WO2003004595A2

WO2003004595A2 - Materials and methods relating to protein and nucleic acid evolution

Info

Publication number: WO2003004595A2
Application number: PCT/GB2002/003145
Authority: WO
Inventors: Paul Anthony Dalby
Original assignee: University College London
Priority date: 2001-07-05
Filing date: 2002-07-05
Publication date: 2003-01-16
Also published as: GB0116478D0; AU2002317313A1; WO2003004595A3; WO2003004595A9

Abstract

The present invention concerns materials and methods relating to protein and nucleic acid evolution. Particularly, but not exclusively, the invention relates to new methods of PCR error-prone mutagenesis of nucleic acid sequences encoding for peptides or proteins having improved specificity or other functional nucleic acid sequences having improved function.

Description

Materials and Methods Relating to Protein and Nucleic Acid Evolution

Field of the Invention

The present invention concerns materials and methods relating to protein and nucleic acid evolution. Particularly, but not exclusively, the invention relates to new methods of PCR error-prone mutagenesis of nucleic acid sequences encoding for peptides or proteins having improved specificity or functional nucleic acid sequences having improved function.

Background of the Invention

Mutagenesis of proteins is of paramount importance for the understanding of protein structure and function. The structural and functional roles of different amino acid residues can be studied by comparing the mutant proteins containing the modified amino acids to the wild-type protein. To obtain the modified proteins, the mutant genes have to be created and expressed. In the past, the only way to obtain mutations was to isolate naturally occurring mutants with strain-screening methods. However, the rate of natural mutation is very low, and mutants'had to be generated by treatment with mutagenic agents such as chemical mutagens or UV light. Moreover, even if an accurate screening test was available, isolation of mutants that were lethal or that did not produce observable changes was not possible (Zaccolo, M.,Williams, D.M., Brown,.D.M., and Gherardi, R, J.Mol.Biol., 255, 589-603, 1996).

Random mutagenesis is now, usually, achieved by PCR methods. These methods have revolutionised the means by which mutants are obtained because they are more precise and more efficient, yielding mutations in 50-100% of the proteins created, than that of phenotypic screening (Smith, M., Biochimie, 67, 717-723, 1985 & Zoller, M., Curr.Opin.Biotech., 2, 526-531, 1999).

h contrast to natural evolution, directed evolution has a defined goal, which is to generate large pools of molecular variants at the DNA level from which proteins with the desired properties are selected. As opposed to protein engineering by design, directed evolution does not require a prior knowledge of the protein structure (Manuela Zaccolo and Ermanno Gherardi., J.Mol.Biol., 285, 775-783, 1999). Furthermore, it relies on the fact that proteins can tolerate a number of amino acid residue substitutions without dramatic effect on folding or stability (Axe et al, Proc.Natl.Acad.Sci. USA, 93, 5590-5594, 1996; Bowie et al., Methods Enzymol., 266, 598-616, 1996). The other fact is that natural evolution has only screened for a subset of potentially useful sequences and therefore an unexplored sequence space can reveal better solutions to biological problems (Manuela Zaccolo and Ermanno Gherardi., J. Mol. Biol., 285, 775-783, 1999). hi practice, the mutational load representing the number of mutations induced into the gene cannot exceed a certain rate and the size of the mutant DNA libraries are limited to about 10⁶to 10¹³ clones depending on the mode of screening or selection.

Random mutations have previously been introduced into a stretch of DNA using non-PCR methods such as chemical mutagenesis, UV irradiation, mutator strain and poisoned nucleotides (Kuchner and Arnold., Trends Biotechnol., J 5, 523-530, 1997). These methods are reported to be successful but they suffer from a number of disadvantages. First, the mutations can affect any gene in the organism's genome and the average number of mutated genes of interest can be very low. Second, prior to DNA cloning and sequencing technologies there was no way of knowing the location and the type of mutations being induced into the genome.

Therefore there was a huge necessity for biologists to refine mutagenesis techniques and combine it to PCR amplification methods in order to generate higher numbers of mutants with more precise mutations. These methods have revolutionized the means by which mutants are obtained (Smith M et al., Biochimie, 67, 717-723, 1985 & Zoller, M.J., Curr. Opin. Biotech., 2 (4), 526-531, 1991).

Among the methods that introduce random mutations in the entire gene is Error-prone PCR (epPCR). The approach has successfully been applied to engineer new protein functions and to improve the catalytic activity. Another technique is combinatorial cassette mutagenesis, which uses oligonucleotides containing randomised codons as mutagenic cassettes for their introduction into the gene of interest by PCR methods (Reidhaar-Olson et al., Methods Enzymol., 208, 564-586, 1999). Moreover, a DNA shuffling method developed for random in vitro DNA recombination represents a significant advance in the applications of directed evolution methods (Stemmer et al., Nature, 370, 389-391, 1994).

A typical cycle of directed evolution starts with the selection of DNA sequences encoding for proteins that involve to some extent the sought after property. The diversity of the sequences is then increased through the mutagenesis step by introducing random point nucleotide mutations and amplifying the DNA fragment using Error-prone PCR. These DNA sequences are then cloned into an expression vector and transformed into competent E.coli cells. A screening procedure is then employed to isolate the transformant E.coli cells containing the mutated PCR amplified DNA fragments encoding for proteins with improved characteristics.

The selected sequences are then amplified again so that the mutagenesis, amplification and screening are repeated many times until the proteins with the desired properties or functions are obtained.

A remarkable success has been achieved in the industrial application of directed evolution to improve the activities and thermostabilities to vaccines and pharmaceuticals (Schmith-Dannert & Arnold, Trends Biotechnol., J 7, 135-136, 1999). These successful applications have proved the different possibilities for future uses of directed evolution in understanding protein functions and the production of novel biocatalysts (Gregory L.Moore and Costas D.Maranas, J.Theor.BioL, 205 (3), 483-503, 2000).

The technique of DNA shuffling creates gene libraries, containing combinations of mutations derived from a set of homologous DNA sequences or arise as a result of point mutations (Kuchner and Arnold., Trends Biotechnol., 15, 523-530, 1997). Recombination serves to promote positive traits and eliminate negative traits in the progeny, resulting in a rapid accumulation of beneficial mutations in separate genes. hi the Error-prone PCR method, the gene of interest is amplified after many PCR cycles under conditions that increase normal mis-incorporation errors. The error prone PCR replication process (Cadwell & Joyce, PCRMeth. Appl., 3(6), S136-S140, 1994) intentionally introduces copying errors by imposing mutagenic reaction conditions, based on the parameters below: 1 . The error rate (fidelity) of the Taq polymerase.

2 . The length of the mutagenised gene and the number of effective doubling cycles.

3 . The concentration of MnCl₂ affecting the rate of mutations induced by the Taq polymerase.

The first step of PCR is the denaturation of the DNA into 2 single strands. The second step is the annealing of a primer to the DNA single strands. The third step is the extension by Taq polymerase.

Nucleotides complementary to the single strand template are added by using the original sequence as a template, extending the complementary strands till the normal DNA double strands are recovered. Most mutations occur in this step where the non-complementary nucleotides are incorporated into the chain. The mutation rates induced by Taq range from 10^"7up to 10^~3 per nucleotide polymerized, as reported by Eckert & Kunkel, Nucleic Acids Research, 18, 3739-3744, 1990. However these mutations are nucleotide dependent (Cadwell & Joyce., (supra); Shafikhani et al., Biotechniques, 23, 304-310, 1997). Therefore the monitoring of these variable replication errors is crucial for the mutagenesis.

Examples of error prone PCR akeady in use, include improved solvent, thermostability and enhanced specific activity of enzymes and proteins (Chen and Arnold., P. Natl. Acad.Sci.USA, 90 (12), 5618-5622, 1993; Giordano et al, Biochemistry-US, 38 (10), 3043-3054, 1999; Heneke and Bornscheuer., Biol.Chem., 380, (7-8), 1029-1033, 1999; Moore and Arnold., Nat. Biotechnol., 14, 458-467, 1996; Shibata et al., Protein.Eng, ϋ (6), 467-472, 1998; You and Arnold,. Protein Eng., 9 (1), 77-83, 1996). Therefore specific protein activities and functions that never occur in nature can be easily generated.

The method of error-prone PCR is subject to several disadvantages. Firstly, there is a tendency for neutral and deleterious mutations to accumulate in the selected progeny sequences, increasing with the number of cycles of error-prone PCR. Secondly, the random mutagenesis is directed across the entire gene sequences in the hope that a mutation resulting in the desired improvement will be found within the generated library. As a result, most mutants that are formed, are non-functional (deleterious), or have no affect on the desired property (neutral). Given the restrictions on library size that can be searched in a practical manner, these redundant mutations further limit the useful sequence space that can be searched.

Combinatorial cassette mutagenesis is sometimes used to mutate selected sequences and therefore reduces the sampling of redundant sequence space. However, the level of mutation can no longer be varied using this method, unless new DNA primers are synthesized for each mutational load required. Control of mutational load is desired in order to control the generated sequence space, and hence the library size, to within a practical limit. It is also extremely difficult to generate combinatorial cassettes that contain only non-disruptive mutations, i.e. encoding only for amino acids with similar physico-chemical properties to that of the original wild-type amino acid. Altering an amino acid in a protein sequence frequently disrupts the structure or function of the protein, resulting in the need to search again through redundant sequence space.

Summary of the Invention

The present inventors have determined a novel approach to error-prone PCR which allows hyper-mutation by "focused error-prone PCR" at a specific and selected active site of a nucleic acid or polypeptide. The approach is analogous to phage-display libraries and the natural repertoire of antibodies, in that only the active site of the displayed protein is randomised. Phage-display has been used successfully to obtain tighter ligand binding, and also to obtain "catalysis" of a reaction. However, phage-display relies on the linkage between catalysis and a binding property in order to select variants from a large library, thus limiting its potential to the selection of single turnover events and not true catalysis. Focused error-prone PCR is a hybrid approach that combines a direct assay for nucleic acid or polypeptide function and the focused sequence randomization of phage display.

Focused error-prone PCR is a novel method based on error-prone PCR in which a nucleic acid fragment is amplified by PCR using Taq polymerase. Taq polymerase has a low fidelity of replication due to a lack or reduction of 3 '-5' exonuclease proof-reading activity. The rate of mutagenesis and hence the mutational load can be altered by varying the concentration of Mn²⁺ in the PCR reaction according to previously established equations (Fromant et al., Anal.Biochem., 224, 347-353, 1995). During amplification, the sequence between the two PCR primers becomes mutated at random and the sequence of the primers can also become mutated at random, although generally to a lesser degree than the sequence between the primers.

Focused error-prone PCR takes advantage of the fact that the majority of residues important for nucleic acid function (e.g. promoter or enhancer activity) or polypeptide function (e.g. catalysis or substrate binding) make up only a small proportion of the entire nucleic acid sequence or protein in most cases (Clackson et al., J. Mol. Biol., 277, 1111-1128, 1998). The primers for PCR are thus chosen to complement the sequence at either side of these short and specific regions to be randomised. The primers may complement the sequences within the short and specific regions or may complement sequences just outside the short specific regions to be amplified. The result is that only those regions of the nucleic acid or polypeptide comprising the active site are randomized.

Thus, at its most general, the present invention provides a method of randomly modifying a specific region of a functional nucleic acid sequence or polypeptide sequence while maintaining the remaining sequence so as to arrive at a functional nucleic acid or polypeptide with improved characteristics. In a first aspect, there is provided a method of producing a modified polypeptide with improved characteristics comprising the steps of

(a) obtaining nucleic acid primers which flank an active site within a parent nucleic acid sequence encoding a parent polypeptide;

(b) carrying out a polymerase chain reaction (PCR) using said primers and the parent nucleic acid sequence as a template under suitable conditions for introducing mutations into the amplified active site sequence;

(c) isolating said mutated active site;

(d) introducing said mutated active site into the parent nucleic acid sequence to replace the non-mutated active site thereby producing a modified nucleic acid sequence, or introducing said mutated active site into a template nucleic acid sequence to produce a modified nucleic acid; and

(f) expressing said modified nucleic acid sequence to produce a modified polypeptide.

The method of the first aspect of the present invention may further comprise the step of screening the expressed polypeptide for improved characteristics.

The term polypeptide as used herein refers to a polymer of amino acids that has a measurable function affected by the sequence of polypeptide and does not refer to a specific length of the product; thus peptides, oligopeptide and protein are included in the term polypeptide.

In a second aspect, there is provided a method of producing a modified functional nucleic acid with improved characteristics comprising the steps of

(a) obtaining nucleic acid primers which flank an active site within a functional parent nucleic acid sequence;

(c) isolating said mutated active site;

(d) introducing said mutated active site into the parent nucleic acid sequence to replace the non-mutated active site thereby producing a modified nucleic acid sequence or introducing said mutated active site into a template nucleic acid sequence to produce a modified nucleic acid.

The method of the second aspect of the present invention may further comprise the step of screening the modified nucleic acid sequence for improved characteristics.

The term functional nucleic acid as used herein means any nucleic acid such as DNA, cDNA, RNA, niRNA, etc. that has a measurable function affected by the sequence of the nucleic acid.

The method of the first aspect and second aspect of the present invention may as a first step additionally comprise determining the active site within the parent nucleic acid sequence.

A specific example of the method according to the first and second aspect of the present invention is shown schematically in figure 1.

With respect to the method of the first aspect of the present invention, the active site may be any region or multiple regions within a polypeptide sequence that is responsible for a particular property of the protein. For example, if the polypeptide is an enzyme the active site of interest maybe the region associated with catalysis. Alternatively the active site may be a binding site, folding nucleus, hydrophobic core, epitope etc.

With respect to the method of the second aspect of the present invention the active site may be any region or multiple regions within a functional nucleic acid sequence that is responsible for a particular property of the nucleic acid. For example if the functional nucleic acid sequence is a promoter the active site may be the region exhibiting the most similarity to the promoter consensus sequence. Alternatively, the active site may be a ligand binding site of an enhancer etc., the catalytic region of catalytic RNA, the region of a transposable DNA element involved in movement of the element, etc. Once the active site of interest has been selected or determined, e.g. been sequenced, primers are designed that flank this particular region. During PCR the active site between and/or complementary to the flanking primers is amplified. However, by altering the conditions of the PCR, random mutagenesis of the amplified nucleic acid can be achieved to different degrees depending on the conditions. It is preferable that the optimal conditions for PCR ^"are firstly established. Then the levels of mutagenesis required can be achieved by increasing the levels of Mn²⁺ in the reaction mixture above those considered optimal. It is important that the level of mutagenesis occuring is sufficient so that mutations are occuring in the relatively short active site that is being amplified. Previously, large nucleic acid sequences are mutated using error prone PCR and the level of mutagenesis is relatively low so that only a few mutations occur within the nucleic acid sequence. The present invention requires that the level of mutagenesis is higher than in previous methods of error prone PCR because less nucleic acid is being amplified and it is desirable to generate at least one mutation in the relatively short amplified nucleic acid. It has been found that the necessary level of mutagenesis can be obtained by carefully controlling the PCR conditions and in particular by controlling the concentration of Mn²⁺ in the PCR reaction mixture. It was surprising that a sufficiently high level of controlled mutagenesis could be obtained to produce the necessary level of mutagenesis in the relatively short sequences being amplified.

Preferably the concentration of Mn²⁺ in the PCR reaction mixture is between about O.OlmM and about 2 mM, more preferably between about 0.05mM and about ImM, most preferably between about 0.2 mM and ImM.

The mutated amplified nucleic acid sequence corresponding to the selected active site is isolated using standard techniques. The level of mutagenesis maybe tested using restriction enzymes and comparing a digestion profile of the active site before and after amplification.

The amplified and mutated nucleic acid relating to the active site is then incorporated into the wild type nucleic acid . This maybe done by standard digestion and ligation techniques or it may be achieved by using the mutated active site nucleic acid as a template against the wild type template, e.g. by the commercially available Stratagene Quikchange ™ method. When there is more than one mutation in the mutated active site nucleic acid, it may be desirable to use the Stratagene Quikchange ™ Multi Site-Directed Mutagenesis Kit, which is described in WO 01/25483.

Alternatively, the amplified and mutated nucleic acid relating to the active site is incorporated into a template to test the effect of the mutated nucleic acid. This maybe done by standard digestion and ligation techniques. The template is a nucleic acid into which the mutated nucleic acid sequence can be inserted in order to test the effect of the mutated nucleic acid. For example, the template may encode the structural parts of a protein wherein the mutated nucleic acid encodes an active site of the protein. Alternatively, the template may be an expression vector wherein the mutated nucleic acid is a promoter which is inserted into the expression vector so that it is operable linked to an expressible gene.

When the method of the first aspect of the present invention is performed and the mutated active site has been incorporated into the wild type sequence, the modified nucleic acid may be expressed and the resulting polypeptide may be screened for improved characteristics. In the case of enzymes, the improved characteristics maybe increased or decreased catalysis. In the case of receptors or ligands, improved or lowered binding may be achieved. Other polypeptides may be altered to change the way they fold resulting in the disguise or new appearance of epitopes. Further examples of improved characteristics which may be obtained by the method according to the first aspect of the invention include improved protein folding either as faster or slower folding, or via fewer or more intermediate stages; improved protein stability may be achieved as either more or less stable, for example by mutation of hydrophobic core residues or metal ion binding residues; improved enzyme regulation may be obtained as either tighter or weaker regulation, or regulation by a new regulator molecule, by mutation of the regulatory site; improved protein expression maybe obtained by mutation of unfavoured sequence regions; improved aggregational properties could be obtained as either more or less aggregation in vivo as inclusion bodies or in vitro, by mutation of, for example, hydrophobic surface patches; and improved protein secretion properties maybe obtained by mutation of signal peptide, pre- or pro-peptide regions. Other examples of improved characteristics will be apparent to the skilled person.

When the method of the second aspect of the present invention is performed and once the mutated active site has been incorporated into the wild type sequence, the modified functional nucleic acid may be screened for improved characteristics. In the case of promoters, the improved characteristics may be increased or decreased transcription of an operably linked gene. In the case of enhancers, the improved characteristics may be increased or decreased enhancer activity. In the case of ligand responsive nucleic acids, the specificity of ligand binding may be altered, which may effect transcription of an operably linked gene. In the case of catalytic RNA, the improved characteristics may be increased or decreased activity, or altered specificity. In the case of transposable elements, the improved characteristics maybe an increased or decreased level of transposition, or the specificity of the sites of insertion of the transposable element may be altered. Other examples of improved characteristics will be apparent to the skilled person and will depend on the function of the functional nucleic acid.

As mentioned above, the polypeptide or functional nucleic acid in question may have multiple active sites. It may be desirable to modify each of these active sites. This can be achieved by carrying out independent PCR methods each using primers flanking the active site in question. However, the incorporation of the mutated active sites maybe achieved in one step by using the mutated active site in a PCR or extension reaction with the wild type sequence as a template.

The method according to the first aspect of the present invention may be applied to any polypeptide/protein where improved characteristics or properties would be desirable, e.g. enzymes, receptors, ligands, antibodies, antigens, cytokines, etc. The method according to the second aspect of the present invention maybe applied to any functional nucleic acid where improved characteristics or properties would be desirable, e.g. gene regulation sequences such as promoters, enhancers, transcription termination sequences, etc., ligand responsive elements, catalytic RNA, nucleic acid that forms tertiary structures such as hairpin loops, transposable elements, etc. The nucleic acid encoding the polypeptide/protein of interest or the functional nucleic acid is used as a template and may be between fifty base pairs and several thousand base pairs. The active site(s) may be of any length but are preferable between 10 and lOOObp, more preferable between 10 and lOObp and even more preferable between 10 and 50bp or 10 and 20bp.

The primers (3' and 5') are designed to flank the active site requiring modification. The primers may complement the sequences of the active site or may complement the sequences adjacent to the active site. As the primers themselves can incorporate mutations during amplification, there does not have to be any intervening sequence between the primers, instead the primers may complement the entire active site. Preferably there is an intervening sequence between the primers. It is further preferred that the intervening sequence is the active site requiring modification. Knowledge of at least the sequence flanking the active site must be known or must be determined as part of the method according to the invention. The design of primers is well known to the skilled person. The primers may incorporate a restriction enzyme site to help with the isolation of the amplified product. The primers are preferably between 8bp and 50bp in length, more preferably between 10 and 30bp in length.

The modified polypeptides/protems or functional nucleic acids produced by the methods of the invention may be used for research purposes or preferably they may be used as pharmaceuticals or diagnostic tools. More preferably, they may be formulated into pharmaceutical compositions. These compositions may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. 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. Physiological saline solution, dextrose or other saccharide solution or glycols such as 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, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

In a third aspect of the present invention, there is provided a kit consisting of a container containing substances and instructions required to carry out the method of the first or second aspect of the present invention. The kit may comprise one or more of the following substances or components: Taq polymerase, MnCL solution, MgCi2 solution, dNTP's, 10 x polymerase buffer, control plasmid DNA, control primers 5' and 3', control restriction enzyme, lOx restriction enzyme buffer, Dpnl restriction enzyme, and competent cells such as E.coli. The last two components are used in the Stratagene Quikchange ™ .

The methods according to the first and second aspects of the present invention have numerous applications. For example, the methods can be used to produce DNA libraries and consequently can make RNA, peptide and protein libraries. The libraries can be used for phage display, ribosome display, plasmid display, other display techniques or in vitro screening techniques in order to screen for desirable characteristics. A further advantage of the methods of the present invention is that the same primers can be used to create multiple library types with different mutational loads by altering the level of mutagenesis that occurs during the amplification stage of the methods.

Specific uses of the methods of the present invention include:

1. Altering enzyme substrate specificity by randomising residues in or close to the enzyme active-site that are responsible for substrate binding.

2. Altering enzyme product selectivity by randomising residues in or close to the enzyme active-site that are responsible for substrate binding. _N

3. Altering the secretion properties of a protein by random mutation of a signal peptide, or pre- or pro- peptide regions.

4. Altering ligand or cofactor binding properties of a protein by randomising residues in or close to the binding site that are responsible for ligand or cofactor binding.

5. Introducing new ligand binding properties into a protein by randomising residues on the surface of the protein.

6. Introducing new catalytic properties into a protein by randomising residues within a ligand binding pocket.

7. Altering protein folding rates or mechanism by randomising residues responsible for condensation-nucleation, nucleation, hydrophobic collapse, folding core, molten-globule stabilisation, stability of folding intermediate(s) or stabilisation of folding transition state(s).

8. Altering protein stability by randomisation of hydrophobic core residues, metal ion binding residues, secondary structure elements, ligand binding residues or cofactor binding residues. 9. Altering stability of protein to degradation by proteases, oxidants, or other reactive chemicals (aldehydes, sulphides, peroxides, azides etc), by randomisation of surface residues, or solvent-exposed residues.

10. Altering enzyme regulation by randomisation of the regulator binding site.

11. Altering protein expression by randomisation of unfavoured sequence regions, such as tryptophan residues, less favoured codons for a given host cell, residues that destabilise the protein, or residues that contribute to protein aggregation or inclusion body formation.

12. Altering protein aggregation properties (in vivo as inclusion bodies or in vitro), by randomisation of hydrophobic surface patches, beta-sheet regions, or other protein surface residues known to interact with regions of the same protein.

13. Altering activity of catalytic RNA by randomising DNA bases corresponding to RNA bases responsible for catalysis.

14. Altering substrate specificity of catalytic RNA by randomising DNA bases corresponding to RNA bases responsible for substrate binding.

15. Altering product selectivity of catalytic RNA by randomising DNA bases corresponding to RNA bases responsible for substrate binding.

16. Altering ligand binding of RNA (Aptamers) by randomising DNA bases corresponding to RNA bases responsible for ligand binding.

17. Introducing catalytic activity or ligand binding into RNA.

18. Altering promoter regions by randomising DNA bases responsible for regulating transcription of a gene. 19. Altering gene regulation by randomising DNA bases responsible for transcription effector binding and regulating transcription of a gene.

20. Altering transposon properties by randomising DNA bases within transposable elements and/or transposons.

Brief Description of the Drawings

Figure 1 shows schematically the method for rapid introduction of sequence diversity into short stretches of plasmid DNA, wherein (A) the target sequence is amplified by error-prone PCR causing incorrect bases (1) to be randomly incorporated into the growing strands of DNA (dotted lines); (B) the resulting array of mutant dsDNA fragments; (C) the mutant fragments are used as primers for the QuikChange™ protocol in a second PCR step using wild-type plasmid DNA as the template (thick lines); and

(D) non-mutated methylated template DNA is removed by digestion with Dpnl.

Figure 2 shows a 2.5 % agarose gel used to analyse the PCR amplified products.

Figure 3 shows a 2.5% agarose gel for the Error-prone PCR amplified products.

Figure 4 shows a 2.5% agarose gel for mutation analysis of the Error-prone PCR amplified products after treatment with BstEJJ enzyme.

Figure 5 shows a graph illustrating the mutation probability in the restriction site versus the number of random mutations induced in the whole DNA fragment (12 bp).

Figure 6 shows the 0.8% agarose gel analysis of the PCR amplified plasmids.

Figure 7 shows the 0.8% agarose gel DNA digest analysis.

Figure 8 shows the TktA sequence data for 10 colonies (S1-S10), wherein sequences from the beginning of TKF1 to the end of TKF2 are shown. The 12 bp target is shaded in grey and the 7 bp ifatEJJ. restriction site is boxed in black. Dots indicate non-mutated sequence.

Figure 9 shows the mutation frequency across an active site, wherein the primers are at nucleotide positions 1 to 34 and 47 to 81, and the active site is at nucleotide positions

35 to 46.

Detailed Description of the Invention

Focussed error-prone PCR according to the present invention targets the random mutations to regions of the DNA sequence that are identified as being most likely to affect the desired property. This reduces the number of mutants generated that are neutral or deleterious to the protein structure or function desired, and consequently improves the efficiency of searching relevant sequence space. This could be achieved using combinatorial cassette mutagenesis, but without the further improvements below afforded by focused error-prone PCR.

As opposed to combinatorial cassette mutagenesis, focused error-prone PCR creates mutations at first by predominantly single base changes, therefore, in the case of creating a modified protein, creating codon changes that encode for amino acids with similar physico-chemical properties to the wild-type amino acids. This results in the generation of fewer disruptive mutations, and again improves. the efficiency of searching relevant sequence space. More extreme mutations may be generated using a second round of the same focused error-prone PCR procedure.

Error-prone PCR allows libraries with different mutational loads to be created using the same PCR primers, by simply altering the concentration of Mn²⁺ used in the PCR reaction. For the combination cassette mutagenesis, new primers must be synthesised to define each mutational load.

A flexible and fast method of DNA mutagenesis and cloning of the Transketolase (TK) gene is described below as an illustration of the method of the first aspect of the present invention. This technique is very useful and efficient for both the lab and the industrial scale because it produces a large number of mutants in a very short time.

MATERIALS AND METHODS

PLASMID PURIFICATION

The recombinant plasmid pQR711 containing the TK gene, is purified from the E-coli cells using QIA prep spin Miniprep Kit (50) from QIAGEN. 5ml of the recombinant pQR711 strain of E-coli cells are centrifuged at 1400 rpm for 10 minutes and the supernatant discarded. They are resuspended in 250μl of buffer PI . Then 250 μl of buffer P2 is added to each sample and mixed. 350μl of buffer N3 is also added and mixed, then the samples are centrifuged for 10 minutes at 13,000 rpm. The supernatant u is gently applied to QIAprep column and centrifuged for 30-60 seconds at 13,000 rpm. QIAprep spin column is washed by adding 0.5ml of buffer PB and centrifuged for 30-60 seconds at the same speed. QIAprep spin column is washed again by adding 0.75ml of buffer PE and centrifuge for another 30-60 seconds. The flow-through is discarded, and centrifuged for an additional minute to remove residual wash buffer.

To elute the DNA, 50μl of buffer EB is added to the centre of each QIAprep column and centrifuge for 1 minute. Then the purified plasmid DNA in the flow through is keρt at -20C.

The polymerase chain reaction (PCR)

PCR mixtures were prepared under sterile conditions using Gilson pipettors and sterile pipettors tips that were kept exclusively for use in setting up PCR in order to minimise DNA contamination. The primer sequences and PCR amplified DNA fragment generated are given in the Table below.

The mixture of the amplification reaction consists of 10 μl of lOx Taq polymerase buffer, a varying amount of MgCl₂, 2 μl deoxynucleotide triphosphate "dNTP" mixture (lO M each dNTP), 2μl of template DNA at different dilutions, lμl of primer TKF1 and lμl of primer TKF2 at a concentration of 250 ng/μl each (amersham pharmacia biotech). The PCR reaction mixtures are made up to a final volume of 100 μl with dd H₂0. Finally lμl of Taq polymerase is added to each PCR sample immediately after the first 2 minutes in the PCR cycling.

The PCR reaction for each sample is performed using the cycling parameters outlined in the following Table:

However, the Error-prone PCR is performed using the method described above but also including different concentrations of MnCk, ranging from 0.05 mM to ImM in the PCR reactions.

Agarose gel electrophoresis:

The PCR products and the DNA markers were analysed by agarose gel electrophoresis in order to determine whether the DNAs were amplified and also verify that there was no contamination. The 0.8% agarose gel was prepared by heating 0.96g of agarose (Sigma Chemicals Co, St Louis, M.O, USA) in 120ml of 0.5X TBE (5X TBE was made up with 54g Tris Base, 27.5g Boric acid, 20 ml 0.5M EDTA and the solution was made up to 1000ml with distilled water). After the agarose was dissolved, Ethidium bromide was added to a final concentration of 0.5μg/ml in the gel and then poured into a gel tank and left to set for 30 minutes. A lOμl aliquot of each PCR product was mixed with 2μl of agarose gel loading buffer and then loaded in the well slots. A DNA molecular weight marker (lkb or 100 bp ladder with 2μl of agarose gel loading buffer) was electrophoresed alongside the products in order to evaluate the size of the PCR products. Electrophoresis was performed at 80V for 60-90 minutes (depending on the size of the PCR products) and the gels were viewed under a UV transilluminator. The percentage of agarose gel was dependent on the size of the DNA of interest (the percentage increased with decreasing DNA fragment size. A 0.8% agarose gel (made up with 0.96 g of agarose in 120ml of 0.5X TBE) was used for the analysis of the plasmid DNA. Whereas the PCR products (0.9kb) were analysed in a 2.5% agarose gel (made up with 3.92g of agarose in 120 ml of 0.5X TBE buffer).

DNA restriction digest analysis:

1 μl of the BstEJJ restriction enzyme (Sigma) is added to 20μl of each PCR sample as well as pQR711 plasmid not treated with BstEJJ, which is used as a control reaction. Each digestion reaction also contains 8μl of BstELI buffer and the total volume is made up to 80μl with dd H₂ O. The samples in each tube are mixed immediately and incubated at 37C for 1 hour to digest the non-mutated PCR fragments at the BstEJJ restriction sites. However the enzyme BstEJJ does not cut the TK gene if its restriction site is mutated. The DNA digests are then analysed by agarose gel electrophoresis.

E-coli competent cells preparation (CaCl₂ method):

For the preparation of 100ml of JM107 strain of E-coli competent cells, the culture is grown in LB medium at 37C so that they reach an absorbance of 0.5 - 0.7 at A550. The cells are placed in ice to chill down and then centrifuged at 6000 rpm for 10 minutes at 4C. The cells are washed with 40 ml of 0.1M MgCl₂ and centrifuged again at 6000 rpm for 10 minutes at 4C.

The cells are resuspended in 40ml of 0.1M CaCl₂ and incubated in ice for 30 minutes. Finally 2ml of 50% glycerol is added and the competent cells are then stored at -70C.

Preparation of DNA library using PCR : 1 . PCR amplification

The recombinant plasmid DNA pQR711 is amplified using the PCR QuickChange™ technique from Stratagene, where the mutated fragments in the TK gene from the previous Error-prone PCR are used as primers. These mutagenic primers mutate the TK gene in the recombinant plasmid DNA as the PCR cycles proceed. The reaction mixture in this PCR is made of 5 μl of 1 Ox reaction buffer, (5-50 ng) of ds DNA template, 125 ng of ss oligonucleotide primer 1 (the 1^st strand of the mutated fragment from TK gene), 125 ng of ss oligonucleotide primer 2 (the 2^nd strand of the same mutated fragment), 1 μl of 10 mM dNTP mix and the final volume of the reaction is made up to 50 μl with dd H₂O. Then lμl of Pfu DNA polymerase (2.5 U/μl) is added to each sample reaction. The reaction is performed using the cycling parameters outlined in the following table:

The PCR products are then analysed by taking aliquots from the PCR reactions and running them in agarose gel electrophoresis.

1 . Enzyme digestion of the PCR products:

1 μl of the Dpnl restriction enzyme (lOU/μl) is added to each PCR sample, as well as the wild type plasmid, which is used as a control reaction.

The samples in each tube are mixed immediately and incubated at 37C for 1 hour to digest the methylated parental supercoiled ds DNA.

The DNA digests are then analysed by agarose gel electrophoresis.

2 . PCR product transformation into the competent E-coli cells:

1 μl of the Dpnl-treated DNA is taken from the control reaction (Dpnl treated pQR711) plasmid and the PCR sample reactions, added into separate 200 μl aliquots of the competent E-coli cells (JM107 strain) and stored on ice for 30 minutes. The samples are then heat shocked at 42°C for 90 seconds and then chilled down in ice for 1-2 minutes. 800μl of autoclaved LB medium is added to each tube and the cultures are incubated for 45 minutes at 37C to allow the bacteria to recover and express the antibiotic resistance encoded by the plasmid. Then lOOμl of the transformed competent cells at different dilutions are plated on agar + LB medium plates containing ampicillin at a concentration of 50 μg ml. The plates are incubated overnight at 37C and the colonies grown on the plates are counted the following day.

RESULTS AND DISCUSSION

Analysis of PCR amplified TK fragment:

The standard PCR amplification method using Taq polymerase is carried out to amplify the DNA fragment of interest (12 bp) in the TK gene. Different concentrations of Template DNA and MgCl₂ are used in each reaction to optimise the PCR amplification. Figure. 2 shows the agarose gel analysis of the different PCR products. Lane 2 shows the most concentrated PCR product, where 1/10 diluted DNA is added. However, as the concentration of the template DNA added to the PCR reaction is reduced, the amount of the PCR product decreases as shown in lane 3, and the PCR product disappears as the template DNA concentration is further reduced as shown in lane 4. Similarly in lane 5 & 7 where the same PCR sample is loaded, no PCR product is obtained because the concentration of the starting DNA is too low, although 1.5 mM of MgCl₂ is added. But when a suitable template DNA concentration is added together with 1.5 mM of MgCl₂to the reaction, a PCR product appears on the gel (lane 8). The amount of PCR product in lane 8 is much less than the one in lane 2, showing the negative effect of the added MgCl₂ on the efficiency of the PCR amplification in this experiment.

In lane 5 & 7 where 1/250 diluted template DNA is added to the PCR reaction in conjunction with 1.5mM of MgCl₂, no PCR product is obtained, meaning that the amount of the template DNA added is below the threshold value for this particular PCR amplification. All the PCR products obtained are 90 bp long (containing the DNA fragment of interest and the two primers), as indicated by the size of the marker DNA bands. However, the bands below the PCR products bands on the gel are due to the excess of oligonucleo tides used. In lane 9, the lack of the PCR product is due to the absence of the Taq polymerase in the PCR reaction. Analysis of Error-prone PCR amplified products (TK fragment):

The Error-prone PCR amplification method is carried out using the same conditions as the optimum standard PCR amplification in the presence of MnCl₂ to induce random mutations in the DNA fragment of interest (12 bp) within the TK gene. Different concentrations of MnCl₂ are used in each reaction to vary the mutational load in the PCR products. Figure 3 shows the agarose gel analysis of the different PCR products. In this gel the mutated PCR fragments all show bands of 90 base pairs, as expected. In lanes 2, 3 and 4 the density of the band for the PCR product increases as the concentration of the MnCl₂ added to the PCR reaction is augmented. However in lane 4, where 0.4 mM of MnCl₂ is added to the reaction, the amount of amplified PCR product reaches the maximum, and starts to gradually decrease till it disappears as the concentration of MnCl₂is further increased, as shown in lane 5, 6 and 7. In lane 8, the presence of a concentrated PCR product indicates the reproducibility of PCR reaction 1.

Digest analysis of the mutated PCR amplified products:

In order to estimate the rate of mutations being randomly induced in the DNA fragment of interest (12bp), the restriction enzyme BstEJJ is used to digest the different PCR products.

BstEII recognises its restriction site of 5 base pairs long within the 12 base pairs. Figure 4 shows the agarose gel analysis of the different BstEII treated PCR products. In lane 2,3 the faint bands shown represent the wild type DNA fragment after being cut by BstEII on its restriction site, resulting in two BstEII cut DNA fragments which merge with each other on the gel because the difference between them is only 6 base pairs. In lane 4, the appearance of the two distinct bands is due to the induction of mutations in the BstEII restriction site.

Firstly the heavier band containing 90 base pair, represent the uncut DNA fragment because the BstEJJ restriction site has been mutated, and therefore BstEII could not recognise it. Secondly, the lighter band which run further on the gel, contains the 2 DNA fragments generated after BstEII digestion. From the results shown in lane 4, it is assumed that the mutations in the BstEII restriction site have been induced in 50% of the DNA population after the Error-prone PCR reaction. By contrast, lane 4 only contains the uncut PCR product, meaning that all of the PCR product has a mutated BstEII restriction site. Lane 5, however, only contains the BstEII cut PCR product as no mutations are induced due to the absence of MnCl₂ in the PCR reaction.

Mutations probability in the BstEII restriction site in the tk gene:

In order to calculate the probability of a random mutation hit induced in the BstEII restriction site within the TKPCR fragment, the probabilities_Nof all induced mutations in the rest of the DNA fragment (7 bp) are calculated.

-7bp- -][- -5bp-

The probability of a mutation hit in the BstEII restriction site is calculated using the equation below:

Probability of a mutated BstEII restriction site = 1 - ( 7/12 )ⁿ , where n = the number of mutations induced.

The graph in Figure 5 shows the mutation probability in the BstEII restriction site versus the number of random mutations induced in the whole DNA fragment (12 bp).

The probability results shown on the graph are only applicable if the mutations are induced at random and therefore the mutations can occur in the same site again. The graph shows that the mutation hit in the restriction site goes up with respect to the increased mutation number, from 0 to 5 mutation hits in the whole gene (12 base pairs).

The probability of the mutation hit in the restriction site achieves its maximum "1" after the induction of approximately 8 mutations in the whole gene. Based on the genetic code, the number of possible amino acid substitutions possible on average is 5.7 per single base pair mutation. Therefore the number of likely Transketolase mutants generated is 5.7⁴= 1055.60 mutants.

PCR amplification of the pQR711 plasmid DNA:

The recombinant plasmid DNA pQR711 is amplified using QuickChange™ PCR technique, where the two mutated fragments in the TK gene from the second Enor prone PCR (lane 2 & 5 in figure 5) are used as primers. These mutagenic primers mutate the TK gene bom in the plasmid DNA as the PCR cycles proceed. Figure 7 shows the agarose gel analysis of the 2 amplified plasmids using the QuickChange™ PCR protocol.

This PCR technique uses the nonstrand-displacing action of Pfu DNA polymerase, extends and incorporates the mutagenic primers resulting in the amplified pQR711 plasmid DNA carrying the mutated TK genes. Figure 6 shows the agarose gel analysis of these 2 PCR products.

In lane 1, where the mutagenic primers used are taken from Error-prone PCR reaction 3, the concentration of the PCR product is little as shown by the very faint bands, due to experimental errors when preparing the PCR reactions.

In lane 2, however, where the mutagenic primers used are taken from Error-prone PCR reaction 4, the concentration of the PCR product is significantly higher. The 2 distinct bands seen on this lane have different sizes ranging from 4,324 base pairs to 6,369 base pairs due to variations in the DNA extension cycle. By contrast, the purified recombinant pQR711 plasmid DNA loaded in lane 3 shows 3 bands, representing the 3 different forms of the plasmid DNA. The relaxed circular form of the plasmid runs slowest on the gel because it contains bulky DNA, making the movement of the DNA through the gel very slow. Then the linear form of the plasmid shows a band of roughly 6 kb long and finally comes the supercoiled form of the plasmid, which runs fastest in the gel.

Dpnl digestion analysis for PCR amplified recombinant pQR711 plasmid DNA After the production of PCR amplified pQR711 plasmid DNA carrying the mutant TK genes, the parental template DNA is digested using Dpnl restriction enzyme, which cuts the DNA on the methylated, non-mutated C residues. The 2 different PCR amplified JM107 PQR711 plasmids as well as the non-amplified pQR711 plasmid DNA are digested with Dpnl. Figure 7 shows 0.8% agarose gel digest analysis of the different samples. In lane 1, the very faint band corresponding to the size of 5.8 KB represents the new mutated pQR711 plasmid by PCR because it has not been cut by the restriction enzyme Dpnl . The band representing the mutant plasmid is not very clear due to the inefficiency of the PCR amplification reaction.

However the other 4 bands running further on the gel are due to the Dpnl digestion of the original plasmid, which remained after the PCR amplification reaction. The original pQR711 plasmid DNA contains 21 Dpnl restriction sites, resulting in 21 fragments after a complete digestion with Dpnl enzyme.

By contrast, only 4 bands are shown for the Dpnl digested original plasmid. This is due to the absence of the small Dpnl cut fragments because 0.8% gel can not detect fragments smaller than about 500 base pairs. In lane 2, the band representing the mutant amplified plasmid is significantly more dense that its counterpart in lane 1. Whereas the concentration of the Dpnl generated fragment from the remaining original plasmid is lower.

In other words, the PCR amplification reaction was more efficient for the PCR product loaded in lane 2 than lane 1, because more of the template DNA was mutated during the PCR reaction.

Lane 3 shows the original plasmid digested with Dpnl, where the whole plasmid is cut by Dpnl generating populations of DNA fragments at different sizes, as shown with the remaining original plasmid being cut by Dpnl from both PCR samples. The different DNA fragment bands seen in lane 3 have the sizes: 1310, 995, 734, 536 and a smear of 17 fragments. However the densities of the fragments seen in lane 3 are significantly higher than the bands seen in lane 1 & 2 since all the DNA is digested in the original plasmid, unlike in the PCR samples where only the remaining original template plasmid DNA are digested. Lane 3 shows 3 bands representing the three different forms of the non-amplified original plasmid DNA: circular, linear and supercoiled forms respectively.

Transformation of mutated plasmid in the competent JM107 E-coli cells:

The mutant plasmids pQR711 by PCR reaction using the mutagenic primers are introduced into the competent JM107 E-coli cells. The efficiency of the competent JM107 cells is calculated after the transformation the original plasmid pQR711. The table below shows the different results achieved after using different DNA samples.

The number of colonies (100 colonies) seen when the JM107 were transformed with the intact plasmid indicates that the transformation efficiency of these competent cells is 37000 colonies/mg. However the absence of colonies in the plates, where the competent JM107 cells are transformed with the Dpnl treated original plasmid is due to the complete digestion of the plasmid with Dpnl. Therefore the cells do not have any plasmid DNA to uptake. Whereas the transformation of the JM107 cells with 30 ng or 3ng of PCR 1 amplified plasmid, led to growth of 1 colony due to the low efficiency of the competent cells and also the tiny amount of plasmid DNA added to the cells. The low transformation efficiency is mainly due to the double-nicked mutated plasmid PQR711.

On the other hand, 3 colonies of JM107 E-coli cells were transformed after the addition of 30ng and 3ng of PCR2 amplified plasmid DNA. The results show that the JM107 cells could take up the mutated plasmid DNA, resulting in transformed JM107 colonies that are ampicillin resistant, and therefore can survive on the agar plates containing the ampicillin.

The number of the transformants can be significantly raised by increasing the amount of plasmid DNA added to the competent JM107 cells, and also by improving the transformation efficiency of the competent cells.

The method of introducing sequence diversity into the active site of the TK gene described above is again performed and a number of the obtained mutants are sequenced in order to determine the changes to the sequence.

The plasmid pQR711 carries the bacterial transketolase gene tktA. A 12 bp portion of the gene, corresponding to the enzyme's active site, was the target for random mutagenesis in this study. By introducing sequence diversity in this region it is possible to create mutants of transketolase with markedly different substrate specificites to the wild-type. Primers TKF1 and TKF2 were used to amplify the target sequence. The error prone PCR step itself (schematically shown in Figure 1) involved eight 100 μl reactions, all containing the following: 5 U Taq polymerase (Roche Diagnostics, Lewes, East Sussex, UK), Taq polymerase buffer, 30 ng ρQR711 DNA, 0.5 mM of each primer (tktAlF and tktAIR), 0.2 mM of each deoxynucleoside-triphosphate (dATP, dCTP, dGTP, and dTTP), and 1.5 mM magnesium chloride. To reduce the fidelity of the Taq polymerase, and thus achieve "ercor-prone" PCR, manganese (II) chloride was added to the reactions. A range of mutation densities were investigated by increasing MnCl₂ concentration across the reactions in 0.2 mM increments from 0 mM in Rl (reaction 1) to 1.4 mM in R8. The samples were submitted to the following program of thermal cycling in a TechGene® (Techne, Cambridge, UK): 94°C for 3 min; 25 cycles of 94°C for 1 min, 55°C for 1 min, 68°C for 1 min; a final extension step of 68°C for 10 min. After amplification, the samples were purified using a QIAquick® PCR Purification Kit (Qiagen, Crawley, West Sussex, UK). Agarose gel electrophoresis revealed that the 80 bp fragment had been amplified in only the first five reactions (Rl-5) with MnCl₂ concentrations higher than 0.8 mM severely inhibiting the polymerase. It will be apparent to those skilled in the art that the concentration of MnCl₂ that will work will vary on a case to case basis due to the variation in the other components of the method. Accordingly, higher concentrations of MnCl₂ may be used in the method of the present invention.

The presence of a BsiER restriction site in the target sequence (Figure 8) allowed the relative mutation rates of the five successful enor prone PCR reactions to be estimated. Each reaction was digested with 5 U ifatEII (Roche Diagnostics) for 2 hr at 37°C and the fragments were analysed by agarose gel electrophoresis. Rl (0.0 mM MnCl₂) was completely digested, indicating that the restriction site had been faithfully replicated in all of the product. R5 (0.8 mM MnCl₂) was the least digested with approximately 50% of the 80 bp products carrying mutations in the BstEE restriction site. Reactions R2, R3 and R4 appeared to have intermediate levels of digestion. In the next step of the procedure, an undigested aliquot of R5 was used to generate a library of pQR711 mutants using a variation of the QuikChange™ protocol (Figure 1C). The 50 μl PCR reaction contained 2.5 U PfuTurbo polymerase (Stratagene), PfuTurbo polymerase buffer (including magnesium), 150 ng of dsDNA template (pQR711), and 0.2 mM of each deoxynucleoside-triphosphate (dATP, dCTP, dGTP, and dTTP). 250 ng of the R5 product replaced the usual mutagenic primers. The following program of thermal cycling was then used: 18 cycles of 95°C for 30 sec, 55°C for 1 min, 68°C for 12 min. The output from the reaction was digested with 10 U of Dpnl (Stratagene) for 2 hr at 37°C to remove the methylated parental DNA (Figure ID). 1 ml of the digestion was transformed into E. coli XLl-Blue competent cells (Stratagene) by heat-shock. Following plating on ampicillin-agar, the total library size was estimated to be approximately five thousand mutants.

The target region, including flanking DNA, was sequenced for ten colonies (Figure 8). Five of the colonies were found to have single mutations, with two occurring inside the 12 bp target sequence (S5 and S8) and three within the tktAIR primer region (S3, S4, and S7). A sixth colony was a double mutant with mutations in both the target sequence and in tktAIR (SI).

Only one out of the ten sequenced mutants was found to have a mutation in the BstEII restriction site (SI). BsiEU digestion of the epPCR fragments before library creation revealed a frequency of 50%; this drop in mutational load may have resulted from selection against excessive mutation by the QuikChange™ procedure. The new QuikChange™ Multi Site-Directed Mutagenesis Kit (Stratagene) can be used to reduce this effect.

In summary, this method facilitates the rapid creation of a mutant library in which mutation is hmited to a short target sequence. Mutation density can be adjusted by modulating the concentration of MnCl₂ in the enor prone PCR step and quickly ascertained by digestion of the product with a restriction enzyme. The mutational load could potentially be increased further by repeating the method with plasmid DNA prepared from the first library, or by repeating the enor prone PCR step with the product from the first enor prone PCR reaction. It should be noted that several mutations occurred in the primer regions, which is not unexpected since the enor prone PCR process does not exclude mutation of these sections. The method allows the generation of a library where sequence diversity is biased towards a nanow region of sequence with some mutagenesis occurring in the primer regions. This contrasts with the highly defined libraries of oligonucleotide mutagenesis, or the virtually undefined libraries typically generated using enor prone PCR of entire genes. The method can also be applied towards target sequences ranging from a few base-pairs to several hundred.

As indicated above, the methods of the present invention can be used to generate libraries of proteins or enzymes with randomised active sites, binding sites or hydrophobic cores etc. Such libraries could be screened for directed evolution studies or selected, for example, by phage-display (Parmley et al., Gene, 73, 305-318, 1988). Aside from the greater ease of library generation by the method of the present invention, one major benefit is that the same primers can be used to create several libraries of varying mutagenic load, in contrast to oligonucleotide cassette mutagenesis where the library diversity is designed into the primers.

Figure 9 is a histogram showing the distribution of mutation between amplified primer sequences and the amplified intervening sequence based on the data shown in Figure 8. The x-axis shows the frequency of mutation within a sliding window of 5 bases across the amplified region. The primers are at nucleotide positions 1 to 34 and 47 to 81, and the active site is at nucleotide positions 35 to 46. It can be seen that mutations generally occur in the interevening sequence, i.e. the active site, more frequently than in primer sequences.

All documents cited above are incorportated herein by reference.

Claims

1. A method of producing a modified polypeptide with improved characteristics comprising the steps of (a) obtaining nucleic acid primers which flank an active site within a parent nucleic acid sequence encoding a parent polypeptide;

(b) carrying out a polymerase chain reaction (PCR) using said primers and the parent nucleic acid sequence as a template under suitable conditions for introducing mutations into the amplified active site sequence; (c) isolating said mutated active site;

(d) introducing said mutated active site into the parent nucleic acid sequence to replace the non-mutated active site thereby producing a modified nucleic acid sequence; and

(e) expressing said modified nucleic acid sequence to produce a modified polypeptide.

2. The method according claim 1 further comprising the step of screening the expressed polypeptide for improved characteristics.

3. The method according to claim 1 or claim 2, wherein the protein is an enzyme, a receptor, a ligand, an antibody, an antigen, or a cytokine.

4. The method according to claim 1 or claim 2, wherein the active site is a region associated with catalysis, a binding site, a folding nucleus, a hydrophobic core or an epitope.

5. A method for producing a modified functional nucleic acid with impro ed characteristics comprising the steps of

(c) isolating said mutated active site; and (d) introducing said mutated active site into the parent nucleic acid sequence to replace the non-mutated active site thereby producing a modified nucleic acid sequence.

6. The method according claim 5 further comprising the step of screening the modified nucleic acid sequence for improved characteristics.

7. The method according to claim 5 or claim 6, wherein the functional nucleic acid is a promoter, an enhancer, catalytic RNA or a transposable DNA element.

8. The method according to claim 5 or claim 6, wherein the active site is a region associated with transcription of an operably linked gene, a ligand binding site or a region involved in the teriary structure of a nucleic acid.

9. The method of any one of the preceding claims which additionally comprises as a first step determining the active site within the parent nucleic acid sequence.

10. The method according to any one of the preceding claims, wherein there is a single active site.

11. The method according to any one of claims 1 to 9, wherein there are multiple active sites.

12. The method according to any one of the preceding claims, wherein the level of mutations introduced into the amplified sequence is controlled by varying the concentration of Mn2⁺ in the PCR.

13. The method according to claim 12, wherein the concentration of Mn²⁺ in the PCR is between about 0.0 ImM and about 2 mM.

14. The method according to claim 7, wherein the concentration of Mn²⁺ in the

PCR is between about 0.05mM and about ImM.

15. The method according to any one of the preceding claims, wherein the parent nucleic acid sequence is between 50 bp and several thousand bp in size.

16. The method according to any one of the preceding claims, wherein the active site is between 10 and lOOObp in size.

17. The method according to any one of claims 1 to 15, wherein the active site is between 10 and lOObp in size.

18. The method according to any one of claims 1 to 15, wherein the active site is between 10 and 50bp or 10 and 20bp in size.

19. The method according to any one of the preceding claims, wherein the primers incorporate a restriction enzyme site to assist with the isolation of the amplified product.

20. The method according to any one of the preceding claims, wherein the primers are between 8bp and 50bp in length.

21. The method according to any one of claims 1 to 19, wherein the primers are between 10 and 30bp in length.

22. A modified protein produced by the method according to any one of claims 1 to 4.

23. A modified nucleic acid produced by the method according to any one of claims 5 to 8.

24. A pharmaceutical composition comprising the modified protein according to claim 22 or the modified nucleic acid according to claim 23, and a pharmaceutically acceptable excipient, carrier, buffer or stabiliser.

25. The pharmaceutical composition according to claim 24 for use in therapy.

26. A kit for performing the method according to any one of claims 1 to 21 comprising one or more of the following substances or components: Taq polymerase, MnCl₂ solution, MgCl₂ solution, dNTP's, 10 x polymerase buffer, control plasmid DNA, control primers 5' and 3', control restriction enzyme, lOx restriction enzyme buffer, Dpnl restriction enzyme, and/or competent cells, and instructions for performing the method according to any one of claims 1 to 21.

27. A method of randomly modifying a specific region of a functional nucleic acid sequence or polypeptide sequence while maintaining the remaining sequence so as to arrive at a functional nucleic acid or polypeptide with improved characteristics.