Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

Abstract

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The present invention relates to a method for the production of polynucleotides conferring a desired phenotype and / or encoding a protein having an advantageous predetermined property which is selectable. In an aspect, the method is used for generating and selecting nucleic acid fragments encoding mutant proteins.[0003]2. Description of the Related Art[0004]The complexity of an active sequence of a biological macromolecule, e.g. proteins, DNA etc., has been called its information content (“IC”; 5-9). The information content of a protein has been defined as the resistance of the active protein to amino acid sequence variation, calculated from the minimum number of invariable amino acids (bits) required to describe a family of related sequences with the same function (9, 10). Proteins that are sensitive to random mutagenesis have a high information content. In 1974, when this definition was coined, protein diversity existe...

AI Technical Summary

Benefits of technology

This approach enables the creation of libraries with a substantial fraction of novel combinations, facilitating the identification of proteins with desired properties, such as enhanced binding affinity or stability, and allows for iterative cycles of shuffling and selection to achieve optimal protein variants.

Problems solved by technology

However, computer simulations have suggested that point mutagenesis alone may often be too gradual to allow the block changes that are required for continued sequence evolution.

The published error-prone PCR protocols do not allow amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting their practical application.

Further, repeated cycles of error-prone PCR lead to an accumulation of neutral mutations, which, for example, may make a protein immunogenic.

This approach does not generate combinations of distant mutations and is thus not combinatorial.

The limited library size relative to the vast sequence length means that many rounds of selection are unavoidable for protein optimization.

This process constitutes a statistical bottleneck, it is labor intensive and not practical for many rounds of mutagenesis.

Error-prone PCR and oligonucleotide-directed mutagenesis are thus useful for single cycles of sequence fine tuning but rapidly become limiting when applied for multiple cycles.

However, the published error-prone PCR protocols (11, 12) suffer from a low processivity of the polymerase.

Therefore, the protocol is unable to result in the random mutagenesis of an average-sized gene.

This inability limits the practical application of error-prone PCR.

Another serious limitation of error-prone PCR is that the rate of down-mutations grows with the information content of the sequence.

Finally, repeated cycles of error-prone PCR will also lead to the accumulation of neutral mutations, which can affect, for example, immunogenicity but not binding affinity.

Thus error-prone PCR was found to be too gradual to allow the block changes that are required for continued sequence evolution (1, 2).

Therefore, the maximum information content that can be obtained is statistically limited by the number of random sequences (i.e., library size).

This constitutes a statistical bottleneck, eliminating other sequence families which are not currently best, but which may have greater long term potential.

Therefore, this approach is tedious and is not practical for many rounds of mutagenesis.

It is becoming increasingly clear that the tools for the design of recombinant linear biological sequences such as protein, RNA and DNA are not as powerful as the tools nature has developed.

However as discussed above, the existing mutagenesis methods that are in widespread use have distinct limitations when used for repeated cycles.

However, their system relies on specific sites of recombination and thus is limited.

Thus the method is limited to a finite number of recombinations equal to the number of selectable markers existing, and produces a concomitant linear increase in the number of marker genes linked to the selected sequence(s).

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