Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-06-02
2002-03-26
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S069100, C530S350000, C536S023100, C536S024200, C536S025300
Reexamination Certificate
active
06361943
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to a molecule assigning a genotype to a phenotype. More specifically, it relates to a molecule assigning a genotype to a phenotype, comprising a nucleic acid portion having a nucleotide sequence reflecting the genotype and a protein portion comprising a protein involved in exhibition of the phenotype. The molecule assigning the genotype to the phenotype of the present invention is a highly useful substance that can be utilized in evolutionary molecular engineering such as in the modification of enzymes, antibodies, ribozymes and other such functional biopolymers and creation of biopolymers having functions not found in living organisms.
Through advances in biochemistry, molecular biology and biophysics, it has been learned that living organisms are molecular machines which function and propagate by interactions among molecules. Among the characteristics of earth's living organisms, the fundamentals are their preservation of genetic information in DNA nucleotide sequences and their ability to translate this information into functional proteins through the medium of mRNA. Owing to progress in genetic engineering, biopolymers with given sequences, like nucleotides and peptides, can now be easily synthesized. Protein engineering and RNA engineering, today a focus of attention, owe their existence to genetic engineering. The aims of protein engineering and RNA engineering are to solve the puzzle of the three-dimensional structures required for proteins and RNA fulfilling specific functions and to enable humans to freely design proteins and RNA possessing desired functions. Because of the diversity and complexity of these structures and the difficulty of a theoretical approach to their three-dimensional structures, however, current protein engineering and RNA engineering are still at the stage of modifying some of residues at active sites and observing changes in the structure and functions. Human knowledge has thus not yet reached the stage of designing proteins and RNA.
Understanding the functions of biopolymers in their relationship to the elemental processes of higher life phenomena will require elucidation of the correlation between protein molecular structure and function. The line of thought we take in the following is not only to make the best of “human knowledge” but also to take advantage of the “wisdom of nature.” This is because we concluded that we would have to acquire the ability to put both to work in order to overcome the current difficulties of protein engineering and move forward with the design and production of functional biopolymers. When the classical methods are diverted to the design of proteins with new functions and activities, the difficulty of protein design by site-specific mutations can sometimes be avoided. This can be called “taking advantage of the wisdom of nature.”
Although the drawback of this method is the difficulty of screening to identify mutants with new functions and activities, this difficulty is overcome by the RNA catalysts that have recently come into the spotlight. Attempts have been made to select an RNA with specific characteristics from among RNAs synthesized to have an extremely large number of random sequences (about 10
13
types) (Ellington, A. D. & Szostak, J. W. (1990) Nature, 346, 818-822).
This is an example of evolutionary molecular engineering. As typified by this example, the primary goal in the evolutionary molecular engineering of proteins is to find out optimum sequences by searching an expansive sequence space of a scale unimaginable in conventional protein engineering. By “making the best of human knowledge” to devise a screening system for this, it will be possible to discover numerous quasi-optimum sequences around the optimum sequences and thus to construct an experimental system for studying “sequence vs function.”
The remarkable functions of living bodies were acquired through the process of evolution. Therefore, if evolution can be replicated, it should be possible to modify enzymes, antibodies, ribozymes and other functional biopolymers and, further, to create biopolymers with functions not found in living organisms in the laboratory. Needless to say, research on protein modification and creation is an object of utmost importance to various aspects of biotechnology such as utilization of enzymes as industrial catalysts, biochips, biosensors and sugar-chain engineering.
Given the fact that molecular design utilizing structural theory is, as symbolized by the continuing high regard for “screening,” still in an unperfected state, the evolutionary technique has a practical value for utilization in selecting useful proteins as a more efficient strategy. Building a “time machine” capable of more efficiently producing evolution in a laboratory, if such were possible, would not only enable modification of enzymes, antibodies (vaccines, monoclonal antibodies etc.) and other existing proteins but also open the way to the creation of enzymes for decomposing environmental contaminants, purifiers and others and new proteins not present in the biological world. If an experimental system for protein evolution can be established, therefore, it can be expected to be aggressively utilizable for application in a wide range of fields including power saving and energy preservation in industrial processes, energy production and environmental preservation. The assigning molecule of the present invention is a highly useful substance in protein modification and other aspects of evolutionary molecular engineering.
2. Description of the Related Art
Evolutionary molecular engineering is a field of study that attempts to conduct molecular design of functional polymers by utilizing high-speed molecular evolution in the laboratory, i.e., by laboratory investigation and optimization of the adaptive locomotion of biopolymers in sequence space. It is a completely new molecular biotechnology that first produced substantial results in 1990 (Yuzuru Husimi (1991) Kagaku, 61, 333-340; Yuzuru Husimi (1992) Koza Shinka, Vol. 6, University of Tokyo Publishing Society).
Life is a product of molecular evolution and natural selection. The evolution of molecules is a universal life phenomenon but its mechanism is not something that can be elucidated by studies that track the history of past evolution. Rather, the approach of constructing and studying the behavior of simple molecules and life systems that evolve in the laboratory better provides fundamental knowledge regarding molecular evolution and enables establishment of a verifiable theory applicable in molecular engineering.
It is known that a polymer system will evolve if it satisfies the following five conditions: (1) an open system far out of equilibrium, (2) a self-replicative system, (3) a mutation system, (4) a system with genotype and phenotype assignment strategy, and (5) a system with appropriate adaptation topography in sequence space. (1) and (2) are conditions for occurrence of natural selection and (5) is determined beforehand by the physicochemical properties of the biopolymer. The genotype and phenotype assignment of (4) is a prerequisite for evolution by natural selection.
The following three strategies are adopted in both the natural world and evolutionary molecular engineering: (a) ribozyme-type in which the genotype and the phenotype are carried on the same molecule, (b) virus-type in which the genotype and the phenotype form a complex, and (c) a cell-type in which the genotype and the phenotype are contained in a single compartment (FIG.
1
).
As the ribozyme-type (a) in which the genotype and the phenotype are carried on the same molecule is a simple system, success with RNA catalysts (ribozymes) has already been reported (Hiroshi Yanagawa (1993) New Age of RNA, pp.55-77, Yodosha).
Conceivable problem points of the cell-type (c) are (1) the averaging effect, (2) the eccentricity effect and (3) the random replication effect. The averaging effect arises because the assignment of the genotype to the ph
Husimi Yuzuru
Miyamoto Etsuko
Nemoto Naoto
Yanagawa Hiroshi
Davis Katharine F
Mitsubishi Chemical Corporation
Schwartzman Robert A.
Wenderoth , Lind & Ponack, L.L.P.
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