] [Google Scholar]
  23. Soukup G. A., Breaker R. R. Allosteric nucleic acid catalysts. Curr Opin Struct Biol. 2000 Jun;10(3):318–325. doi: 10.1016/s0959-440x(00)00090-7. [DOI] [PubMed] [Google Scholar]
  24. Soukup G. A., Breaker R. R. Nucleic acid molecular switches. Trends Biotechnol. 1999 Dec;17(12):469–476. doi: 10.1016/s0167-7799(99)01383-9. [DOI] [PubMed] [Google Scholar]
  25. Tang J., Breaker R. R. Examination of the catalytic fitness of the hammerhead ribozyme by in vitro selection. RNA. 1997 Aug;3(8):914–925. [PMC free article] [PubMed] [Google Scholar]
  26. Vaish N. K., Heaton P. A., Eckstein F. Isolation of hammerhead ribozymes with altered core sequences by in vitro selection. Biochemistry. 1997 May 27;36(21):6495–6501. doi: 10.1021/bi963134r. [DOI] [PubMed] [Google Scholar]
  27. Wedekind J. E., McKay D. B. Purification, crystallization, and X-ray diffraction analysis of small ribozymes. Methods Enzymol. 2000;317:149–168. doi: 10.1016/s0076-6879(00)17013-2. [DOI] [PubMed] [Google Scholar]
  28. Wilson D. S., Szostak J. W. In vitro selection of functional nucleic acids. Annu Rev Biochem. 1999;68:611–647. doi: 10.1146/annurev.biochem.68.1.611. [DOI] [PubMed] [Google Scholar]
  29. Wright M. C., Joyce G. F. Continuous in vitro evolution of catalytic function. Science. 1997 Apr 25;276(5312):614–617. doi: 10.1126/science.276.5312.614. [DOI] [PubMed] [Google Scholar]

Articles from RNA are provided here courtesy of The RNA Society

RESOURCES