Continuous in vitro evolution of catalytic function

Science

Volumn 276, Issue 5312, 1997, Pages 614-617

  Wright, Martin C ^a   Joyce, Gerald F ^a  

^a SCRIPPS RESEARCH INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

RNA;

ARTICLE; CATALYSIS; GENE AMPLIFICATION; GENE MUTATION; MOLECULAR EVOLUTION; POPULATION AND POPULATION RELATED PHENOMENA; PRIORITY JOURNAL; RNA SYNTHESIS;

NASA DISCIPLINE EXOBIOLOGY; NASA DISCIPLINE NUMBER 52-20; NASA DISCIPLINE NUMBER 93-10; NASA PROGRAM EXOBIOLOGY; NASA PROGRAM NSCORT; NON-NASA CENTER;

BASE SEQUENCE; CATALYSIS; DIRECTED MOLECULAR EVOLUTION; DNA-DIRECTED RNA POLYMERASES; EVOLUTION, MOLECULAR; MOLECULAR SEQUENCE DATA; MUTATION; NUCLEIC ACID CONFORMATION; PROMOTER REGIONS (GENETICS); RNA, CATALYTIC; SACCHAROMYCES CEREVISIAE; TEMPLATES, GENETIC; TRANSCRIPTION, GENETIC; VIRAL PROTEINS;

EID: 0030901131     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5312.614     Document Type: Article

References (19)

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2. For recent reviews see P. K. R. Kumar and A. D. Ellington, FASEB J. 9, 1183 (1995); R. R. Breaker, Curr. Opin. Biotechnol. 7, 442 (1996); A. J. Hager, J. D. Pollard, J. W. Szostak, Chem. Biol. 3, 717 (1996).
3. For recent reviews see P. K. R. Kumar and A. D. Ellington, FASEB J. 9, 1183 (1995); R. R. Breaker, Curr. Opin. Biotechnol. 7, 442 (1996); A. J. Hager, J. D. Pollard, J. W. Szostak, Chem. Biol. 3, 717 (1996).
4. For recent reviews see P. K. R. Kumar and A. D. Ellington, FASEB J. 9, 1183 (1995); R. R. Breaker, Curr. Opin. Biotechnol. 7, 442 (1996); A. J. Hager, J. D. Pollard, J. W. Szostak, Chem. Biol. 3, 717 (1996).
5. R. R. Breaker and G. F. Joyce, Proc. Natl. Acad. Sci. U.S.A. 91, 6093 (1994).
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9. -1. and was incubated at 37°C for 2 hours. The protein enzymes were inactivated by heating to 65°C for 5 min in the presence of 15 mM disodium EDTA; 8 μl of this mixture was used to initiate the next round. After rounds 5, 10, and 15, the population was treated with reverse transcriptase, selectively amplified by PCR, and transcribed in order to reduce amplification artifacts.
10. 2, 50 mM KCl, 2 mM spermidine, and 50 mM EPPS (pH 8.5). Ribozyme was present in excess of substrate to ensure that a constant amount reacted during each round. Reverse transcription was carried out at 37°C for 5 min, then T7 RNA polymerase was added and transcription was continued for 30 to 45 min. After heat inactivation of the polymerase proteins, 5 μl of the amplification mixture was diluted into 20 μl of a fresh reaction mixture. The population was purified every 10 rounds by PCR amplification of the complementary DNA and polyacrylamide gel electrophoresis of the RNA.
11. -1 could not be determined by manual pipetting methods. The proportion of correctly folded ribozymes depended on the method of purification. The values reported were obtained with in vitro transcribed RNA that was purified by phenol extraction and Sephadex chromatography, reacting to 60% completion.
12. A disabled form of the ribozyme was constructed by reverse transcription of reacted RNA with a mutagenic DNA primer (5′-GCTGAGCCTGCGATTGGCTTTTAGGTTCAGTGTAATGTGTTGAGAACGCTGGC-3′, mutations underlined), followed by PCR amplification. The double-stranded DNA product was cloned and sequenced to confirm the presence of the desired mutations. The mutant ribozyme could be amplified nonselectively in the presence of reverse transcriptase and T7 RNA polymerase with the use of primer 1 (7) and an alternative version of primer 2 that did not require attachment of the substrate to the 5′ end of the RNA.
13. J. F. Milligan, D. R. Groebe, G. W. Witherell, O. C. Uhlenbeck, Nucleic Acids Res. 15, 8783 (1987).
14. E. H. Ekland and D. P. Bartel, Nature 382, 373 (1996).
15. 2, 200 mM KCl, 0.6 mM disodium EDTA, and 30 mM tris-HCl (pH 8.0). The RNA was dephosphorylated prior to reaction so as to prevent its direct ligation to the substrate.
16. A. A. Beaudry and G. F. Joyce, Science 257, 635 (1992); J. Tsang and G. F. Joyce, Biochemistry 33, 5966 (1994); J. Mol. Biol. 262, 31 (1996).
17. A. A. Beaudry and G. F. Joyce, Science 257, 635 (1992); J. Tsang and G. F. Joyce, Biochemistry 33, 5966 (1994); J. Mol. Biol. 262, 31 (1996).
18. A. A. Beaudry and G. F. Joyce, Science 257, 635 (1992); J. Tsang and G. F. Joyce, Biochemistry 33, 5966 (1994); J. Mol. Biol. 262, 31 (1996).
19. We thank R. Breaker for helpful suggestions and D. Bartel and J. Szostak for making available the sequence of the b1-207 ribozyme prior to publication. Supported by NASA and the NASA Specialized Center for Research and Training in Exobiology.