Method for creating polynucleotide and polypeptide sequences

Details Method for creating polynucleotide and polypeptide sequences Method for creating polynucleotide and polypeptide sequences

1998-12-04

2003-03-25

Jones, W. Gary (Department: 1643)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S091100, C435S091200, C435S069100, C435S320100, C536S027400

Reexamination Certificate

active

06537746

ABSTRACT:

TECHNICAL FIELD
The invention resides in the technical field of genetics, and more specifically, forced molecular evolution of polynucleotides to acquire desired properties.
BACKGROUND
A variety of approaches, including rational design and directed evolution, have been used to optimize protein functions (1, 2). The choice of approach for a given optimization problem depends, in part, on the degree of understanding of the relationships between sequence, structure and function. Rational redesign typically requires extensive knowledge of a structure-function relationship. Directed evolution requires little or no specific knowledge about structure-function relationship; rather, the essential features is a means to evaluate the function to be optimized. Directed evolution involves the generation of libraries of mutant molecules followed by selection or screening for the desired function. Gene products which show improvement with respect to the desired property or set of properties are identified by selection or screening. The gene(s) encoding those products can be subjected to further cycles of the process in order to accumulate beneficial mutations. This evolution can involve few or many generations, depending on how far one wishes to progress and the effects of mutations typically observed in each generation. Such approaches have been used to create novel functional nucleic acids (3, 4), peptides and other small molecules (3), antibodies (3), as well as enzymes and other proteins (5, 6, 7). These procedures are fairly tolerant to inaccuracies and noise in the function evaluation (7).
Several publications have discussed the role of gene recombination in directed evolution (see WO 97/07205, WO 98/42727, U.S. Pat. No. 5,807,723, U.S. Pat. No. 5,721,367, U.S. Pat, No. 5,776,744 and WO 98/41645 U.S. Pat. No. 5,811,238, WO 98/41622, WO 98/41623, and U.S. Pat. No. 5,093,257).
A PCR-based group of recombination methods consists of DNA shuffling [5, 6], staggered extension process [89, 90] and random-priming recombination [87]. Such methods typically involve synthesis of significant amounts of DNA during assembly/recombination step and subsequent amplification of the final products and the efficiency of amplification decreases with gene size increase.
Yeast cells, which possess an-active system for homologous recombination, have been used for in vivo recombination. Cells transformed with a vector and partially overlapping inserts efficiently join the inserts together in the regions of homology and restore a functional, covalently-closed plasmid [91]. This method does not require PCR amplification at any stage of recombination and therefore is free from the size considerations inherent in this method. However, the number of crossovers introduced in one recombination event is limited by the efficiency of transformation of one cell with multiple inserts. Other in vivo recombination methods entail recombination between two parental genes cloned on the same plasmid in a tandem orientation. One method relies on homologous recombination machinery of bacterial cells to produce chimeric genes [92]. A first gene in the tandem provides the N-terminal part of the target protein, and a second provides the C-terminal part. However, only one crossover can be generated by this approach. Another in vivo recombination method uses the same tandem organization of substrates in a vector [93]. Before transformation into
E. coli
cells, plasmids are linearized by endonuclease digestion between the parental sequences. Recombination is performed in vivo by the enzymes responsible for double-strand break repair. The ends of linear molecules are degraded by a 5′′3′ exonuclease activity, followed by annealing of complementary single-strand 3′ ends and restoration of the double-strand plasmid [94]. This method has similar advantages and disadvantages of tandem recombination on circular plasmid.
SUMMARY OF THE INVENTION
The invention provides methods for evolving a polynucleotide toward acquisition of a desired property. Such methods entail incubating a population of parental polynucleotide variants under conditions to generate annealed polynucleotides comprises heteroduplexes. The heteroduplexes are then exposed to a cellular DNA repair system to convert the heteroduplexes to parental polynucleotide variants or recombined polynucleotide variants. The resulting polynucleotides are then screened or selected for the desired property.
In some methods, the heteroduplexes are exposed to a DNA repair system in vitro. A suitable repair system can be prepared in the form of cellular extracts.
In other methods, the products of annealing including heteroduplexes are introduced into host cells. The heteroduplexes are thus exposed to the host cells' DNA repair system in vivo.
In several methods, the introduction of annealed products into host cells selects for heteroduplexes relative to transformed cells comprising homoduplexes. Such can be achieved, for example, by providing a first polynucleotide variant as a component of a first vector, and a second polynucleotide variant is provided as a component of a second vector. The first and second vectors are converted to linearized forms in which the first and second polynucleotide variants occur at opposite ends. In the incubating step, single-stranded forms of the first linearized vector reanneal with each other to form linear first vector, single-stranded forms of the second linearized vector reanneal with each other to form linear second vector, and single-stranded linearized forms of the first and second vectors anneal with each to form a circular heteroduplex bearing a nick in each strand. Introduction of the products into cells thus selects for cirular heteroduplexes relative to the linear first and second vector. Optionally, in the above methods, the first and second vectors can be converted to linearized forms by PCR. Alternatively, the first and second vectors can be converted to linearized forms by digestion with first and second restriction enzymes.
In some methods, polynucleotide variants are provided in double stranded form and are converted to single stranded form before the annealing step. Optionally, such conversion is by conducting asymmetric amplification of the first and second double stranded polynucleotide variants to amplify a first strand of the first polynucleotide variant, and a second strand of the second polynucleotide variant. The first and second strands anneal in the incubating step to form a heteroduplex.
In some methods, a population of polynucleotides comprising first and second polynucleotides is provided in double stranded form, and the method further comprises incorporating the first and second polynucleotides as components of first and second vectors, whereby the first and second polynucleotides occupy opposite ends of the first and second vectors. In the incubating step single-stranded forms of the first linearized vector reanneal with each other to form linear first vector, single-stranded forms of the second linearized vector reanneal with each other to form linear second vector, and single-stranded linearized forms of the first and second vectors anneal with each to form a circular heteroduplex bearing a nick in each strand. In the introducing step selects for transformed cells comprises the circular heteroduplexes relative to the linear first and second vector.
In some methods, the first and second polynucleotides are obtained from chromosomal DNA. In some methods, the polynucleotide variants encode variants of a polypeptide. In some methods, the population of polynucleotide variants comprises at least 20 variants. In some methods, the population of polynucleotide variants are at least 10 kb in length.
In some methods, the polynucleotide variants comprises natural variants. In other methods, the polynucleotide variants comprise variants generated by mutagenic PCR or cassette mutagenesis. In some methods, the host cells into which heteroduplexe

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