Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2000-04-12
2002-03-19
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C435S006120
Reexamination Certificate
active
06358713
ABSTRACT:
TECHNICAL FIELD
The invention relates to selection of variant rRNA molecules catalyzing formation of non-peptidyl products when incorporated into large ribosomal subunits.
BACKGROUND
Catalysis of peptide bond formation requires precise juxtaposition by the ribosome of the acceptor ends of P (peptidyl) and A (aminoacyl) site bound tRNAs in its “active site”. Accumulating evidence points to a functionally important role for rRNAs in the various steps of the translational cycle, and in particular in peptidyl transferase. See, for example, Noller et al.,
Science,
1992, 256:1416-1419; and Samaha et al.,
Nature,
1995, 377:309-314. A number of approaches have begun to identify specific nucleotides in 23S rRNA that are located proximal to the tRNA substrates of the ribosome. See review by Green and Noller,
Ann. Rev. Biochem.,
1997, 66:679-716. Studies by Moazed and Noller identified a small number of nucleotides in domain V of 23S rRNA whose protection from chemical modification is dependent on the presence of the CCA acceptor ends of A and P-site bound tRNAs. Moazed and Noller,
Cell,
1989, 57:585-597. While these critical nucleotides are predominantly found in the central loop structure of domain V, several are located in peripheral elements of this 23S RNA domain including the 2250 and the 2555 loops. Discovery of a base-pairing interaction between C74 of P-site-bound tRNA and G2252 of domain V established a direct functional role for this region of 23S rRNA in peptidyl transferase (PT). Samaha et al., 1995, supra. Recent experiments describing an interaction between C75 of A site tRNA and G2553 of 23S rRNA further substantiate this role. Kim and Green,
Mol. Cell,
1999, 4:859-864. Characterization of a P-site-specific peptidyl transferase-reactive crosslink between a benzophenone derivatized peptidyl-tRNA and A2451/C2452 in the central loop of domain V of 23S rRNA identified this region as another likely component of the active site of 23S rRNA. Steiner et al.,
EMBO J.,
1988, 7:3949-3955.
SUMMARY
The invention is based on a method for evolving or redirecting the standard peptidyl transferase chemistry that is performed by the ribosome. Specifically, an iterative, in vitro selection system is described that allows for the isolation of variant major rRNAs of large ribosomal subunits with novel properties. The selection system allows rRNA variants to be isolated with enriched catalytic activity on altered peptidyl and aminoacyl ribosome substrates such as D-amino acids, methyl phosphinyl derivatized substrates, N-derivatized, and &bgr;-amino acid substrates. The coupling of such evolved ribosomes with RNA-peptide fusion technology allows for the generation of combinatorial chemical libraries that can be screened and deconvoluted to identify novel and biologically stable target compounds.
The invention features a method for selecting rRNA variant molecules catalyzing formation of non-peptidyl products. The method includes crosslinking a peptidyl substrate to ribosomes, wherein the major RNA of the large ribosomal subunit in a plurality of the ribosomes is an rRNA variant molecule. The ribosomes can be eukaryotic or prokaryotic ribosomes, such as
Escherichia coli
or
Bacillus stearothermophilus
ribosomes, and the rRNA variant can be a 28S or 23S rRNA variant molecule.
The crosslinked ribosomes and a labeled, derivatized aminoacyl substrate are reacted under conditions such that the labeled, derivatized aminoacyl substrate is transferred to the rRNA variant molecule to form labeled ribosomes. The rRNA variant molecules are selected from labeled ribosomes. The peptidyl substrate can be a benzophenone derivatized peptidyl substrate, and the labeled, derivatized aminoacyl substrate can be N-derivatized, a &bgr;-amino acid, or a D-amino acid. The labeled, derivatized aminoacyl substrate can be biotinylated or can include a thiol moiety. The method further can include repeating the method steps with the selected rRNA variant molecules.
The invention also features a method for selecting rRNA variant molecules catalyzing formation of non-peptidyl products that includes crosslinking an aminoacyl substrate to ribosomes, wherein the major RNA of the large ribosomal subunit in a plurality of the ribosomes is an rRNA variant molecule. The rRNA variant molecule is thr major RNA of the large ribosomal subunit as described above. The crosslinked ribosomes and a labeled, derivatized peptidyl substrate are reacted under conditions such that the labeled, derivatized peptidyl substrate is transferred to the rRNA variant molecule to form labeled ribosomes. The labeled rRNA variant molecules are selected from the labeled ribosomes. The aminoacyl substrate can be 4-thio-dT-p-C-p-Puromycin, and the labeled, derivatized peptidyl substrate can include a methyl phosphinyl derivatized peptidyl substrate or a D-amino acid. The method further can include repeating the method steps with the selected rRNA variant molecules.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
REFERENCES:
patent: 4743535 (1988-05-01), Carrico
patent: 5643722 (1997-07-01), Rothschild et al.
patent: 5872104 (1999-02-01), Vermeulen et al.
patent: 5962244 (1999-10-01), Lynch et al.
patent: 5965695 (1999-10-01), Simon et al.
patent: WO 98/31700 (1998-07-01), None
patent: WO 00/09737 (2000-02-01), None
Lohse et al.,“Ribozyme-catalysed amino-acid transfer reactions”; Nature 1996, vol. 381, pp. 442-444.*
Schumacher et al., “Identification of D-Peptide Ligands Through Mirror-Image Phage Display”, 1996, vol. 271, pp. 1854-1857.*
Steiner et al.,“Photo-affinity labelling at the peptidyl transferase center reveals two different positions for the A- and P-sites in domain V of 23S rRNA”;The EMBO Journal, 1988, vol. 7, No. 12, pp. 3949-3955.*
Green et al., “Localization of The A Site On The Ribosome”; The Second Annual Meeting of the RNA Society, 1997, May 27-Jun. 1, Banff Centre, Banff, Alberta.*
Kuyl-Yeheskiely,“An expeditious route to methylphosphonate analogues of DNA”, Recl. Trav. Chim. Pays-Bas, 1993, 113, pp. 040-044.*
Green, et al.,RNA '97,1997, p. 87.
Green, et al.,Science,1998, 280:286-289.
Lohse and Szostak,Nature,1996, 381:442-444.
Cadwell, R., et al., “Randomization of Genes by PCR Mutagenesis,” PCR Methods and Applications, (1992) vol. 2, No. 1, pp. 28-33.
Green, R., et al., “Reconstitution of Functional 50S Ribosomes from in Vitro Transcripts ofBacillus stearothermophilus23S rRNA,” Biochemistry (1999) vol. 38, No. 6, pp. 1772-1779.
Kuechler, E., et al., “Aromatic Ketone Derivatives of Aminoacyl-tRNA as Photoaffinity Labels for Ribosomes,” Methods in Enzymology (1977) vol. 46, pp. 676-683.
Lorsch, J., et al., “In vitro evolution of new ribozymes with polynucleotide kinase activity,”, Nature (1994) vol. 371, No. 6492, pp. 31-36.
Green, R., et al., “In vitro genetic analysis of the Tetrahymena self-splicing intron,” Nature (1990) vol. 347, No. 6290, pp. 406-408.
Roberts, R., et al., “RNA-peptide fusions for the in vitro selection of peptides and proteins,”Proc. Natl. Acad. Sci, USA(1997) vol. 94, pp. 12297-12302.
Atkinson, T., et al., “Solid-phase Synthesis of Oligodeoxyribonucleotides by the Phosphite-triester Method,” Oligonucleotide Synthesis: a practical approach, Oxford: IRL Press (1985) pp. 35-81.
Green, R.,et al., “In vitro complementation analysis localizes 23S rRNA pos
Campbell Eggerton A.
Fish & Richardson P.C.
Johns Hopkins University
LandOfFree
In vitro ribosome evolution does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with In vitro ribosome evolution, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and In vitro ribosome evolution will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2846798