Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2001-09-14
2003-07-08
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S004000, C435S069100, C435S069700, C436S501000, C436S518000, C436S536000, C536S023100, C536S023400, C536S024500
Reexamination Certificate
active
06589741
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to methods for identifying nucleic acid molecules encoding (poly)peptides that interact with target molecules. The method of the present invention is particularly characterized by an in vitro translation step under conditions that allow formation of polysomes in the presence of antisense oligonucleotides complementary to the tag-coding sequence of ssrA-RNA. The present invention further relates to kits that are useful for carrying out the method of the invention.
Evolutionary methods may bring the refinement to protein engineering which is beyond the powers and accuracy of rational design today. Evolution can be defined as a succession of “generations”, cycles of genetic diversification, followed by Darwinian selection for a desired phenotypic property. In classic experiments, nucleic acids have been evolved for physical properties (Saffhill,. R., Schneider-Bernloehr, H., Orgel, L. E. & Spiegelman, S. (1970)
J. Mol. Biol
. 51, 531-539) in vitro, and in this case, the substance conferring the phenotype was identical to the genetic material. Oligonucleotide ligands, usually single stranded RNA, have been identified for many targets by SELEX (Gold, L., Polisky, B., Uhlenbeck, O. & Yarus, M. (1995)
Annu. Rev. Biochem
. 64, 763-797; Irvine, D., Tuerk, C. & Gold, L. (1991)
J. Mol. Biol
. 222, 739-761), in which a synthetic DNA library is transcribed, the RNA selected for binding, reverse transcribed and amplified over several rounds. Early experiments with proteins as the carrier of the phenotype, clearly of much broader applicability, had relied on living cells for effecting the coupling between gene and protein, either directly or via the production of phages or viruses (Phizicky, E. M. & Fields, S. (1995)
Microbiol. Rev
. 59, 94-123). Since in this type of experiment the DNA library as the information carrier for encoded protein diversity has to be transformed or transfected into bacterial or eukaryotic cells, the available diversity was severely limited by the low efficiency of DNA uptake (Dower, W. J. & Cwirla, S. E. (1992) in
Guide to Electroporation and Electrofusion
, eds. Chang, D. C., Chassy, B. M., Saunders, J. A. & Sowers, A. E. (Academic Press, San Diego), pp. 291-301). Furthermore, in each generation, the DNA library had to be first ligated into a replicable genetic package by which diversity was again decreased. In addition, many promising variants would have to be selected against in the host environment. Only very few studies (Yang, W. P., Green, K., Pinz-Sweeney, S., Briones, A. T., Burton, D. R. & Barbas 3rd., C. F. (1995)
J. Mol. Biol
. 254, 392-403) have carried protein optimization through more than one generation using methods such as phage display, since this requires repeated switching between in vitro diversification and in vivo screening—a laborious process.
With the goal of circumventing or improving this process, a number of laboratories have designed novel systems that are based on the immediate vicinity and physical connection of mRNA and corresponding (poly)peptides during translation. Thus, a series of studies have shown that specific mRNAs can be enriched by immunoprecipitation of polysomes (Schechter, I. (1973)
Proc. Natl. Acad. Sci. U.S.A
. 70, 2256-2260; Payvar, F. & Schimke, R. T. (1979)
Eur. J. Biochem
. 101, 271-282; Kraus, J. P. & Rosenberg, L. E. (1982)
Proc. Natl. Acad. Sci. U.S.A
. 79, 4015-4019). Recently, Mattheakis and coworkers reported an affinity selection of a short peptide from a library using polysomes, in order to connect genotype and phenotype in vitro (Mattheakis, L. C., Bhatt, R. R. & Dower, W. J. (1994)
Proc. Natl. Acad. Sci. U.S.A
. 91, 9022-9026; WO95/11922).
This system employs an in vitro translation system that is preferentially coupled to an in vitro transcription system. The translation system allows the simultaneous isolation of mRNA and (poly)peptide in a polysome complex after a suitable screening step for the (poly)peptide. Preferably, the (poly)peptides in Mattheakis' system are comprised of two components, one of which is the peptide to be screened and the second is a tether segment that binds to the mRNA. Co-isolation of mRNA and (poly)peptide in the polysome complex can possibly be improved with the help of a translation stalling sequence even though the existence of such sequences is still unclear for
E. coli
. This sequence possibly enhances the overall stability of the polysome complex by decreasing the translation rate and thus allows for suitable conditions for the concomitant screening and isolation of (poly)peptide and corresponding mRNA.
Similar work has earlier been reported by Gold and colleagues (WO 93/03172) and Kawasaki and co-workers (WO 91/05058). Although the above-described systems have established a means of characterizing a nucleic acid via the identification of a protein encoded by said nucleic acid, there are practical limitations with respect to the efficiency of the ribosome displays of the nascent (poly)peptide. The technical problem underlying the present invention was therefore to increase the efficiency of (i) synthesis of a collection of stable RNA molecules and (ii) translation of said RNA molecules, and thereby to achieve an increased efficiency of the use of polysomes in screening. The solution to said technical problem is achieved by providing the embodiments characterized in the claims.
SUMMARY OF INVENTION
Accordingly, the present invention relates to a method for identifying a nucleic acid molecule encoding a (poly)peptide that interacts with a target molecule comprising the following steps:
(a) translating a population of mRNA molecules devoid of stop codons in the correct reading frame in an in vitro translation system, said translation system either comprising antisense oligonucleotides complementary to the tag-coding sequence of ssrA-RNA or being free of ssrA-RNA, under conditions that allow the formation of polysomes;
(b) bringing the polysomes so formed into contact with said target molecules under conditions that allow the interaction of the (poly)peptides encoded by said mRNA molecules and displayed by said polysomes with said target molecules;
(c) separating polysomes displaying (poly)peptides that interact with said target molecules from polysomes displaying no such (poly)peptides; and
(d) identifying the nucleic acid molecule encoding a (poly)peptide displayed in a polysome that interacts with said target molecules.
The term “(poly)peptide” as used in the present invention relates both to peptides as well as to polypeptides. Said (poly)peptides may either comprise a natural or a recombinantly engineered amino acid sequence. The latter alternative also includes fusion proteins.
According to the present invention, the term “polysome” refers to a complex formed by at least one, preferably several ribosomes and mRNA during translation.
The population of mRNA molecules may be of varying origin. For example, it may be derived from a cDNA library. In an alternative embodiment, it may be directly derived from cells or tissue. Particularly advantageous is also the use of the present invention in mutagenized (poly)peptides to find improved variants. Alternatively, synthetic protein or peptide libraries or antibody libraries can be used.
The term “tag-coding sequence of ssrA-RNA” relates to a nucleic acid sequence encoding the amino acid sequence AANDENYALAA (SEQ ID NO: 1). This sequence has been described in Keiler et al.,
Science
221 (1996), 1990-1993.
The antisense oligonucleotides comprised in the translation system employed in the method of the invention are of a suitable length to hybridize to the tag-coding sequence of ssrA-RNA and block translation thereof under conditions that allow the formation of polysomes.
A translation system being free of ssrA-RNA can, for example, be derived from
E. coli
strains lacking a functional ssrA gene such as X90 ssrA1::cat (Keiler et al.,
Science
221 (1996), 1990-1993), N2211 or NM101 (Tu et al.,
J. Biol. Chem
. 270 (1995), 9322-9326), W3110 &Dgr;ssrA (Ko
Hanes Jozef
Jermutus Lutz
Plückthun Andreas
Guzo David
Heller Ehrman White and McAuliffe
Leffers, Jr. Gerald G.
University of Zurich
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