AU2007234569C1 - Reassortment by fragment ligation - Google Patents

Description

REASSORTMENT BY FRAGMENT LIGATION z FIELD OF THE INVENTION The present invention provides methods for preparing polynucleotide variants.

Also provided are methods of making a polynucleotide and/or polypeptide having a Sdesired property.

Cc BACKGROUND OF THE INVENTION The natural evolution of genes and their encoded proteins occurs through an equilibrium between recombination or mutation and selection. While evolution in nature takes millions of years, in vitro methods and compositions have been developed to evolve proteins, with improved and novel functions much more rapidly.

Directed evolution refers to biotechnological processes devoted to the optimization of the protein activity by means of changes introduced into selected respective genes. Directed evolution includes the generation of a collection of mutated genes followed by the selection of mutants encoding proteins with desired features.

These processes can be iterative when gene products having an improvement in a desired property are subjected to further cycles of mutation, selection and screening.

The concept of mutant or mutation is used here in the wide sense of "change". Directed evolution provides a way to adapt natural proteins to work in new chemical or biological environments, and/or to elicit new functions.

Proteins intrinsically possess an enormous potential plasticity, which allows them to face new challenges, such as a new environment and a desired new or altered activity. It is possible to take advantage of this plasticity to generate new proteins with altered activity. In a sufficiently large pool of modified mutant proteins, there is a chance of finding an appropriately modified protein having a desired activity.

Problems arise, however, in generating and identifying a modified protein having a desired activity. Among the practical approaches intended to tackle these problems, two types can be distinguished. One is a purely predictive approach that is based on the assumption that the optimized proteins can be rationally designed in a predictable manner. This approach, however, requires sufficient information regarding the physiochemical properties of individual amino acids and amino acid sequences that govern protein folding, molecular interactions, intra-molecular forces and other dynamics of protein activity. The predictive approach is extremely dependent on a number of variables and parameters that are not known, even if the secondary and/or tertiary structures of a protein are available.

Homologous recombination is defined as the exchange of homologous or similar z DNA sequences between two DNA molecules. An essential feature of homologous recombination is that the enzymes responsible for the recombination event can pair any homologous sequences as substrates. The ability of homologous recombination to transfer genetic information between DNA molecules makes targeted homologous \recombination a very powerful method in genetic engineering and gene manipulation.

t Homologous recombination can be used to add subtle mutations at known sites, replace Cc wild type genes or gene segments or introduce completely foreign genes into cells.

However, homologous recombination efficiency is very low in living cells and is dependent on several parameters, including the method of DNA delivery, how it is packaged, its size and conformation, DNA length and position of sequences homologous to the target, and the efficiency of hybridization and recombination at chromosomal sites. These variables severely limit the use of conventional homologous recombination approaches for gene evolution in cell based systems.

In contrast to the predictive approach, random or stochastic approaches have also been employed. One random approach requires synthesis of all possible protein sequences or a statistically sufficient large number of proteins followed by the screening to identify proteins having a desired activity or property. Other random approaches are based on gene shuffling methods, such as, for example, PCR-based methods that generate random rearrangements between or among two or more sequence-related genes to randomly generate variants of the original gene.

Common in vitro gene evolution methods utilize repeated cycles of random mutagenesis, or random non-specific cleavage, and mixing of related genes containing mutations in PCR-based random recombination. These methods couple multiple rounds of in vitro mutagenesis with screening systems to produce and identify the desired mutants or recombinants (Stemmer, 1994; Arnold, 1996; US 6,579,678).

The DNA shuffling methods of Maxygen, Inc. (see, for example, US 5,605,793, US 6,117,679 and US 6,132,970) comprise the steps of fragmenting at least one kind of double-stranded DNA to be shuffled and conducting polymerase chain reactions (PCR) with-the combined fragments, wherein the homologous fragments -from differentparent DNAs are annealed with each other to form partially overlapping DNA segments and DNA synthesis occurs by employing the respective DNA fragments as a template and concurrently as a primer for each other to produce a random recombinant DNA library.

However, DNase I used in the fragmentation process has to be removed from the resulting DNA fragments so as not to disturb the subsequent polymerization process.

Further, the application of the method is limited by the property of the DNase I. For 3 example, DNase I widely used for the purpose is liable to cleave a phosphodiester z bond having a pyrimidine base rather than a purine base at its terminus, which is a serious obstacle to get a completely randomized pool of DNA fragments. One problem with this procedure is the considerable effort required to get the level of DNase fragmentation "just right". Too little fragmentation and very little if any shuffling will occur, too much and the gene will not reassemble and the process has to be repeated n each time the shuffling is repeated. Also, even when done properly, there are typically Cc, only a couple of recombination events per gene, so the process requires repeated cycles of shuffling to exhaust the combinatorial potential and get the best recombinant. In addition, the size of the gene being shuffled is limited by the size of product that can c. reasonably and efficiently be obtained by PCR.

The Gene Reassembly method of Diversa Corporation (US 5,965,408) comprises the steps of synthesizing DNA fragments by a polymerization process employing at least one kind of double-stranded DNAs to be shuffled as templates and conducting polymerase chain reactions (PCR) with the combined fragments to produce a random recombinant DNA library. It employs partially synthesized fragments produced by UV treatment or adduct formation on the template DNA, thus preventing a complete polymerization on the template DNA. Despite of the randomness of the constructed DNA library, there are still problems for the method of Diversa Corporation in view of mutagenic potential of used reagents and tediousness to optimize the reaction conditions for the treatment of polymerization terminating reagent to obtain the desired size of fragments. In addition, when pyrimidine bases exist contiguously on the DNA strand, UV treatment induces pyrimidine dimers such as a thymidine dimer, which makes the template DNA distorted and prevent the progress of polymerase along the strand. As a result, polymerizations are likely to end up at the site ofa pyrimidine dimer, thus DNA fragments obtained have insufficient randomness.

DNA shuffling and Gene Reassembly methods are characterized by the formation of partially overlapping DNA segments as a prerequisite step and each DNA fragment derived from starting DNAs to be shuffled serves as not only a template but a primer.

There is a need for alternate methods of evolving a polynucleotide toward acquisition of a desired property.

>SUMMARY OF THE INVENTION z The present invention provides new avenues for generating polynucleotide variants.

In a first aspect, the present invention provides a method of preparing polynucleotide variants, the method comprising exposing a pool of two or more related polynucleotides to at least one nicking enzyme, wherein at least some of the polynucleotides are partially and/or fully double Sstranded, b) removing, and/or inhibiting the activity of, the at least one nicking enzyme, c) denaturing the polynucleotides, d) allowing the denatured polynucleotides to form at least partially double stranded polynucleotides, e) exposing the double stranded polynucleotides formed in step d) to a ligase.

In a particularly preferred embodiment, steps d) and e) are conducted simultaneously. In this embodiment, the ligase will preferably be a thermostable ligase.

In a further aspect, the present invention provides a method of preparing polynucleotide variants, the method comprising a) exposing a pool of two or more related single stranded polynucleotides to at least one nicking enzyme, b) removing, or inhibiting the activity of, the at least one nicking enzyme, c) allowing the polynucleotides to form at least partially double stranded polynucleotides, d) exposing the double stranded polynucleotides formed in step c) to a ligase.

In a particularly preferred embodiment, steps c) and d) are conducted simultaneously. In this embodiment, the ligase will preferably be a thermostable ligase.

In another aspect, the present invention provides a method of preparing polynucleotide variants, the method comprising a) exposing at least a first double stranded polynucleotide, or a first pool of related double stranded polynucleotides, to a first restriction enzyme(s), and exposing a second double stranded polynucleotide, or a second pool of related double stranded polynucleotides, to a second restriction enzyme(s), wherein the first and second restriction enzymes cleave at different recognition sequences, and wherein the polynucleotides are related, b) removing, and/or inhibiting the activity of, the restriction enzymes, c) mixing the products of step b), d) denaturing the polynucleotides,

O

e) allowing the denatured polynucleotides to form double stranded z polynucleotides, f) exposing the double stranded polynucleotides formed in step d) to a ligase.

In a particularly preferred embodiment, steps e) and f) are conducted simultaneously. In this embodiment, the ligase will preferably be a thermostable ligase.

\In another aspect, the present invention provides a library of variant polynucleotides produced by a method of the invention.

c In yet another aspect, the present invention provides a method of making a Spolypeptide having a desired property, the method comprising a) generating polynucleotide variants using a method of the invention, b) expressing the variant polynucleotides produced in step a) to produce variant polypeptides, c) screening the variant polypeptides for a desired property, and d) selecting a polypeptide having a desired property from the variant polypeptides.

Also provided is a polypeptide produced using a method of the invention.

The present invention also provides a method of making a polynucleotide having a desired property, the method comprising a) generating polynucleotide variants using a method of the invention, b) screening the variant polynucleotides for a desired property, and c) selecting a polynucleotide having a desired property from the variant polynucleotides.

Also provided is a polynucleotide produced using a method of the invention.

As will be apparent, preferred features and characteristics of one aspect of the invention are applicable to many other aspects of the invention.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS SFigure 1 provides a schematic representation of reassortment by overlapping nicked fragment ligation.

Figure 2 provides a schematic representation of reassortment by overlapping restriction fragment ligation.

¢Cc Figure 3 provides a map of parental gene variants and resulting progeny from reassortment by fragment ligation (to scale). The complete gene is represented by two parallel lines with the locations of the eight codon differences represented by vertical bars black for those sites diagnostic of parental type 1 and grey for parental type 2 (bars were omitted where sequencing was incomplete). The locations of the Nt.Alwl nicking sites on the upper and lower strands are represented by small lines above or below the gene respectively.

Figure 4 provides PCR resolution of heteroduplexes obtained in Example 1. The sample gene was reamplified using nested vector primers from 1 iL of each reannealled nicked DNA sample containing either Taq ligate T4 ligase or no ligase and amplified for 20 or 25 cycles. M Invitrogen 1 kb ladder.

DETAILED DESCRIPTION OF THE INVENTION Reassortment by Fragment Ligation using a Nicking Enzyme(s) In a first aspect of the invention relates to a method of preparing polynucleotide variants, the method comprising a) exposing a pool of two or more related polynucleotides to at least one nicking enzyme, wherein at least some of the polynucleotides are partially and/or fully double stranded, b) removing, and/or inhibiting the activity of, the at least one nicking enzyme, c) denaturing the polynucleotides, allowing the denatured polynucleotides to form at least partially double stranded polynucleotides, e) exposing the double stranded polynucleotides formed in step d) to a ligase.

An embodiment of this procedure is shown schematically in Figure 1. Two or more related double-stranded polynucleotides of interest are generated randomly by methods such as error-prone PCR and/or using mutator strains of E. coli. Alternatively, the two or more related polynucleotides can be produced by rational design such as site-directed mutagenesis techniques. Preferably the polynucleotides are reduced to z PCR amplicons or restriction fragments so as to eliminate, or minimise, extraneous polynucleotides such as vector sequence.

The two or more related polynucleotides are pooled, either in equimolar amounts or in a ratio designed to bias the representation of particular mutations among \the reassorted products. The pool is digested with a nicking enzyme that introduces a nick in one strand of at least some of the polynucleotides in the reaction. The nicking enzyme should nick the polynucleotide at least once on each strand, but ideally a r" number of times. If necessary, two or more nicking enzymes could be used.

A thermostable ligase is added and the reaction mix is heated at 95 0 C for 30 sec to denature the polynucleotides. This heating step also inactivates the nicking enzyme(s). Single strands are allowed to reanneal at 70C and the temperature slowly ramped down to 40 0 C over several hours, during which time both homo- and heteroduplexes will form and nicks will be repaired by the ligase.

PCR amplification of the full-length polynucleotides is then carried out on the ligated reaction in order to resolve heteroduplexes (namely, ligation products containing mismatches due to opposing strands being derived from different parental mutants). If not already incorporated into the variants, the PCR amplification also affords the opportunity to insert restriction sites into the primers to enable subsequent cloning of the reassorted variant products.

Steps c) to e) can be repeated at least once. More specifically, following a single round of these steps it is possible that at least some partially double stranded polynucleotides may be present in the reaction. Thus, repeating steps c) to e) at least once will typically increase the amount of full length double stranded variants that are produced.

In another embodiment, steps a) to e) are repeated at least once. This procedure will typically result in an increase in the number of different types of variants. In this embodiment, it is preferred that the least one nicking enzyme in step a) is different when this step is repeated.

In a preferred embodiment, all of the related polynucleotides used in step a) are fully double stranded.

Step b) can be performed using any technique known in the art. Typically, the nicking enzyme is inactivated by exposing the enzyme to a sufficiently high temperature. For example, many nicking enzymes will be inactivated by heating the reaction mixture to a temperature of about 90 0 C to about 105 0

C.

8 Furthermore, step c) can be performed using any technique known in the art.

z Typically, this step will comprise heating the polynucleotides to a suitable temperature to form single stranded polynucleotides. In a preferred embodiment, step c) comprises exposing the polynucleotides to a temperature of about 90 0 C to about 105 0 C. Other methods which may be used to denature the nicked polynucleotides generated by step a) include pressure and pH.

SIn a preferred embodiment, steps b) and c) are conducted simultaneously by C exposing the at least one nicking enzyme and polynucleotides to a temperature of about 0 C to about 105°C.

Step d) can also be performed using any technique known in the art. Typically, this step will comprise cooling (following denaturing the polynucleotide by heating) the denatured polynucleotides to allow heteroduplexes to be formed. In a preferred embodiment, step d) comprises exposing the denatured polynucleotides to a temperature of about 75 0 C, and allowing the temperature to cool to about 40 0

C.

The present invention relies on the formation of heteroduplexes by ligating adjacent ends of hybridized fragments. To ensure correct fragment reassembly the annealing step (step should be performed at a sufficient temperature, for example, between about 40 0 C to about 75 0 C. Under these conditions small fragments may not be able hydridize to enable the formation of full length double stranded variants. Thus, in a preferred embodiment, none of the single stranded fragments produced following step c) are less than about 40 nucleotides in length.

In a preferred embodiment, steps d) and e) are conducted simultaneously. In this embodiment, the ligase will preferably be a thermostable ligase.

Full length variant heteroduplexes can be resolved using a number of techniques. In a preferred embodiment, a polynucleotide amplification procedure is performed on the double stranded polynucleotides produced by step Preferably, the amplification is performed using oligonucleotide primers that hybridize to the ends of the two or more polynucleotides.

With regard to this aspect of the invention, the procedure may result in the reformation of parental polynucleotides used in step Repeating steps a) to e) as described above will reduce the number of, or eliminate, such molecules. Nonetheless, variant polynucleotides will still be produced which can be screened for a desired activity as described herein.

In some instances it may be necessary to synthesise at least some of the related polynucleotides in order to incorporate mutations that result in recognition sequences for nicking enzymes. Thus, in an embodiment, at least one polynucleotide in the pool O9

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was produced by introducing a recognition sequence for at least one of the nicking C enzymes into a first polynucleotide to produce a second (related) polynucleotide.

Preferably, the first polynucleotide encodes a polypeptide, and the introduced recognition sequence does not alter the amino acid sequence of the encoded polypeptide.

O Any nicking enzyme known can be used in the method of the invention.

Examples include, but are not limited to, a nicking enzyme selected from the group Sconsisting of: Nt.Alwl, Nb.BbvCI, Nt.BbvCI, Nb.BpulOl, Nb.Bsml, Nt.Bst9l, Nt.BstNBI, Nb.BsrDI, and any combination thereof.

In a preferred embodiment the polynucleotides are DNA or RNA. More c preferably the polynucleotide is DNA.

In a further embodiment, the method further comprises screening the variant polynucleotide(s) obtained for a desired activity (property). As an example, the method comprises producing a polypeptide(s) encoded by the variant polynucleotide(s) and screening the polypeptide(s) for the desired activity (property).

In a further preferred embodiment, the variant polynucleotide(s) are cloned into an expression vector(s). The expression vector can be used to express the polynucleotide in a host cell or a cell-free expression system.

In a further embodiment, the expression vector(s) are introduced into a host cell(s).

Preferably, the method is performed in vitro.

Numerous DNA shuffling techniques require the use of a polymerase to "fill in" gaps between two fragments hybridized to, for example, a template polynucleotide. An advantage of the present invention is that such a polymerization step is not essential to the working of the invention. In fact, in a particularly preferred embodiment the method does not comprise the use of a polymerase to fill in any single stranded gaps of the polynucleotide produced using the method.

Other DNA shuffling techniques require the use of enzymes, such as the Flap endonuclease to trim overhanging flaps of any partially hybridized fragments. A further advantage of the present invention is that such a trimming step is not essential to the working of the invention. In fact, in a particularly preferred embodiment the method does not comprise trimming any overhanging flaps of any partially hybridized fragments.

Furthermore, some DNA shuffling techniques require the addition of a "template" or "scaffold" nucleic acid, typically the full length wild type nucleic acid. A further advantage of the present invention is that such templates and/or scaffolds are not required in the present invention as the products of the step a) are of suitable length z to enable a full length molecule to be constructed. Thus, in a particularly preferred embodiment the method does not comprise the addition of a template and/or scaffold nucleic acid beyond the polynucleotides used as a substrate for cleavage in step a).

In addition, some DNA shuffling techniques require the addition of exogenous Soligonucleotides to assist in the formation of full length variant polynucleotides. A In further advantage of the present invention is that such exogenous oligonucleotides are c not required in the present invention as the products of the step a) are sufficient to enable a full length molecule to be constructed. Thus, in a particularly preferred embodiment the method does not comprise the addition of exogenous 1polynucleotides/oligonucleotides.

Yet further DNA shuffling techniques require an exonuclease to digest at least one strand of a template DNA. A further advantage of the present invention is that such exonucleases are not required. Thus, in a particularly preferred embodiment the method does not comprise the addition of an exonuclease.

Some nicking enzymes are capable of nicking single stranded polynucleotides.

Thus, a variation of the above procedure is a method of preparing polynucleotide variants, the method comprising a) exposing two or more related single stranded polynucleotides to at least one nicking enzyme capable of cleaving a single stranded polynucleotide, b) removing, or inhibiting the activity of, the at least one nicking enzyme, c) allowing the polynucleotides to form at least partially double stranded polynucleotides, d) exposing the double stranded polynucleotides formed in step c) to a ligase.

In this instance, when conducting steps a) to d) for the first time there is no need to denature the polynucleotides.

In one embodiment, step a) comprises exposing at least a first single stranded polynucleotide, or a first pool of related single stranded polynucleotides, to a nicking enzyme(s), and exposing a second single stranded polynucleotide, or a second pool of related single stranded polynucleotides, to a nicking enzyme(s) and mixing the two polynucleotides, pools or combinations thereof before, after or during step The nicking enzyme(s) used to cleave each polynucleotide and/or pool can be the same or different. With regard to this embodiment, it is preferred that the first single stranded polynucleotide or the first pool of related single stranded polynucleotides comprise the "sense" strand(s) and the second single stranded polynucleotide or the second pool of related single stranded polynucleotides comprise the antisense strands, thus preventing 11 the formation of double stranded polynucleotides whilst the nicking reactions are being z performed.

In another embodiment, the two or more related single stranded polynucleotides are in a single pool which comprises sense and antisense strands of the polynucleotide.

In this embodiment, step a) is conducted under conditions, such as at 95'C, so that the two strands do not anneal before the nicking enzyme has cleaved the polynucleotides.

'n In accordance with embodiments of the first aspect, an embodiment in the above Cc aspect is that steps a) to d) are repeated at least once. Furthermore, in an embodiment at least one nicking enzyme in step a) is different when this step is repeated. In another embodiment, the method further comprises e) denaturing the polynucleotides produced ri in step d) and repeating steps c) and In this embodiment, step e) preferably comprises exposing the polynucleotides to a temperature of about 90'C to about 105'C.

In another embodiment, step c) comprises exposing the denatured polynucleotides to a temperature of about 75 0 C, and allowing the temperature to cool to about 40 0 C. In yet another embodiment, none of the single stranded fragments produced following step a) are less than about 15, more preferably less than about 30, and even more preferably less than about 40 nucleotides in length. In a further embodiment, steps c) and d) are conducted simultaneously. In yet a further embodiment, the method further comprises conducting a polynucleotide amplification procedure on the double stranded polynucleotides produced by step d).

Following, or in conjunction with, step b) the polynucleotides may be exposed to a denaturing step to ensure that any polynucleotides have not annealed, either to another polynucleotide or themselves (for example to form hair-pin structures etc).

An advantage of the present method when compared to at least some other shuffling procedures is that because fragmentation is achieved by complete digestion of the template by the nicking enzymes, virtually no optimisation of conditions are required and certainly no repeated optimization each time a shuffle is carried out.

Another advantage of the present method when compared to at least some other shuffling procedures is that because the method is ligase rather than polymerase based there is the potential for more per gene recombination events (each ligation event or replication fork is a potential recombination event, to ligate a whole gene back together requires several ligation events, but a whole gene can be recovered with as few as one replication fork) therefore fewer shuffles are required in order to explore the entire combinatorial space using our method.

Another advantage of the present method when compared to at least some other shuffling procedures is that by selecting different nicking enzymes or combinations thereof, one can readily control the rate of recombination per round of shuffling if 0 desired.

Another advantage of the present method when compared to at least some other shuffling procedures is that the rate of recombination in specific regions of the gene can be controlled by selecting nicking enzymes that target those regions.

~Another advantage of the present method when compared to at least some other t shuffling procedures is that because the critical steps of the process do not rely upon a polymerase, theoretically much larger genes can be efficiently shuffled.

Another advantage of the present method when compared to at least some other shuffling procedures is that because the method requires very little PCR (theoretically a c1 single cycle of replication can resolve the heteroduplexes) there is much less chance for extraneous mutations to enter the process which may sometimes be desired.

Another advantage of the present method when compared to at least some other shuffling procedures is that the process entails only a couple of critical steps, each in themselves requiring very little optimisation. Thus the method is simple to implement and repeat. In contrast, more-or-less all other methods where several critical steps are necessary and some of these steps need optimization either once or every time the process is repeated. For example the PCR based methods require the fragmentation of the mutant pool by mechanical, enzymatic, chemical or photochemical means, which requires optimisation each and every time. The optimal PCR condition to reamplify the target from the complex pool then needs to be determined. Finally the process generally requires repeating several times. Furthermore, when compared to other ligation based methods, first the scaffold needs to be prepared and steps taken to ensure that the scaffold does not contribute significantly to the final product (for example, synthesis of dTUP containing scaffolds) and later, the complementary process of removing the scaffold needs to be undertaken. Meanwhile the assembly process requires three distinct enzymatic steps; exonuclease, polymerase and ligase, each needs to be implemented in the appropriate order using three different enzymes in order to work.

Reassortment by Fragment Ligation using Restriction Enzymes In a further aspect, the present invention provides a method of preparing polynucleotide variants, the method comprising a) exposing at least a first double stranded polynucleotide, or a first pool of related double stranded polynucleotides, to a first restriction enzyme(s), and exposing a second double stranded polynucleotide, or a second pool of related double stranded polynucleotides, to a second restriction enzyme(s), wherein the first and second z restriction enzymes cleave at different recognition sequences, and wherein the polynucleotides are related, b) removing, and/or inhibiting the activity of, the restriction enzymes, c) mixing the products of step b), O d) denaturing the polynucleotides, t e) allowing the denatured polynucleotides to form double stranded C polynucleotides, f) exposing the double stranded polynucleotides formed in step d) to a ligase.

An embodiment of this aspect is of the invention is shown schematically in c NI Figure 2. Two or more related polynucleotides of interest are generated randomly by methods such as error-prone PCR or using mutator strains of E. coli. Alternatively, the two or more related polynucleotides can be produced by rational design such as sitedirected mutagenesis techniques. Preferably the polynucleotides are reduced to PCR amplicons or restriction fragments so as to eliminate, or minimise, extraneous polynucleotides such as vector sequence.

The two or more related polynucleotides are pooled, either in equimolar amounts or in a ratio designed to bias the representation of particular mutations among the reassorted products. The pooled polynucleotides are divided into two sub-pools and digested with different restriction enzymes. Each restriction enzyme should cut the polynucleotides at least once, but ideally a number of times. If necessary, more than one restriction enzyme may be used in each pool.

The restriction enzyme(s) are then removed and/or inactivated and the pools placed back into a single pool. A thermostable ligase is added and the reaction mix is heated at 95 0 C for 30 sec to denature the polynucleotides. Single strands are allowed to reanneal at 70 0 C and the temperature slowly ramped down to 40 0 C over several hours, during which time both homo- and heteroduplexes will form and nicks will be repaired by the ligase.

The over-hangs generated by restriction enzyme cleavage do not allow the formation of stable complexes at- these high temperatures. The--heating/cooling steps are repeated several times, incrementally lowering the annealing temperature to 40 0

C,

so that complete complementary fragments (namely, products of the same restriction enzyme digest) that have annealed (unproductive unions) will be re-cycled into productive unions as described above. In this scenario, ligated restriction products themselves become templates for further productive unions in subsequent cycles.

14 PCR amplification of the full-length polynucleotides is then carried out on the z ligated reaction in order to resolve heteroduplexes (namely, ligation products containing mismatches due to opposing strands being derived from different parental mutants). If not already incorporated into the variants, the PCR amplification also affords the opportunity to insert restriction sites into the primers to enable subsequent cloning of the reassorted variant products.

t~n Step a) may comprise additional pools of related polynucleotides cleaved with Cr yet a different restriction enzyme(s). In one embodiment, the pools of related polynucleotides are identical. In another embodiment, whilst the polynucleotides in the S 10 pools are related, different pools have different populations of related polynucleotides.

C1 The populations may differ in the actual primary sequence of the polynucleotides and/or the relative proportion of each different member may vary.

In one embodiment, the first pool and second pool of polynucleotides are identical.

Preferably, the ligase is a thermostable ligase.

Steps d) to f) can be repeated at least once. More specifically, following a single round of these steps it is possible that at least some partially double stranded polynucleotides may be present in the reaction. Thus, repeating steps d) to f) at least once will typically increase the amount of full length double stranded variants that are produced.

In another embodiment, steps a) to f) are repeated at least once. This procedure will typically result in an increase in the number of different types of variants. In this embodiment, it is preferred that different restriction enzymes, or different mixtures of restrictions enzymes are used in step a) when this step is repeated.

Step b) can be performed using any technique known in the art. Typically, the restriction enzyme is inactivated by exposing the enzyme to a sufficiently high temperature. For example, many restriction enzymes will be inactivated by heating the reaction mixture to a temperature of about 90°C to about 105 0

C.

Steps b) and or steps b) to can be performed simultaneously.

Furthermore, step d) can be performed using any technique known in the art.

Typically, this step will comprise heating the polynucleotides to a suitable temperature to form single stranded polynucleotides. In a preferred embodiment, step d) comprises exposing the polynucleotides to a temperature of about 90°C to about 105 0 C. Other methods which may be used to denature the polynucleotide fragments include pressure and pH.

Step e) can be performed using any technique known in the art. Typically, this z step will comprise cooling the denatured polynucleotides to allow heteroduplexes to be formed. In a preferred embodiment, step e) comprises exposing the denatured polynucleotides to a temperature of about 75 0 C, and allowing the temperature to cool to about 40 0

C.

This aspect of the present invention also relies on the formation of l heteroduplexes by ligating adjacent ends of hybridized fragments. To ensure correct C fragment reassembly the annealing step (step should be performed at a sufficient temperature, for example, between about 40°C to about 75 0 C. Under these conditions small fragments may not be able hybridize to enable the formation of full length double stranded variants. Further, as outlined above, these conditions are not suitable for any re-formed sticky overhangs to be a substrate for the re-establishment of an already cleaved restriction enzyme recognition site. Thus, in a preferred embodiment, none of the single stranded fragments produced following step d) are less than about 15, more preferably less than about 30, and even more preferably less than about 40 nucleotides in length.

In a preferred embodiment, steps e) and f) are conducted simultaneously. In this embodiment, the ligase will preferably be a thermostable ligase.

Full length variant heteroduplexes can be resolved using a number of techniques. In a preferred embodiment, a polynucleotide amplification procedure is performed on the double stranded polynucleotides produced by step Preferably, the amplification is performed using oligonucleotide primers that hybridize to the ends of the two or more polynucleotides used in step a).

In a preferred embodiment the polynucleotides are DNA or RNA. More preferably the polynucleotide is DNA.

In a further embodiment, the method further comprises screening the variant polynucleotide(s) obtained for a desired activity (property). As an example, the method comprises producing a polypeptide(s) encoded by the variant polynucleotide(s) and screening the polypeptide(s) for the desired activity (property).

In a further preferred embodiment, the variant polynucleotide(s) are cloned into an expression vector(s). The expression vector can be used to express the polynucleotide in a host cell or a cell-free expression system.

In a further embodiment, the expression vector(s) are introduced into a host cell(s).

Preferably, the method is performed in vitro.

16 As outlined above, numerous DNA shuffling techniques require the use of a z polymerase to "fill in" gaps between two fragments hybridized to, for example, a template polynucleotide. An advantage of this aspect of the present invention is that such a polymerization step is not essential to the working of the invention. In fact, in a particularly preferred embodiment the method does not comprise the use of a polymerase to fill in any single stranded gaps of the polynucleotide produced using the t method.

C€ Other DNA shuffling techniques require the use of enzymes, such as the Flap endonuclease to trim overhanging flaps of any partially hybridized fragments. A further advantage of this aspect of the present invention is that such a trimming step is "1 not essential to the working of the invention. In fact, in a particularly preferred embodiment the method does not comprise trimming any overhanging flaps of any partially hybridized fragments.

Furthermore, some DNA shuffling techniques require the addition of a "template" or "scaffold" nucleic acid, typically the full length wild type nucleic acid. A further advantage of this aspect of the present invention is that such templates and/or scaffolds are not required in the present invention as the products of the step a) are of suitable length to enable a full length molecule to be constructed. Thus, in a particularly preferred embodiment the method does not comprise the addition of a template and/or scaffold nucleic acid beyond the polynucleotides as a substrate for cleavage in step a).

In addition, some DNA shuffling techniques require the addition of exogenous oligonucleotides to assist in the formation of full length variant polynucleotides. A further advantage of this aspect of the present invention is that such exogenous oligonucleotides are not required in the present invention as the products of the step a) are sufficient to enable a full length molecule to be constructed. Thus, in a particularly preferred embodiment the method does not comprise the addition of exogenous polynucleotides/oligonucleotides.

Yet further DNA shuffling techniques require an exonuclease to digest one strand of a template DNA. A further advantage of this aspect of the present invention is that such exonucleases are not required. Thus, in a particularly preferred embodiment, the method does not comprise the addition of an exonuclease.

An advantage of the present method when compared to at least some other shuffling procedures is that because fragmentation is achieved by complete digestion of the template by the restriction enzymes, virtually no optimisation of conditions are required and certainly no repeated optimization each time a shuffle is carried out.

17 Another advantage of the present method when compared to at least some other 0 shuffling procedures is that because the method is ligase rather than polymerase based there is the potential for more per gene recombination events (each ligation event or replication fork is a potential recombination event, to ligate a whole gene back together requires several ligation events, but a whole gene can be recovered with as few as one replication fork) therefore fewer shuffles are required in order to explore the entire t combinatorial space using our method.

Cc Another advantage of the present method when compared to at least some other shuffling procedures is that by selecting different restriction enzymes or combinations thereof, one can readily control the rate of recombination per round of shuffling if desired.

Another advantage of the present method when compared to at least some other shuffling procedures is that the rate of recombination in specific regions of the gene can be controlled by selecting restriction enzymes that target those regions.

Another advantage of the present method when compared to at least some other shuffling procedures is that because the critical steps of the process do not rely upon a polymerase, theoretically much larger genes can be efficiently shuffled.

Another advantage of the present method when compared to at least some other shuffling procedures is that because the method requires very little PCR (theoretically a single cycle of replication can resolve the heteroduplexes) there is much less chance for extraneous mutations to enter the process which may sometimes be desired.

Another advantage of the present method when compared to at least some other shuffling procedures is that the process entails only a couple of critical steps, each in themselves requiring very little optimisation. Thus the method is simple to implement and repeat. In contrast, more-or-less all other methods where several critical steps are necessary and some of these steps need optimization either once or every time the process is repeated. For example the PCR based methods require the fragmentation of the mutant pool by mechanical, enzymatic, chemical or photochemical means, which requires optimisation each and every time. The optimal PCR condition to reamplify the target from the complex pool then needs to be-determined. Finally the process generally requires repeating several times. Furthermore, when compared to other ligation based methods, first the scaffold needs to be prepared and steps taken to ensure that the scaffold does not contribute significantly to the final product (for example, synthesis of dTUP containing scaffolds) and later, the complementary process of removing the scaffold needs to be undertaken. Meanwhile the assembly process requires three distinct enzymatic steps; exonuclease, polymerase and ligase, each needs >to be implemented in the appropriate order using three different enzymes in order to 0 work.

General Techniques and Definitions Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art in cell culture, molecular genetics, immunology, Simmunohistochemistry, protein chemistry, and biochemistry).

lUnless otherwise indicated, the recombinant protein, cell culture, and 010 immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley Sons (including all updates until present).

As used herein, a "nicking enzyme" cleaves a single strand of a polynucleotide at a defined recognition site (sequence). These enzymes may also be called a single strand cutting restriction enzyme (SSCREase) or a nickase. The term "nick" in a double-stranded polynucleotide refers to the absence of a phosphodiester bond between two adjacent nucleotides on one strand. Nicking enzymes have non-palindromic recognition sequences and can occur on either strand of a double stranded polynucleotide. Examples of nicking enzymes that cleave double stranded DNA include; but are not limited to, Nt.AlwI, Nb.BbvCI, Nt.BbvCl, Nb.Bpul01, Nb.Bsml, Nt.Bst9l, Nt.BstNBI and Nb.BsrDI. Examples of nicking enzymes that cleave single stranded DNA include, but are not limited to, AccI, AcclI, Avail, BspRI, CfoI, Ddel, Haell, HapII, Hhal, Hinfl, MboI, Mboll, Mspl, Sau3AI, SfaI and TthHB8I. Further examples of nicking enzymes, and their recognition sites, can be found on the rebase database (rebase.neb.com) (Roberts et al., 2005) As used herein, the term "restriction enzyme" refers to an enzyme which cleaves 0 Z a double stranded polynucleotide (or the double stranded portion of a partially double stranded polynucleotide) by recognizing specific sequences internal to the molecule and subsequently cutting the polynucleotide in both strands at sites either within or outside of the recognition site (sequence). Alternate names used in the art for \restriction enzymes include restriction endonucleases. Restriction enzymes occur naturally in bacteria. When they are purified away from other contaminating bacterial C components, restriction enzymes can be used to break polynucleotide molecules into precise fragments. Restriction enzymes act by recognizing and binding to particular sequences of nucleotides (the "recognition sequence", "recognition site" or "restriction site") along the polynucleotide molecule. Once bound, they cleave the molecule within, or to one side of, the sequence. Different restriction enzymes have affinity for different recognition sequences. Restriction enzymes with symmetrical recognition sequences generally cleave symmetrically within or adjacent to the recognition site, while those that recognize asymmetric sequences tend to cleave at a distance of from 1 to 18 nucleotides away from the recognition site. Over two hundred unique restriction endonucleases have been identified among several thousands of bacterial species that have been examined to date (see for example, Aggarwal (1995); Roberts et al. (2005), as well as the rebase database (rebase.neb.com)). The term "restriction site" refers to a recognition sequence that is necessary for the manifestation of the action of a restriction enzyme, and includes a site of catalytic cleavage. It will be appreciated that a site of cleavage may or may not be contained within a portion of a restriction site that comprises a low ambiguity sequence (namely, a sequence containing the principal determinant of the frequency of occurrence of the restriction site). Thus, in many cases, relevant restriction sites contain only a low ambiguity sequence with an internal cleavage site G/AATTC in the EcoRI site) or an immediately adjacent cleavage site /CCWGG in the EcoRII site). In other cases, relevant restriction enzymes contain a low ambiguity sequence the CTGAAG sequence in the Eco571 site) with an external cleavage site in the N16 portion of the Eco57I site).

When a restriction enzyme or nicking enzyme is said to "cleave" a polynucleotide, it is understood to mean that the enzyme catalyzes or facilitates a cleavage of a polynucleotide.

As used herein, "ligating" or "ligation" refers to covalently attaching polynucleotide sequences together to form a single continuous polynucleotide. This is performed in the methods of the invention by treatment with a "ligase" which catalyzes the formation of a phosphodiester bond between the 5' end of one sequence and the 3' 0 2 end of the other. The ligase catalyses the formation of a phosphodiester bond at the site 0 of a single-stranded break in a duplex polynucleotide. As used herein, the term "thermostable ligase" refers to a ligase which maintains enzymatic activity at high temperatures at least above 40 0 C, at least above 75 0 C, and/or at least above 90 0

C.

Thermostable ligases useful for the invention include, but are not limited to, Tth DNA O ligase, Pfu DNA ligase (Stratagene, Cat 600191), Taq DNA ligase (New England

J

Biolabs, Cat# M02085) Thermus filiformis ligase, marinus DNA ligase, Thermus C scotoductus DNA ligase and Bacillus stearothermophilus DNA ligase.

7- As used herein, the terms "adjacent ends" or "adjacent fragments" refers to hybridized fragments whose ends are flush against each other and separated only by nicks, not by gaps, and thus being a target for ligase activity.

As used herein, the term "related polynucleotides" refers to two or more polynucleotides which share a region of primary sequence identity. In particular, regions of identity should be sufficient to support annealing of polynucleotides such that stable heteroduplexes can be formed. There should be at least sufficient diversity between the related polynucleotides such that reassortment can generate more diverse products than there are starting materials. Thus, there must be at least two polynucleotides differing in at least two positions. The degree of diversity depends on the length of the polynucleotide being reassorted and the extent of the functional change to be evolved. Diversity at between 0.1 -25% of positions is typical.

Reassortment of mutations from very closely related genes or even whole sections of sequences from more distantly related genes or sets of genes can enhance the rate of evolution and the acquisition of desirable new properties. Reassortment to create chimeric or mosaic genes can be useful in order to combine desirable features of two or more parents into a single gene or set of genes, or to create novel functional features not found in the parents. In one embodiment, the polynucleotides to be reassorted are at least 80% identical, more preferably 90% identical, more preferably 95% identical, and even more preferably at least 99% identical, to each other. In another embodiment, one strand of a first related polynucleotide is capable of hybridizing to the opposite strand of a second related polynucleotide under stringent conditions.

The present invention provides methods for preparing "polynucleotide variants".

In this context, this term refers to at least some of the polynucleotide products of the invention differing in primary nucleotide sequence when compared to any of the "related polynucleotides" used at the start of the procedures.

>As used herein, the term "hybridizes" refers to the ability of two single stranded 0 z nucleic acid molecules being able to form at least a partially double stranded nucleic acid through hydrogen bonding.

The term "wild-type" means that the polynucleotide does not comprise any mutations when compared to that found in nature, or polynucleotide derived therefrom Ssuch as a cDNA. Pools or populations to be reassorted using a method of the invention may include wild-type polynucleotides as well as mutants/variants/derivatives thereof.

SA mutant or variant polynucleotide has a sequence which varies from a wild type or reference sequence at one or more positions.

As used herein, the term "heteroduplex" refers to a hybrid (or chimeric) polynucleotide generated by base pairing between complementary single strands derived from the different parental molecules.

As used herein, the term "polynucleotide amplification" refers to any in vitro method for increasing the number of copies of a nucleic acid molecule with the use of a polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a polynucleotide molecule or primer thereby forming a new polynucleotide molecule complementary to a polynucleotide template. The newly formed polynucleotide molecule can be used a template to synthesize additional polynucleotide molecules.

"Operably linked" as used herein refers to a functional relationship between two or more nucleic acid DNA) segments. Typically, it refers to the functional relationship of transcriptional regulatory element (promoter) to a transcribed sequence.

For example, a promoter is operably linked to a coding sequence, such as a variant polynucleotide produced using a method of the invention, if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell. Generally, promoter transcriptional regulatory elements that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, they are cis-acting.

However, some transcriptional regulatory elements, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

As used herein, the term "gene" is to be taken in its broadest context and includes the deoxyribonucleotide sequences comprising the protein coding region of a structural gene and including sequences located adjacent to the coding region on both the 5' and 3' ends for a distance of at least about 2 kb on either end. The sequences which are located 5' of the coding region and which are present on the mRNA are referred to as 5' non-translated sequences. The sequences which are located 3' or downstream of the coding region and which are present on the mRNA are referred to as non-translated sequences. The term "gene" encompasses both cDNA and genomic 0 z forms of a gene. A genomic form or clone of a gene contains the coding region which may be interrupted with non-coding sequences termed "introns" or "intervening regions" or "intervening sequences." Introns are segments of a gene which are transcribed into nuclear RNA; introns may contain regulatory elements such as \enhancers. Introns are removed or "spliced out" from the nuclear or primary transcript; introns therefore are absent in the messenger RNA (mRNA) transcript. The mRNA

C

functions during translation to specify the sequence or order of amino acids in a nascent Spolypeptide.

As used herein, "in vitro" refers to any location outside a living organism.

The terms "protein" and "polypeptide" are used herein interchangeably.

Polynucleotides The terms "polynucleotide" and "nucleic acid" are used herein interchangeably and refer to a polymer of nucleotides. The polymer may include natural nucleosides adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), nucleoside analogs 2aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2thiocytidine), chemically modified bases, biologically modified bases methylated bases), intercalated bases, modified sugars 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), or modified phosphate groups phosphorothioates and N-phosphoramidite linkages). In a preferred embodiment, the related polynucleotides are DNA or RNA or a combination thereof. In a particularly preferred embodiment, the related polynucleotides are DNA.

An oligonucleotide is typically known in the art as a relatively short polynucleotide, such as a polynucleotide that is less than about 40 nucleotides in length.

The identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, the query sequence is at least nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 z nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length. With respect to the present invention, the two sequences are related polynucleotides.

The polynucleotides for use in the invention can vary in length from about S250, 1000, 10,000, 100,000, 10 6 or more bases.

As used herein, the phrase "stringent conditions" refers to conditions under which related polynucleotides will hybridize. Stringent conditions are sequence- Sdependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures than shorter sequences. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific related sequences at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which of the related polynucleotides will hybridize to each other at equilibrium.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion (or other salts) at pH to 8.3 and the temperature is at least about 60°C for polynucleotides greater than residues in length. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

Stringent conditions are known to those skilled in the art and can be found in Ausubel et al. (supra), Current Protocols In Molecular Biology, John Wiley Sons, N.Y. (1989), 6.3.1-6.3.6, as well as the Examples described herein. Preferably, the conditions are such that related polynucleotides at least about 80%, 90%, 95%, 98%, or 99% identical to each other typically remain hybridized to each other. A non-limiting example of stringent hybridization conditions are hybridization in a high salt buffer comprising 6xSSC, 50 mM Tris-HCI (pH 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 mg/ml denatured salmon sperm DNA at 65 0 C, followed by one or more washes in 0.2.xSSC, 0.01% BSA at 50 0

C.

Exemplary conditions for denaturation and renaturation of double stranded polynucleotides are as follows. Equimolar concentrations (1.0 5.0 nM) of the polynucleotides are mixed in 1 x SSPE buffer (180 mM NaCI 1.0 mM EDTA, 10 mM NaH 2

PO

4 pH The mixture is then incubated at 68 0 C for 2-6 hr. Denaturation and reannealing can also be carried out by the addition and removal of a denaturant such as NaOH. The process is the same for single stranded DNA substrates, except that the denaturing step may be omitted.

24 The annealing of polynucleotide strands can be accelerated by techniques 0 known in the art such as the addition of polyethylene glycol (PEG) or salt. The salt concentration is preferably from 0 mM to 200 mM, more preferably the salt concentration is from 10 mM to 100 mM. The salt may be KCI or NaCI. The concentration of PEG is preferably from 0% to 20%, more preferably from 5% to u A pool of related polynucleotides may possess, when compared to naturally t occurring (wild-type) molecules, one or more mutations which are deletions, insertions, CM or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source and be alleles of the same gene of one species of organism and/or be orthologous genes from different species etc) or synthetic (for example, obtained by performing site-directed mutagenesis on the nucleic acid).

Mutations can be introduced in a starting polynucleotide (such as a wild-type molecule) to produce two or more related polynucleotides. Such mutants can be produced by, but not limited to, oligonucleotide-directed mutagenesis, error-prone PCR, random chemical mutagenesis, in vivo random mutagenesis, or by combining genes from gene families within the same or different species. Error-prone PCR uses low-fidelity polymerization conditions to introduce a low level of point mutations randomly over a long sequence. In oligonucleotide-directed mutagenesis, a short sequence of the polynucleotide is removed from the polynucleotide using restriction enzyme digestion and is replaced with a synthetic polynucleotide in which various bases have been altered from the original sequence. Chemical mutagens include, for example, sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.

Other agents which are analogues of nucleotide precursors include nitrosoguanidine, bromouracil, 2-aminopurine. or acridine. Generally, these agents are added to a PCR reaction in place of the nucleotide precursor thereby mutating the sequence.

Intercalating agents such as proflavine, acriflavine, quinacrine and the like can also be used. Random mutagenesis of the polynucleotide sequence can also be achieved by irradiation with X-rays or ultraviolet light.

In some instances it may be desirable to introduce mutations into at least some of the related polynucleotides before a method of the invention is preformed to incorporate recognition sites for the nicking or restriction enzyme that is to be used in the method. Such mutations can be introduced using any technique known in the art such as site directed mutagenesis.

The number of different related polynucleotides to be reassorted can vary widely in size from two to 10, 100, 1000, to more than 105 members.

0

O

Expression Vectors and Host Cells Z Typically, variant polynucleotides produced using a method of the invention will be screened for a desired activity. To achieve this end, in a preferred embodiment individual variants are individually cloned into an expression vector. An expression vector for use on the invention comprises a variant polynucleotide operably linked to a O suitable expression regulatory element(s). The phrase operably linked refers to Sinsertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids.

Expression vectors of the present invention include any vectors that function direct gene expression) in recombinant cells including in bacterial, fungal, endoparasite, arthropod, animal, and plant cells. Vectors of the invention can also be used to express the variant polynucleotide in a cell-free expression system, such systems are well known in the art.

In particular, expression vectors contain regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant (host) cell and that control the expression of polynucleotide molecules. In particular, expression vectors include transcription control sequences. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription.

Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences.

Suitable transcription control sequences include any transcription control sequence that can function in at least one type of host cell or cell-free expression system. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, arthropod and mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha-mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription 26 0 control sequences as well as other sequences capable of controlling gene expression in 0 z prokaryotic or eukaryotic cells.

The vector may include further polynucleotide sequences so that the protein encoded for by a variant polynucleotide is produced as a fusion and/or nucleic acid encoding secretion signals so that the protein produced in the host cell is secreted from a cell.

As used herein, "host cell" refers to a cell that comprises a recombinant polynucleotide molecule, typically a recombinant plasmid or other expression vector.

Thus, for example, host cells can express genes that are not found within the native (non-recombinant) form of the cell. The host cell may be prokaryotic or eukaryotic, including bacterial, mammalian, yeast, aspergillus, and insect cells.

The vectors containing a variant polynucleotide of the invention can be transferred into the host cell by standard methods, depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells. Lipofection, or electroporation may be used for other cellular hosts.

Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, liposomes, electroporation, and microinjection, and biolisitics (see, generally, Sambrook et al., supra). Viral vectors can also be packaged in vitro and introduced by infection. The choice of vector depends on the host cells. In general, a suitable vector has an origin of replication recognized in the desired host cell, a selection maker capable of being expressed in the intended host cells.

A polypeptide encoded by a variant polynucleotide may be expressed by methods well known in the art. Conveniently, expression may be achieved by growing a host cell in culture, containing such a vector, under appropriate conditions which cause or allow expression of the polypeptide.

Polynucleotide Amplification As noted above, "amplification" refers to any in vitro method for synthesizing one or both strands of a polynucleotide template sequence a target DNA molecule) with-the use ofa polymerase. Polynucleotide amplification results in the-incorporation of nucleotides into a polynucleotide DNA) molecule or primer thereby forming a new polynucleotide molecule complementary to the polynucleotide template. The formed polynucleotide molecule and its template can be used as templates to synthesize additional polynucleotide molecules.

Amplification reactions include, but are not limited to, the polymerase chain reaction (PCR), ligase chain reaction (LCR), polynucleotide sequence based 27

O

amplification (NASBA), Q-Beta Replicase reaction, transcription-based amplification z system (TAS), and strand displacement amplification.

As used herein, an "amplified product" or an" amplified polynucleotide product" refers to the double strand and/or single strand polynucleotide population generated during or at the end of an amplification reaction. The amplified product contains the O original polynucleotide template and polynucleotide synthesized by DNA polymerase t using the polynucleotide template during the amplification reaction.

C Performing the amplification reaction with error-prone polymerases can introduced yet further mutations into the variant polynucleotides produced using a method of the invention.

c The term "primer" is used herein especially in connection with a PCR reaction is an oligonucleotide (especially a "PCR-primer") defined and constructed according to general standard specification known in the art ("PCR A practical approach" IRL Press, (1991)).

Amplification reactions as employed in the methods of the invention may be carried out with a wide range number of amplification cycles required to produce the desired quantity of variant polynucleotides. Preferably the number of cycles in the amplification reaction step is 10-60 cycles, more preferably 20 to 40 cycles are performed, and even more preferably the number of cycles is between 25 and Compositions The polypeptides or polynucleotides generated by a method of the invention and identified as having desirable characteristics can be formulated in composition. These compositions may comprise, in addition to one of the above substances, an acceptable excipient, carrier, buffer, stabilizer or other materials well known to those skilled in the art.

In one embodiment, the composition is a pharmaceutical/veterinarial composition. For a pharmaceutical or veterinarial compositions such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

28 Physiological saline solution, dextrose or other saccharide solution or glycols such as z ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, 'n isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required.

Whether it is a polypeptide, e.g. an antibody or fragment thereof, or a polynucleotide identified following generation by the present invention that is to be given to an individual, administration is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.

Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. 1980.

Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons; for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be produced in the target cells by expression from an encoding gene introduced into the cells, e.g. in a viral vector (a variant of the VDEPT technique). The vector could be targeted to the specific cells to be treated, or it could contain regulatory elements which are switched on more or less selectively by the target cells.

Alternatively, the agent could be administered in a precursor form, for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. This type of approach is sometimes known as ADEPT or VDEPT; the former involving targeting the activating agent to the cells by conjugation to a cell- 29 specific antibody, while the latter involves producing the activating agent, e.g. an 0 enzyme, in a vector by expression from encoding DNA in a viral vector (see for example, EP-A-415731 and WO 90/07936).

A pharmaceutical/veterinarial composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

c Uses The present invention is particularly useful for evolving industrially or medically useful molecules or biochemical pathways, wherein the variant c, polynucleotide is itself a useful molecule (for example, promoter, aptamer, catalyst, enhancer, ribozyme, DNAzyme, or dsRNA for RNA interference) or wherein the variant polynucleotide encodes a useful product.

Examples of industrially or medically useful polypeptides or polynucleotides are well known in the art. Specific examples of medically useful polypeptides include, but are not limited to: insulin, erythropoietin, interferons, colony stimulating factors such as granulocyte colony stimulating factor, growth hormones such as human growth hormone, analogs, LHRH antagonists, tissue plasminogen activator, somatostatin analog, Factor IX, calcitonin, alpha, polysaccharides, AG337, bone inducing protein, bone morphogenic protein, brain derived growth factor, gastrin 17 immunogen, interleukins such as PEF superoxide, permeability increasing protein-21, platelet derived growth factor, stem cell factor, thyrotropin alfa and somatomedin C.

Examples of industrially useful molecules include enzymes that synthesize polyketides, transform small molecules, olfactory receptors for use in biosensors, hydrolyze substrates, replace steps in organic synthesis reactions or degrade pollutants such as aromatic hydrocarbons benzene, xylene, toluene and naphthalene), polychlorinated biphenyls and residual herbicides (such as glyphosate) and pesticides (such as organophosphates, pyrethroids, atrazine and carbamates). Catabolic pathways can be evolved using the present invention such that enzyme pathways are produced that degrade manmade pollutants that otherwise are not, or are only slowly catabolized by wild type organisms.

The characteristics to be altered according to the present invention include, but are not limited to, structural motif, stability, half-life, enzyme activity, enzyme specificity, binding affinity, binding specificity, toxicity, antigenicity, interaction with an organism and interaction with components of an organism of said polynucleotide or said encoded polypeptide. Structural motifs for proteins/polypeptides include, for example, alpha helices, beta sheets, solvent exposed loops, leucine zippers, scaffolds Z and the like. Structural motifs for nucleic acids/polynucleotides include, for example, quadraplex, bDNA, triple helices, stem loop, hairpin, protein binding sites and the like.

A functional characteristic can be altered according to the present invention such that the activity of said functional characteristic is enhanced at a higher or lower Stemperature compared to a reference molecule. Furthermore, said functional activities can be enhanced in various physical or chemical environments as described above.

CC Methods for determining activity under desired conditions include standard methods well known in the art. The nature of screening or selection depends on the desired property sought to be acquired. As noted above, desirable properties of enzymes include high catalytic activity, capacity to confer resistance to drugs, high stability, the ability to accept a wider (or narrower) range of substrates, or the ability to function in nonnatural environments such as organic solvents. Other desirable properties of proteins include capacity to bind a selected target, secretion capacity, capacity to generate an immune response to a given target, lack of immunogenicity and toxicity to pathogenic microorganisms. Desirable properties of DNA or RNA polynucleotides sequences include capacity to specifically bind a given protein target, and capacity to regulate expression of operably linked coding sequences. Some of the above properties, such as drug resistance, can be selected by plating cells on the drug.

Other properties, such as the influence of a regulatory sequence on expression, can be screened by detecting appearance of the expression product of a reporter gene linked to the regulatory sequence. Other properties, such as capacity of an expressed protein to be secreted, can be screened by FACS T M using a labelled antibody to the protein.

Other properties, such as immunogenicity or lack thereof, can be screened by isolating protein from individual cells or pools of cells, and analyzing the protein in vitro or in a laboratory animal. One of ordinary skill in the art can readily determine the activity of an enzyme encoded by a variant polynucleotide produced by a method of the invention and select those having the desired characteristics. Enzymes include, but are not limited to, fermenting enzymes, proteases, lipases, esterases, phosphotriesterases, oxidoreductases such as alcohol dehydrogenase, polymerases, hydrolases and luciferase.

EXAMPLES

Example 1 DNA shuffling using nicking enzymes Two variants of the same sample gene, carboxylesterases E3 (1.7 kb) (WO 95/019440), were designed and synthesized to contain eight codon differences 31 distributed along the gene's length (Figure In addition, Nt.Alwl sites were z incorporated into the gene sequence to satisfy the following criteria: i) The introduction of the site did not change the translated sequence, and ii) The nicks introduced by any two Nt.Alwl sites were no closer than 20 bp. This resulted in eight Nt.Alwl sites being located on the top strand, distributed relatively evenly along the length of the gene, and Sonly two Nt.AlwI sites being located on the lower strand close to each other (Figure 3).

These two constructs were cloned into a standard plasmid vector and the two gene variants were then PCR amplified using vector-specific primers ensuring that any Nt.Alwl sites in the flanking vector sequence also satisfied criterion ii) above. The PCR

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amplicons were purified using a commercially available PCR clean up kit and the resulting DNA was quantified spectrophotometrically.

To nick the gene variants in preparation for reassortment, approximately 220 ng of each amplicon was digested with 10 U of Nt.Alwl (NEB) in a 15 1 iL reaction volume at 37°C for 2 hr. To test that digestion was successful, 2.5 L of digested DNA and an equivalent dilution of undigested DNA were each denatured by the addition of 10 upL of formamide and heating to 85 0 C for 15 min prior to separation on a 1% agarose/TAE gel.

The reassortment was then effected by setting up three reaction tubes in parallel; into each tube was added 2 utL of both nicked gene variants and the samples were heated to 80°C for 20 min to inactivate any remaining Nt.Alwl. To each tube, 13 p.L of purified water was added, and the samples heated to 95 0 C to denature the DNA. The samples were immediately cooled to 80 0 C and then allowed to cool slowly at a rate of 0 C/min for 30 min to a temperature of 65 0 C to permit reannealing of the overlapping nicked fragments. At this point 2 gL of 10x Taq ligase buffer and 1 p.L (40 U) of Taq ligase (NEB) were added to reaction tube and then all three tubes were cooled further at the slower rate of 0.2°C/ min for 100 min to a temperature of 45 0 C. Reaction tube was maintained at 45C for a further 2 hr. To the remaining two reaction tubes, 2 g.L of 1Ox T4 ligase buffer was added, as well as 1 pL (400 U) of T4 ligase (NEB) to reaction tube #2 and 1 U.L of purified water to reaction tube Reaction tubes #2 and #3-were then both incubated at 37 0 C for 2 hr.

The contents of all three reaction tubes were then separated on a 0.7% agarose /TAE gel and a faint band of-2 kb (consistent with the full size construct) was excised from all three reactions and purified on a commercial gel purification column and eluted the DNA into 20 pL of elution buffer.

The heteroduplexes formed during the reassortment process were then resolved by using a second pair of nested vector-specific primers and a reduced number of PCR >cycles: The 50 tL reactions contained Ix PCR buffer (Invitrogen), 50 mM MgCI 2 zmM each dNTP's, 10 M each of forward and reverse primer and 1 IL of template.

Reactions were heated to 95°C for 1 min then cooled to 80°C prior to adding 2.5 U of Taq polymerase (Invitrogen). The reactions were then cycled 20 or 25 times at 95 0

C

for 30 sec, 53°C for 30 sec and 72 0 C for 30 sec. To check the efficacy of the PCR reactions, 2 pL samples were separated on a 1% agarose/TAE gel. The resulting gel (Figure 4) clearly showed an amplicon of the correct size kb) where a ligase (Taq or T4) was added to reannealled nicked DNA (reaction tubes #1 and with an increased yield from additional thermo cycles, but not where ligase was omitted from

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the reannealled nicked DNA (reaction tube The remaining PCR-resolved DNA from reactions #1 and #2 were then gel purified by running the samples out on a 0.7% agarose gel, excising the desired bands and purifying the DNA using a commercial gel clean up kit and the DNA eluted into .tL of buffer. 4ul of the purified DNA was then cloned into pCR®2.1-TOPO using the TOPO TA cloning kit (Invitrogen) as per the manufacture's instructions.

Six plasmid DNAs were purified from each of the resulting Taq ligase and T4 ligase reassorted clones; these were then sequenced from each end using the same primers used to resolve the heteroduplexes. The resulting reassorted progeny are summarized in Figure 3. These progeny showed clear evidence of reassortment, with only five clones possessing a parental combination of variable sites. Six of the remaining seven clones showed evidence of recombination occurring between the fourth and fifth variable sites. The high preponderance of this recombination event and the parental type constructs is explicable by the fact that Nt.Alwl nicked the lower strand only twice, both times between the fourth and fifth variable sites. With half the recombinants expected to come from reassorted lower strands, a high proportion of this simple recombination and reformed parental molecules are expected amongst the progeny. In fact the large disparity in nicking sites between the upper and lower strands (eight versus two) is expected to favour the complete ligation of the lower strand over the upper strand, which would then bias progeny in favour of the lower strand.

Example 2 DNA shuffling using nicking enzymes (prophetic) Mutations of the gene of interest are generated randomly (for example by errorprone PCR or using mutator strains of E. coli) or by rational design site-directed mutagenesis techniques. The mutant constructs are reduced to PCR amplicons or restriction fragments so as to eliminate, or minimise, extraneous DNA such as vector 33 sequence. The mutant constructs are pooled, either in equimolar amounts or in a ratio z designed to bias the representation of particular mutations among the reassorted products.

The mutant pool (for example, 1 pg of DNA) is digested with Nt.Alwl (New England Biolabs) in reaction volumes of 20 ul, according to the enzyme manufacturer's O conditions. It may be necessary to synthesise the gene in order to incorporate silent t mutations that result in recognition sequences for nicking enzymes. Providing at least C one suitable recognition sequence is present in the pool of dsDNA Nt.AlwI will nick the DNA.

Nt.Alwl is then heat inactivated (as per the manufacturer's recommendation), Taq DNA ligase (New England Biolabs) is added and the reaction mixes heated at 0 C for 30 sec to denature the DNA. Single strands are allowed to reanneal at 70 0

C

and the temperature slowly ramped down to 40 0 C over several hours, during which time both homo- and heteroduplexes will form and nicks will be repaired by the ligase.

PCR amplification of the full-length gene is then carried out on the ligated reaction in order to resolve heteroduplexes ligation products containing mismatches due to opposing strands being derived from different parental mutants). If not already incorporated into the gene, the PCR amplification also affords the opportunity to insert restriction sites into the primers to enable subsequent cloning of the reassorted gene products.

Example 3 Reassortment by overlapping restriction fragment ligation (prophetic) Mutations of the gene of interest are generated randomly by error-prone PCR or using mutator strains of E. coli) or by rational design site-directed mutagenesis techniques. The mutant constructs are reduced to PCR amplicons or restriction fragments so as to eliminate, or minimise, extraneous DNA such as vector sequence. The mutant constructs are pooled, either in equimolar amounts or in a ratio designed to bias the representation of particular mutations among the reassorted products.

One aliquot of the resultant mutant pool (1 jg) is digested with BamHI (Promega) and another aliquot of the resultant mutant pool (1 p.g) is digested with EcoRl (Promega) in reaction volumes of 20 il, according to the enzyme manufacturer's conditions. The procedure is designed such that each restriction enzyme cuts the DNA at least once, but ideally a number of times.

Restriction enzymes are then heat inactivated (as per the manufacturer's recommendation), and the pools combined. Taq DNA ligase (New England Biolabs) is 34 added and the reaction mix heated at 95 0 C for 30 sec to denature the DNA. Single 0 z strands are allowed to reanneal at 70'C for 5 min, during which time both homo- and heteroduplexes will form and nicks between adjacent fragments annealed to an overlapping complementary strand (productive union) will be repaired by the ligase.

The heating cooling steps are repeated several times, incrementally lowering \tthe annealing temperature to 40 0 C, so that complete complementary fragments (i.e.

t products of the same restriction enzyme digest) that have annealed (unproductive unions) will be re-cycled into productive unions as described above (note that ligated restriction products themselves become templates for further productive unions in subsequent cycles).

PCR amplification of the full-length gene is then carried out on the ligated reaction in order to resolve heteroduplexes ligation products containing mismatches due to opposing strands being derived from different parental mutants). If not already incorporated into the gene, the PCR amplification also affords the opportunity to insert restriction sites into the primers to enable subsequent cloning of the reassorted gene products.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

All publications discussed above are incorporated herein in their entirety.

This application claims priority from US 60/838,098, the entire contents of which are incorporated herein by reference.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

REFERENCES

Aggarwal (1995) Curr Opin Struct Biol. 5:11-19.

Arnold (1996) Chemical Engineering Science 51:5091-5102.

Needleman and Wunsch (1970) J. Mo!. Biol. 48:443-453.

Roberts et al. (2005) Nucl Acids Res 33:D230-D232.

Stemmer (1994) Nature 370:389-39 1.

Claims (43)

1. A method of preparing polynucleotide variants, the method comprising 00 a) exposing a pool of two or more related polynucleotides to at least one nicking enzyme, wherein at least some of the polynucleotides are partially and/or fully double stranded, b) removing, and/or inhibiting the activity of, the at least one nicking enzyme, c) denaturing the polynucleotides, CN d) allowing the denatured polynucleotides to form at least partially double 010 stranded polynucleotides, e) exposing the double stranded polynucleotides formed in step d) to a ligase.

2. The method of claim 1, wherein steps a) to e) are repeated at least once.

3. The method of claim 1 or claim 2, wherein at least one nicking enzyme in step a) is different when this step is repeated.

4. The method according to any one of claims 1 to 3, wherein steps c) to e) are repeated at least once. The method according to any one of claims 1 to 4, wherein step d) comprises exposing the denatured polynucleotides to a temperature of about 75 0 C, and allowing the temperature to cool to about 40 0 C.

6. The method according to any one of claims 1 to 5, wherein none of the single stranded fragments produced following step c) are less than about 40 nucleotides in length.

7. The method according to any one of claims 1 to 6, wherein step c) comprises exposing the polynucleotides to a temperature of about 90 0 C to about 105'C.

8. The method according to any one of claims 1 to 7, wherein steps b) and c) are conducted simultaneously by exposing the at least one nicking enzyme and polynucleotides to a temperature of about 90 0 C to about 105 0 C. 37 C

9. The method according to any one of claims 1 to 8, wherein steps d) and e) are a conducted simultaneously. 00

10. The method according to any one of claims 1 to 9 which further comprises conducting a polynucleotide amplification procedure on the double stranded polynucleotides produced by step e).

11. A method of preparing polynucleotide variants, the method comprising C a) exposing a pool of two or more related single stranded polynucleotides to at 0 10 least one nicking enzyme, N b) removing, or inhibiting the activity of, the at least one nicking enzyme, c) allowing the polynucleotides to form at least partially double stranded polynucleotides, d) exposing the double stranded polynucleotides formed in step c) to a ligase.

12. The method of claim 11, wherein steps a) to d) are repeated at least once.

13. The method of claim 11 or claim 12, wherein at least one nicking enzyme in step a) is different when this step is repeated.

14. The method according to any one of claims 11 to 13, wherein the method further comprises e) denaturing the polynucleotides produced in step d) and repeating steps c) and d). The method of claim 14, wherein step e) comprises exposing the polynucleotides to a temperature of about 90 0 C to about 105 0 C.

16. The method according to any one of claims 11 to 15, wherein step b) comprises exposing the nicking enzyme and polynucleotides to a temperature of about 90 0 C to about 105 0 C thereby inactivating the nicking enzyme and denaturing the polynucleotide, and step c) comprises exposing the denatured polynucleotides to a temperature of about 75 0 C, and allowing the temperature to cool to about 40 0 C. 38 O O 17. The method according to any one of claims 11 to 16, wherein none of the single a stranded fragments produced following step a) are less than about 40 nucleotides in Slength. 00

18. The method according to any one of claims 11 to 17, wherein steps c) and d) are conducted simultaneously.

19. The method according to any one of claims 11 to 18 which further comprises CN conducting a polynucleotide amplification procedure on the double stranded polynucleotides produced by step d). The method according to any one of claims 1 to 19, wherein the ligase is a thermostable ligase.

21. The method according to any one of claims 1 to 20, wherein at least one polynucleotide in the pool was produced by introducing a recognition sequence for at least one of the nicking enzymes into a first polynucleotide to produce a second polynucleotide.

22. The method of claim 21, wherein the first polynucleotide encodes a polypeptide, and the introduced recognition sequence does not alter the amino acid sequence of the encoded polypeptide.

23. The method according to any one of claims 1 to 22, wherein the nicking enzyme is selected from the group consisting of: Nt.AlwI, Nb.BbvCI, Nt.BbvCI, Nb.Bpul01, Nb.BsmI, Nt.Bst9I, Nt.BstNBI, Nb.BsrDI, and any combination thereof.

24. The method according to any one of claims 1 to 23, wherein the polynucleotides are DNA or RNA. The method according to any one of claims 1 to 24 which further comprises screening the variant polynucleotide(s) obtained for a desired activity.

26. The method of claim 25 which comprises producing a polypeptide(s) encoded by the variant polynucleotide(s) and screening the polypeptide(s) for the desired activity.

27. The method of claim 25 or claim 26, wherein the variant polynucleotide(s) are Scloned into an expression vector(s). 00

28. The method of claim 27, wherein the expression vector(s) are introduced into a host cell(s).

29. The method according to any one of claims 1 to 28, wherein step b) comprises CN exposing the nicking enzymes to a temperature of about 90 0 C to about 105 0 C. O

30. The method of claim 10 or claim 19, wherein the amplification is performed using oligonucleotide primers that hybridize to the ends of the two or more polynucleotides.

31. A method of preparing polynucleotide variants, the method comprising a) exposing at least a first double stranded polynucleotide, or a first pool of related double stranded polynucleotides, to a first restriction enzyme(s), and exposing a second double stranded polynucleotide, or a second pool of related double stranded polynucleotides, to a second restriction enzyme(s), wherein the first and second restriction enzymes cleave at different recognition sequences, and wherein the polynucleotides are related, b) removing, and/or inhibiting the activity of, the restriction enzymes, c) mixing the products of step b), d) denaturing the polynucleotides, e) allowing the denatured polynucleotides to form double stranded polynucleotides, f) exposing the double stranded polynucleotides formed in step d) to a ligase.

32. The method of claim 31, wherein the first pool and second pool of polynucleotides are identical.

33. The method of claim 31 or claim 32, wherein the ligase is a thermostable ligase.

34. The method according to any one of claims 31 to 33, wherein steps d) to f) are repeated at least once. N

35. The method according to any one of claims 31 to 34, wherein step e) comprises d) exposing the denatured polynucleotides to a temperature of about 75 0 C, and allowing Sthe temperature to cool to about 40 0 C. 00

36. The method according to any one of claims 31 to 35, wherein none of the single stranded fragments produced following step d) are less than about 40 nucleotides in length. C1) IN 37. The method according to any one of claims 31 to 36, wherein the polynucleotides are DNA or RNA.

38. The method according to any one of claims 31 to 37 which further comprises screening the variant polynucleotide(s) obtained from step f) for a desired activity.

39. The method according to any one of claims 31 to 38 which comprises producing a polypeptide(s) encoded by the variant polynucleotide(s) and screening the polypeptide(s) for the desired activity. The method of claim 38 or claim 39, wherein the variant polynucleotide(s) are cloned into an expression vector(s).

41. The method of claim 40, wherein the expression vector(s) are introduced into a host cell(s).

42. The method according to any one of claims 31 to 41, wherein step b) comprises exposing the restriction enzymes to a temperature of about 90 0 C to about 105°C.

43. The method according to any one of claims 31 to 42, wherein steps e) and f) are conducted simultaneously.

44. The method according to any one of claims 31 to 43, which further comprises conducting a polynucleotide amplification procedure on the double stranded polynucleotides produced by step f). 41 O c

45. The method of claim 44 wherein the amplification is performed using a oligonucleotide primers that hybridize to the ends of the double stranded polynucleotides used in step a). 00

46. The method according to any one of claims 1 to 45, wherein the method does not comprise the use of a polymerase to fill in any single stranded gaps of the Spolynucleotide produced using the method. C, 47. The method according to any one of claims 1 to 46, wherein the method is performed in vitro.

48. A polynucleotide produced using a method according to any one of claims 1 to 47.

49. A library of variant polynucleotides produced by a method according to any one of claims 1 to 47. A method of making a polypeptide having a desired property, the method comprising a) generating polynucleotide variants using a method according to any one of claims 1 to 47, b) expressing the variant polynucleotides produced in step a) to produce variant polypeptides, c) screening the variant polypeptides for a desired property, and d) selecting a polypeptide having the desired property from the variant polypeptides.

51. A polypeptide produced using a method according to claim

52. A method of making a polynucleotide having a desired property, the method comprising a) generating polynucleotide variants using a method according to any one of claims 1 to 47, b) screening the variant polynculeotides for a desired property, and c) selecting a polynucleotide having the desired property from the variant polynucleotides. t 42 O d 53. A polynucleotide produced using a method according to claim 52. 00