MXPA99010476A - Method for the construction of binomial libraries of mutagenized oligodeoxyribonucleotides at dock level using deoxynucleoside-phosphoramidites - Google Patents
Method for the construction of binomial libraries of mutagenized oligodeoxyribonucleotides at dock level using deoxynucleoside-phosphoramiditesInfo
- Publication number
- MXPA99010476A MXPA99010476A MXPA/A/1999/010476A MX9910476A MXPA99010476A MX PA99010476 A MXPA99010476 A MX PA99010476A MX 9910476 A MX9910476 A MX 9910476A MX PA99010476 A MXPA99010476 A MX PA99010476A
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- Mexico
- Prior art keywords
- dnf
- mutagenesis
- binomial
- construction
- codon
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Links
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Abstract
La invención se refiere a un método de mutagénesis para la construcción de bibliotecas binomiales de oligodesoxirribonucleótidos mutagenizados a nivel de codón que comprende el uso de dos conjuntos de desoxinucleósido-fosforamiditos protegidos en el hidroxilo 51 mediante los grupos protectores ortogonales entre sí, los cuales son combinados durante la síntesis de oligodesoxirribonucleótidos. Este método constituye una valiosa herramienta para el estudio de la relación estructura-función de proteínas e ingeniería de las mismas, ya que se pueden generar de manera controlada y predecible pocos reemplazos de aminoácidos por proteína, permitiendo investigar la importancia individual de cada uno de los aminoácidos silvestres para la función de la proteína y al mismo tiempo evitar su destrucción funcional, a fin de mejorar una innumerable cantidad de proteínas con aplicación comercial, como por ejemplo las enzimas denominadas subtilisinas que se utilizan en los detergentes biodegradables.
Description
Method for the construction of binomial libraries of oligodeoxyribonucleotides, utagenizados at the codon level using deoxynucleoside-phosphoramidites.
TECHNICAL FIELD OF THE INVENTION
The invention object of the present invention relates to a method of mutagenesis for the construction of mutagenized oligodeoxyribonucleotide libraries at the codon level, by means of which the generation of binomial distributions of mutants towards few or many codon replacements can be controlled by mutant oligonucleotide in the form directly proportional to the level of -mutagenesis. This method comprises the use of two sets of deoxynucleoside-phosphoramidites (DNF's) protected at the 5 'hydroxyl by the orthogonal protective groups 4,4' -dimethoxytrityl (DMT) or 9-fluorenylmethoxycarbonyl (Fmoc), which are combined during the synthesis of oligodeoxyribonucleotides.
BACKGROUND OF THE INVENTION
The proteins are constituted mainly by 20 different amino acids, and since the DNA is only composed of 4 different deoxynucleotides, each of the amino acids of a protein is encoded by at least one group of three deoxynucleotides which is called triplet or codon.
It is now known that the function of a protein, such as molecular recognition or catalysis, depends on its characteristic three-dimensional structure, ordered by its primary amino acid sequence. For this reason, many research groups are interested in studying the structure-function relationship of proteins in order to rationally improve a large number of enzymes that may well have a commercial application.
Protein engineering is the structural modification of these materials through the modification of their coding gene, in order to understand the relationships that are established between the amino acid sequence, the final folding of the polypeptide chain and its function.
This work is feasible thanks to advances in recombinant DNA technology and methods of chemical synthesis of DNA, which make it possible to replace, eliminate or add any amino acid in a protein, modifying in its coding gene the respective codon. It is known that a large number of proteins can dramatically alter their properties by changing a single, functionally critical amino acid.
Site-directed mutagenesis (by nucleotide or codon), through synthetic oligodeoxyribonucleotides, is being used extensively in almost all disciplines of biochemistry to explore the relationship between protein structure and function. However, the selective perturbation of individual amino acids requires some understanding at the molecular level of the interacting structures (protein-protein, enzyme-substrate or protein-DNA). That is, you need to have some bases to predict the changes of some amino acid residues that will produce a particular functional consequence in the protein. Therefore, rational attempts in the engineering and evolution of proteins to modify existing proteins are limited to the need to have a high resolution structure of the protein of interest and a good understanding of its molecular mechanism. Unfortunately there are few systems that meet this requirement [Hermes, J.D. 1989].
An alternative method, of a more general nature, both for the understanding of structure-function relationships, and for protein engineering, employs random mutagenesis and in particular one of its versions, combinatorial mutagenesis. In this method a random "mix" of mutants (library) is generated, instead of introducing particular premeditated changes. Once the library of mutant genes is created, they are cloned and the proteins that these genes encode are expressed in an appropriate host. The transformed colonies are then selected or monitored, looking for the appearance of a phenotype caused by the properties of the new proteins (e.g., greater thermostability, greater catalytic power, different specificity, etc.). When the procedure allows to explore combinations of mutants, it is called combinatorial mutagenesis.
In the literature, a large number of random gene mutagenesis protocols have been described, generally classified in chemical methods [Botstein, D. 1985], enzymatic [Lehtovaara,
P.M. _1988] and oligo-directed [Hermes, J.D. 1989].
Chemical methods require exposure of the microorganism to mutagenic reagents [Myers, R.M. 1985 and Kadonaga, J.T. 1985], such as sodium bisulfite, hydroxylamine and nitrous acid, among others, which cause modifications on the deoxynucleotides that make up cellular DNA. In enzymatic methods [Lehtovara, et al. 1988 and Reeve M.A. 1995] it is necessary to make use of polymerases that under certain experimental conditions make mistakes during the nucleotide addition, some examples of these enzymes are the reverse transcriptase of avian myeloblastosis virus and Taq polymerase. In both cases, libraries of mutant genes that present specific nucleotide changes are generated, allowing only between 18% and 40% of all possible amino acid changes to be analyzed, a direct consequence of the degeneracy of the genetic code [Sirotkin, K. 1986] . In both methods, the window of mutagenesis, the representativeness of amino acids, the distribution of amino acids, and the level of mutagenesis are difficult to control.
The method of combinatorial mutagenesis directed by oligonucleotides, recurs. to the use of libraries of synthetic oligodeoxyribonucleotides, produced in a single experiment through mixtures of the four deoxynucleotides [Dunn I.S. 1988 and Del Río, G. 1994]. This type of mutagenesis can be performed "by saturation" or "contamination". In the first one, each wild-type deoxynucleotide of the codons to be mutated is replaced by a mixture of the four deoxynucleotides, generating a library of variant codons of size 64n (32n in case of using the NNG / C system) where n represents the number of codons to replace. Considering a practical transformation efficiency of 107 to 109 colonies per library of oligodeoxyribonucleotides, with this methodology it is only possible to analyze a maximum of 5 amino acids per experiment. However, the main drawback of this methodology is that to obtain a sampling of codons that represent all the amino acids, a greater representativeness of mutants of high multiplicity (several changes of codons per gene) in the sample is incurred, which normally they generate loss in protein function.
The combinatorial mutagenesis by contamination, directed by oligodeoxyribonucleotides, consists in contaminating each of the wild nucleotide couplings during the chemical synthesis of the oligonucleotide, with a small proportion (a) of the 3 non-wild-type deoxynucleotides [Hermes, J.D. 1989 and Ner S.S. 1988]. Although this methodology allows to obtain libraries rich in low multiplicity mutants (few changes of codons per gene) and to explore windows of relatively large mutagenesis, the problem inherent to the degeneracy of the genetic code is still present, that is to say, it continues to favor the replacement of those amino acids whose codons only vary on a basis with respect to wild ones [Sirotkin K., 1986].
The ideal random mutagenesis protocol must meet many requirements. First, the region of n codons or amino acids to be explored (that is, the mutagenesis window) must be easily specified. Second, each codon located in the mutagenesis window must have the same probability of being replaced (homogeneous distribution). Third, the replacement of an amino acid by any of the other 19 must occur with the same probability (homogeneous frequency). Fourth, the system must allow defining immutable positions within the window-of mutagenesis. Fifth, it should be possible to control the rate or level of mutagenesis (a) in order to be able to adjust the density of mutants desired through the combinatorial analysis theory, and sixth, the mutagenesis efficiency must be high so that most of the clones analyzed correspond to mutant sequences [Hermes, JD 1989; Lehtovaara, P.M. 1988 and Sondek, J. & Shortle, D. 1992].
Some authors agree that the ideal method of combinatorial mutagenesis should involve the use of mixtures of trinucleotides to generate mutagenesis at the codon level and not at the nucleotide level in order to explore the sequence space in a better way. These trinucleotides must be able to be coupled in a substoichiometric manner during the conventional synthesis of oligodeoxyribonucleotides to generate libraries that follow a predictable binomial distribution of mutants.
The aforementioned method would avoid the problems associated with the degeneracy of the genetic code and would allow a homogeneous distribution and frequency of variants. However, these reagents are not yet commercially available and their preparation involves a considerable time and cost (U.S. Serial No. 60 / 123,438).
Another method used to perform mutagenesis at the codon level is the so-called "media separation method" (Cormack, BP 1993, Glaser, SM 1992, Hooft van Huijsduijnen, RAM 1992) in which the oligonucleotide is synthesized in a column or reactor to the point where it is desired to initiate mutagenesis, at which time the column is dismantled to initiate the replacement of the codon; the CPG support containing the growing oligonucleotide is separated into two portions (according to the level of mutagenesis defined by the size of the window and the multiplicity of substitutions to be favored), which are repacked within a column called mutant and another column called wild. The mutant column is subjected to three cycles of synthesis with an equimolar mixture of the four deoxynucleoside phosphoramidites (N) in the first two positions of the codon and an equimolar mixture of G / C in the third position to generate a NNG / C combination. which produces 32 codons that code at least once for each of the twenty amino acids. The wild column is subjected to three cycles of synthesis with the DNF's that define the wild sequence. The two columns are reopened, the support of both is combined and separated again repeating the process for each codon to mutate.
Despite the apparent simplicity of the support separation method, it turns out to be very laborious and tedious, requires large amounts of CPG support and therefore large amounts of DNF's to be able to handle low levels of mutagenesis and relatively large mutagenesis windows or otherwise, it can only be applied for high levels of mutagenesis and small windows of mutagenesis.
DETAILED DESCRIPTION OF THE INVENTION
BRIEF DESCRIPTION OF THE FIGURES
Figure 1.- Illustrates the synthesis of Fmoc-deoxynucleosides and the corresponding methyl-phosphoramidites. B = 6N-benzoyladenine, 4N-benzoylcytosine, 2N-isobutyrylguanine or thymine. Fmoc-Cl = 9-fluorenylmethoxycarbonyl chloride, Py = pyridine, DIPEA = N, N-diisopropylethylamine.
Figure 2. - Protocol called "pre-addition" for the assembly of libraries of oligodeoxyribonucleotides by the Orthogonal Combinatorial Mutagenesis Method
(MCO). A) Wild sequence to mutate. This sequence comprises: a) the 3 'flanking region, b) window of mutagenesis including only two amino acids and e) the 5' flanking region. B) Procedure for the assembly of oligodeoxyribonucleotide libraries by the pre-addition protocol. 1) assembly of the wild sequence of the 3 'flanking region with the appropriate A-DNF's; 2) first cycle of mutagenesis with an ordered combination of B-DNF's and A-DNF's; 3) second cycle of mutagenesis and 4) assembly of the wild-type sequence of the 5 'flanking region with A-DNF's. Where:? - - Represents the support CPG, w = wild-type deoxynucleotide, A-DNF = deoxynucleoside-phosphoramidite protected with group A, wA = A-DNF appropriate according to the wild-type sequence, B-DNF-deoxynucleoside-protected phosphoramidite with group B, NB = diluted solution of the four B-DNF's, NB = "concentrated solution of the four B-DNF's and S-A = concentrated solution of A-dG and A-dC-phosphoramidites Figure 3. - Protocol called "mixed in line" for the assembly of oligodeoxyribonucleotide libraries by the MCO method. The only difference with respect to the pre-addition protocol is that the contamination is carried out by the simultaneous release of the diluted mixture containing the four B-DNF's and the first A-DNF that defines the first nucleoside of the wild-type codon to utar. Where: Q ^) -Represents the CPG support, w = wild-type deoxynucleotide, A-DNF = deoxynucleoside-phosphoramidite protected with group A, wA = A-DNF appropriate according to the wild-type sequence, B-DNF = deoxynucleoside-phosphoramidite protected with group B, NB = diluted solution of the four B-DNF's, NB = concentrated solution of the four B-DNF's and SA = concentrated solution of A-dG and A-dC-phosphoramidites.
Figure 4.- Combinatorial libraries of oligodeoxyribonucleotides assembled with the MCO method. Gray represents the distribution of mutants obtained in Ira. library (a = 49.5%), black represents the distribution of mutants obtained in the 2nd. library (a = 78.9%) and the white represents the distribution of mutants obtained in the 3rd. library (a = 10.61%).
As mentioned in the previous section, there are several methods of combinatorial mutagenesis, directed by oligodeoxyribonucleotides, in which some authors have described the use of mixtures of trinucleotides to generate mutagenesis at the codon level instead of mutagenesis at the nucleotide level, reporting that it is possible to generate libraries that follow a predictable binomial distribution of mutants, with advantages such as: a better exploration of the sequence space (by minimizing the problems associated with the degeneracy of the genetic code) and a better distribution and frequency of variants. However, an important limitation of these methods is that the reagents, particularly the trinucleotides are not commercially available and also their preparation involves a considerable time and cost.
On the other hand, several authors describe codon-level mutagenesis methods that are based on the use of conventional DNF's, as is the case of the media separation method, in which the wild-type sequence of the oligonucleotide is assembled in a column, while that in another the mixture of mutant codons is synthesized using conventional DNF 's.
The media separation method is highly manual, laborious, tedious, requires large amounts of CPG support and therefore large amounts of DNF's to handle low levels of mutagenesis and large windows or otherwise, this method is only viable for high levels of mutagenesis and small windows.
The inventors of the present invention propose a solution to these limitations consisting of an alternative method for the construction of binomial libraries of mutagenized oligodeoxyribonucleotides at the codon level, with clear advantages over the aforementioned methods, since by this method it is feasible to handle and control any level of mutagenesis and window size in an almost automated way.
The mutagenesis method of the present invention, called Orthogonal Combinatorial Mutagenesis (MCO), makes use of two sets of deoxynucleoside-phosphoramidites (DNF's) protected in the 5 'hydroxyl with two protective groups orthogonal to each other, that is to say they present stability conditions and / or removal opposite or opposite, as is the case of group 4, 4'-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc), the DMT group is labile to acidic conditions and stable to basic conditions, while the group Fmoc is stable to acidic conditions and labile to basic conditions. On the other hand, the derivatization of the 3 'hydroxyl with the phosphoramidite function favors the incorporation of said DNF's in the automated synthesis of oligodeoxyribonucleotides.
The inventors proposed the use of two sets of deoxynucleoside-phosphoramidites (DNF's) protected at the 5 'hydroxyl with orthogonal protecting groups from each other for the construction of binomial libraries of oligodeoxyribonucleotides mutagenized at the codon level, ie, oligodeoxyribonucleotides mutagenized at the level of codons follow binomial or Gaussian distribution biased according to the level of mutagenesis, characterized because it comprises the following steps:
a) Attach sequentially on a solid support, the deoxynucleoside phosphoramidites (DNF's) protected by a group A (A-DNF's), to assemble the wild-type sequence corresponding to the 3 'flanking zone of the mutagenesis window that it is desired to explore in a determined gene;
b) initiate a cycle of mutagenesis, which comprises the following steps: 1.- coupling to the previously assembled sequence, the deoxynucleotide of Ira. position of the mutant codon by the addition of a mixture containing the protected DNFs at the 5 'hydroxyl group B (B-DNF's); at an appropriate concentration that allows generating a previously defined level of mutagenesis; 2. - coupling to the chains of oligodeoxyribonucleotides that did not react in the previous step, the deoxynucleotide of Ira. position of the wild-type codon by the addition of A-DNF, appropriate according to the pattern of the wild-type sequence; 3.- continue with the coupling of the 2nd. and 3rd. position of both the mutant and wild-type codons, regardless of the coupling order, only keeping the following conditions: I) In the two subsequent couplings for the wild-type codon, A-DNF's are used, according to the pattern of the wild-type sequence; II) for the coupling of the 2nd. position of the mutant codon, a high concentration mixture containing the B-DNF's is used; III) for the coupling of the 3rd. position of the mutant codon, a high concentration mixture containing DNF's protected with group B or A is used.
optionally, the mutagenesis cycle of part b) is repeated, as many times as necessary to conclude the number of codons that one wishes to explore;
d) concluded the mutagenesis cycles, synthesize the wild sequence corresponding to the 5 'flanking zone of the mutagenesis window, using the appropriate A-DNFs according to the wild sequence.
That is, in the Orthogonal Combinatorial Mutagenesis method, illustrated in Figure 2, the wild sequence is assembled with A-DNF's, while para-assembling the Ira. and 2nd. position of the mutant codons, DNF's are used protected with a group orthogonal to that used in the wild sequence, called B-DNF's and particularly, for the assembly of the 3rd. position, DNF's protected either with group A or with group B can be used indiscriminately. It is important to mention that an important feature of the method of the present invention is that protective groups A and B must exhibit stability and removal characteristics. orthogonal to each other. This feature favors the combined coupling of the DNF's to synthesize the wild-type codon and the mutant codons.
There are several protective groups that have been used for the protection of the 5 'hydroxyl of the DNF's, among which we can mention: 4, 4'-dimethoxytrifilo (DMT), 4'-monomethoxytrifilo (MMT), terbutildimetilsilano (TBDMS), terbutildifenilsilano ( TBDPS), 9-fluorenylmethoxycarbonyl
(Fmoc), levulinyl (Lev) and 2-dansylethoxycarbonyl, among others. The DMT group is the most commonly used for the protection of the DNF's that are used in the preparation of normal oligodeoxyribonucleotides, which is why in this invention the inventors have designated it as a conventional group. This group is labile to mild acid conditions. DMT-DNF's are commercially available.
The Fmoc group, orthogonal to the DMT group, has several advantages compared to other orthogonal protective groups, since it is highly regioselective towards the primary hydroxyl of the nucleosides, is commercially available and is quickly removed by a mild basic treatment. On the other hand, although currently the Fmoc-DNF's are not commercially available, their synthesis is relatively simple and proceeds with high yields.
The inventors propose to use the DMT and Fmoc groups, for the protection of the DNF's used for the construction of binomial libraries of mutant oligodeoxyribonucleotides with the method of the present invention, given the characteristics of orthogonality between them and that it is not required to modify the protocols standard of synthesis recommended by the manufacturer of equipment conventionally used for synthesis of oligodeoxyribonucleotides.
The couplings of the DMT-DNF's and Fmoc-DNF 's were made in an automatic DNA synthesizer through a normal synthesis protocol as recommended by the manufacturer. This protocol includes for each coupling the following steps: 1) hydrolysis of the DMT group with an acid solution for example 2% dichloroacetic acid in dichloromethane and / or Fmoc with a basic solution for example 0.1 M DBU in acetonitrile; 2) coupling of DMT-DNF or Fmoc-DNF to the growing oligonucleotide on the solid support in the presence of tetrazole; 3) blockade by acetylation of the 5 'hydroxyls that did not react and 4) oxidation of the newly formed triester phosphite to more stable triester phosphate.
Having a mutagenesis strategy at the codon level, which generates binomial libraries of mutant oligodeoxyribonucleotides, using reagents that are easily synthesized or commercially available and practically automated, constitutes a valuable tool for the study of the structure-function relationship of proteins and engineering thereof. Through this strategy, few amino acid replacements per protein can be generated, which allows to investigate the individual importance of each one of the wild-type amino acids for the function of the protein and at the same time prevent their functional destruction, in order to improve by in vitro evolution, an innumerable amount of proteins with current and / or future commercial application, such as the enzymes called subtilisins, which have been modified to withstand extreme temperature and pH conditions, which give them advantages for use in biodegradable detergents.
Another way to carry out the method for the construction of binomial libraries in a simplified way, is described in Figure 3 and consists of modifying the mutagenesis cycle of part b), performing steps 1 and 2 in a single step, by means of the simultaneous addition of a mixture of the B-DNF's for the Ira assembly. position of the mutant codon and the A-DNF corresponding to the wild-type sequence. In this way, the reduction in the number of steps of the mutagenesis cycle is achieved and, therefore, the method becomes simpler and faster.
A preferred way for the inventors to carry out the method for the construction of binomial libraries is to perform the coupling of the 3rd. position of the codon_mutante, by means of a mixture of high concentration that contains the DNF's protected with a group A.
This alternative allows, on the one hand, to ensure that the coupling of the 3rd is carried out. position of the mutant codon given the high concentration of the mixture and on the other hand, it is favored that at the end of the coupling of the mutant codon, all the sequences are protected with the same protective group ie A, and therefore at the end, with only one Removal step of the protective group, both the wild-type codon and the mutant are deprotected.
Preferably, the method of the present invention uses the protective group A as the DMT and as the protective group B as the Fmoc. This option comprises using DNF's protected with the DMT group for coupling the codons according to the wild-type pattern and DNF's protected with the Fmoc group for the coupling of the mutant codons. This represents certain advantages, since in this way, the reagents that are consumed in greater proportion are the DMT-DNF's, which are currently commercially available.
(Glen Research). However, if the commercial interest in Fmoc-DNF 's is increased in the future and these are commercially produced, the decision to use DMT as group A and F - moc as group B could be reversed based on criteria such as costs.
Another relevant point in the construction of binomial libraries by the method of the present invention consists in the flexibility of the mutagenesis window, since the number of codons to be scanned can be very variable, from a minimum of two codons to the maximum allowed by the particular characteristics of the synthesis team and the economics of the experiment.
Currently available equipment allows the synthesis of oligodeoxyribonucleotides of up to 100 nucleotides so by discounting 10 base pairs for each of the ends adjacent to the window, this allows us to handle a window of mutagenesis up to 80 nucleotides (approximately 27 codons).
It is obvious to an expert in the state of the art that as new devices or new chemical systems allow the synthesis of larger oligodeoxyribonucleotides, the window of matagénesis may grow in the same proportion.
Another important aspect of the method of the present invention is the flexibility in handling the proportions of the DNF's used for the assembly of the mutant codons during the mutagenesis cycles. This means that the DNF's of adenine, guanine, cytokine and thymine, duly protected in the 5 'hydroxyl with a protecting group, used for the coupling of the mutant codons, can be present in equimolar proportions or in different proportions, which allows to bias the distribution of mutant codons to a desired subset of encoded amino acids. On the other hand, the number of DNF's present in the mixtures is also variable, being able to contain from 1 to 4 DNF's. It should also be mentioned that these conditions prevail for each of the couplings of the different positions of the mutant codon.
One of the important and determining aspects in the construction of binomial libraries of mutant oligodeoxyribonucleotides by the method of the present invention, is that the concentration of the mixture of the protected DNF's used for the coupling of the Ira. position of the mutant codon, directs and defines the level of mutagenesis (a).
In this sense, it is important to note that one characteristic of the method of the present invention is that it has a complete control of the level of mutagenesis. However, it is preferable to handle low levels to generate preferably low multiplicity mutants. In these cases, it is necessary that the concentration of the mixture of the protected DNF's that are used for the coupling of the Ira. The position of the mutant codon is preferably handled at low levels, regardless of the number of protected DNFs it contains and the proportions that they keep with each other.
On the other hand, by means of this method it is feasible to carry out the coupling of the mutant codons with a NNG / C combination, thus achieving a distribution of variants equivalent to that obtained by the method of separation of supports, where N represents a "preferably equimolar mixture of the 4 DNF's and G / C a preferably equimolar mixture of deoxyguanosine and deoxycytidine.
Likewise, it is also feasible to favor certain subgroups of amino acids, using the appropriate combinations of DNFs in the coupling of the mutant codons. This according to the distribution table proposed by Arkin & Youvan (1992).
It is important to highlight the flexibility in the handling of the mutagenesis levels of the method of the present invention, and the fact that the olidesoxyrubonucleotide library mutagenized at the codon level have a population that follows a predictable binomial distribution by means of the combinatorial analysis equation:
n! P = a (l -a) x! (n-x)! Where: represents the proportion of a certain type of mutants
(single, double or triple, etc.) in the population. n is the size of the mutagenesis window (expressed as the number of codons to be scanned) x the type of mutant (the simple corresponds to No. 1, to double No. 2, etc.). to the level or rate of mutagenesis.
In view of the fact that by means of this method the production of low multiplicity mutants is feasible, in these particular cases, the mutagenesis level a will be low and will be determined by the concentration of the mixture of the protected DNFs used for the coupling of the . position of the mutant codon.
On the other hand, an additional advantage in the construction of mutant libraries by means of this method is that they can be built in a single support, since DNF's are used protected with orthogonal groups that have high coupling efficiency under conventional working conditions. therefore, it is highly feasible that the process of the present invention be automated, representing a clear advantage compared to the method of separation of supports.
In order to illustrate the application of the method of the present invention, for the construction of binomial libraries of oligodeoxyribonucleotides mutagenized at codon level, using DNF's, some examples are described below for the synthesis of some oligodeoxyribonucleotide libraries, generated at different levels of mutagenesis with 2 different synthesis protocols.
EXAMPLES
In the following examples the present invention is described in order to illustrate it better, but of course without restricting its scope.
Example 1
This example illustrates the chemical synthesis of 4 Fmoc-DNF 's.
The four Fmoc-DNF 's were synthesized by the procedure reported by Lehmann et al. as illustrated in Figure 1, with only minor changes. The 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) was added at room temperature instead of 0 ° C for the N-acyl-deoxynucleosides or thymidine previously dissolved in pyridine and the reaction was carried out in only 5 min. instead of 30 min. In all cases, small amounts of the 3 'and 3' -5 'byproducts were generated, which were removed by rapid column chromatography. Deoxynucleosides protected at the 5 'hydroxyl with the Fmoc group (Fmoc deoxynucleosides) were recovered in high yields (60-70%) using gradients of methanol in dichloromethane for the elution process. The phosphorylation of the Fmoc-deoxynucleosides was carried out with chlorine (diisopropylamino) methoxyphosphine and diisopropylethylamine in tetrahydrofuran as solvent, to give the corresponding Fmoc-DNF 's, which were purified by chromatography, using 5% pyridine in dichloromethane as eluent.
The results show that the 4 Fmoc-DNF 's, were obtained with at least 90% purity (evaluated by HPLC and 31P NMR analysis) in global yields of 50-70%. It should be noted that an important contribution to this protocol to increase the total yield of compounds containing Fmoc, was to eliminate washing with sodium bicarbonate and instead use a wash with brine and water in the presence of pyridine to prevent hydrolysis of the compounds.
Example 2
This example illustrates the relative reactivity of the 4 Fmoc-DNF 's.
To evaluate the relative reactivity of the 4 Fmoc-DNF 's, an equimolar solution of the 4 Fmoc-DNF' s was used at a total concentration of 100 mM (25 mM each) in anhydrous acetonitrile. This mixture was placed in the position of the X-vial of the synthesizer. Three dinucleotides with the XC sequence were synthesized using the conventional DMTdCbz-CPG support as the starting material and the coupling protocol recommended by the manufacturer for β-cyanoethylphosphoramidites. The Fmoc group was removed with a 100 mM solution of DBU in acetonitrile for one min. and the dimers were sequentially subjected to demethylation with thiophenol for 1 hr and complete deprotection with concentrated NH 4 OH for 12 h at 55 ° C. The three dimer mixtures were analyzed by reverse phase HPLC in a manner similar to that reported by Ward and Juehne, using a vydac ODS analytical column (4.6 x 250 mm) and a linear gradient of 10% acetonitrile in water of 30% at 70% in 0.1 M triethylammonium acetate buffer, pH 7 for 20 min., At a flow of 1 ml / min. , with detection at 260 nm.
The relative reactivity of each of the four Fmoc-DNF 's, was calculated by calibration curves by HPLC for each of the dinucleotides, obtaining a relative reactivity of 31.4%, 22.4%, 20.8% and 25.4% for dA, dC , dG and dT respectively. As can be seen, the four Fmoc-DNF 's showed different reactivities and therefore to obtain a homogeneous distribution of the 32 variant codons, it could require an adjustment of concentrations when carrying out the assembly of the libraries, where a greater amount of G and C will be used due to its lower reactivity with respect to the other bases.
Example 3
This example illustrates the reactivity of Fmoc-DNF 's against DMT-DNF's.
In this case two 100 mM solutions containing the phosphoramidites mixed in equimolar proportions were prepared (FmocdA-Me-amidite + FmocdC-Me-amidite + DMTdG-Me-amidite + DMTdT-Me-amidite and DMTdA-Me-amidite + DMTdC-Me -amidito + FmocdG-Me-amidito + FmocdT-Me-amidito) in acetonitrile, with each compound being 25 mM. These solutions were used similarly to Example 2. In each case three dinucleotides with the sequence XC were also prepared and evaluated by
HPLC.
In these experiments, a relative reactivity of 12.7%, 12.2%, 13.3%, 12.5%, 14.2%, 9.9%, 12.2% and 13.0% was determined for DMT-dA, DMT-dC, DMT-dG, DMT-dT, Fmoc -dA, Fmoc-dC, Fmoc-dG and Fmoc-dT respectively. As can be seen, the reactivity of the Fmoc-DNF 's with respect to all the DMT-DNF's show small non-significant differences, in this sense, it was concluded that the level of mutagenesis could easily be controlled using molar equivalents for the level of desired replacement.
Example 4
This example illustrates the correlation between the level of mutagenesis and the binomial distribution of mutant oligodeoxyribonucleotides obtained in two libraries generated by the mutagenesis protocol, which in this work was called pre-addition.
Two oligodeoxyribonucleotide libraries were synthesized with the sequence 5 'C TCC GAG TGA ATT CGA GCT CGG TAC CCG_GGG ATC CTC CTA 3' at different levels of mutagenesis by the protocol called pre-addition. This protocol consists in coupling the first deoxynucleotide of the mutant codons before the first deoxynucleotide of the wild-type codons, through a diluted mixture of the properly protected DNF's.
In this case, a synthesizer labeled A was loaded with solutions of DMT-DNF's 0.1 M in their respective vials, and position X was loaded with a 20 mM solution of the 4 Fmoc-DNF 's (5 mM each) in the case of the first library and a 50 mM solution (12.5 mM each) for the second library. The other auxiliary reagents in the synthesis were conventional. Another DNA synthesizer labeled B was loaded in position 1- with a solution of the 4 Fmoc-DNF 's at a total concentration of 100 mM (25 mM each) and position 2 with an equimolar solution of DMTdG-Me -amidito and DMTdC-Me-amidito at a total concentration of 100 mM. As in the "A" synthesizer, the auxiliary reagents of the synthesis were the same, except for the trichloroacetic acid solution, which was replaced by a solution of 100 mM DBU in acetonitrile. The codons to be substituted randomly were underlined. All the sequences programmed "in both synthesizers were programmed to be protected with the Trityl group The synthesis of the libraries was started with the programming of the sequence 5 'CCG X GGG ATC CTC CTA 3' in the synthesizer I, omitting the step of acetylation during the addition of X. Then, the column synthesis was transferred to synthesizer II and the sequence NG / C was added to complete the first group of mutant codons.The procedure was then repeated three times with the appropriate codons to complete all the mutagenesis window Finally, the addition of the 5 'C TCC GAG TGA ATT CGA 3' sequence was programmed in the synthesizer II The completely protected oligonucleotide still attached to the CPG support was subjected to treatment with thiophenol for 1 h to remove the groups internucleotide methyl and subsequent treatment with concentrated ammonium hydroxide to remove all remaining protective groups. Oligodeoxyribonucleotide libraries were purified in 15% polyacrylamide gels containing 8 M urea and recovered in deionized water after being desalted with n-butane1.
Example 5
This example illustrates Synthesis of oligodeoxyribonucleotide libraries by the mutagenization protocol by online mixing of DMT-DNF defined by the wild sequence for Ira. position of the wild-type codon and the Fmoc-DNF 's that define Anger. position of the mutant codon.
A library of oligodeoxyribonucleotides with the sequence 5 'C TCC GAG TGA ATT CGA GCT CGG TAC CCG GGG ATC CTC CTA 3' was synthesized in order to evaluate the mutagenesis protocol by in-line mixing.
For the assembly of the library, synthesizer I was loaded with 150 mM solutions of the 4 DMT-DNF's in their respective vials and vial X was loaded with a 50 mM solution of the 4 Fmoc-DNF 's (12.5 mM each). ). Synthesizer II was loaded as Example 3. The coupling of this oligodeoxyribonucleotide library was initiated with the synthesis of the 5 'CC (GX) GGG ATC CTC CTA 3' fragment in Synthesizer I, programmed so that the sequence would be tritylated. The bases in parentheses were simultaneously added from vial C and X for this first codon. Then, the column was transferred to synthesizer B and the sequence NG / C was added. This cycle of mutagenesis was repeated three more times with the appropriate in-line mixture of DMT-DNF defining the first position of the codon to be mutated and the 4 Fmoc-DNF's contained in vial X. This oligodeoxyribonucleotide library was terminated as the library of the example 4 and unprotected and purified in the same way.
In order to evaluate the final distribution of mutant oligodeoxyribonucleotides in each of the three libraries, each one, independently, were mixed with the 5 'primer oligonucleotide CTCCGAGTGAATTCG
3 'and subjected to extension with the klenow polymerase to generate libraries of mutant cassettes, which were digested with the restriction enzymes EcoRI and BamH I and linked to pUCld plasmids and expressed in the E. coli strain.
JM101. The mutant colonies were sequenced by analyzing
42, 38 and 49 colonies for the first, second and third libraries respectively.
The analysis of the codon replacements in Figure 4 showed that all the libraries follow approximately a binomial distribution of variants according to the level of mutagenesis, that is to say that mutants of high multiplicity were generated at high levels of mutagenesis (many changes of codon per gene), whereas for low levels of mutagenesis, low multiplicity mutants were preferably generated (few codon changes per gene). These results clearly indicate that the proposed method of mutagenesis is highly predictable and corresponds to the expected distribution of variants. In Table I, all the results are concentrated.
Table I
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Claims (16)
1. - Method for the construction of binomial libraries of oligodeoxyribonucleotides mutagenized at codon level using two sets of deoxynucleoside phosphoramidites (DNF's) protected at the 5 'hydroxyl with orthogonal groups to each other, characterized in that it comprises the following steps: a) Sequentially coupling on a solid support, the deoxynucleoside-phosphoramidites protected in the 5 'hydroxyl by a group A (A-DNF's), to assemble the wild sequence corresponding to the 3' flanking zone of the mutagenesis window that it is desired to scan in a given gene; b) initiate a cycle of mutagenesis, which comprises the following steps: 1.- coupling to the previously assembled sequence, the deoxynucleotide of Ira. position of the mutant codon by the addition of a mixture containing the DNF's protected at the 5 'hydroxyl with the group B (B-DNF's); at an appropriate concentration that allows generating a previously defined level of mutagenesis; 2. - coupling to the chains of oligodeoxyribonucleotides that did not react in the previous step, the deoxynucleotide of Ira. position of the wild-type codon by the addition of A-DNF, appropriate according to the pattern of the wild-type sequence; 3.- continue with the coupling of the 2nd. and 3rd. position of both the mutant and wild-type codons, regardless of the coupling order, only keeping the following conditions: I) In the two subsequent couplings for the wild-type codon, A-DNF's are used, according to the pattern of the wild-type sequence; II) for the coupling of the 2nd. position of the mutant codon, a high concentration mixture containing the B-DNF's, III) is used for the coupling of the 3rd. position of the mutant codon, a high concentration mixture containing the protected DNFs with group B or A is used. Optionally, the mutagenesis cycle of part b) is repeated, as many times as necessary to conclude the number of codons that are want to explore; d) concluded the mutagenesis cycles, synthesize the wild sequence corresponding to the 5 'flanking zone of the mutagenesis window, using the appropriate A-DNF's according to the wild sequence.
2. - Method for the construction of binomial libraries according to claim 1, characterized in that in the mutagenesis cycle of part b), steps 1 and 2 are preferably carried out in a single step, by the simultaneous addition of A-DNF defined by the wild sequence to assemble the Wrath. position of the wild codon and the mixture of the B-DNF's that define the Anger. position of the mutant codon.
3. - Method for the construction of binomial libraries according to any of claims 1 or 2, characterized in that the coupling of the 3rd. position of the mutant codon, is carried out by means of a high concentration mixture containing the DNF's preferably protected with a group A.
4. Method for the construction of binomial libraries according to claim 1 or 2, characterized in that the protective groups orthogonal to each other are preferably 4,4'-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc).
5. - Method for the construction of binomial libraries according to claim 4, characterized in that the protective group A is preferably 4,4'-dimethoxytrityl (DMT) and the protective group B is preferably 9-fluorenylmethoxycarbonyl (Fmoc)
6. - Method for the construction of binomial libraries according to any of claims 1 or 2, characterized in that preferably the window of mutagenesis is flexible to any number of codons per library.
7. - Method for the construction of binomial libraries according to any of claims 1 or 2, characterized in that the mixture containing the B-DNF's, used in step 1 of the mutagenesis cycle of part b), can contain from 1 to 4 B-DNF's, regardless of the proportions they keep with each other.
8. - Method for the construction of binomial libraries according to claim 7, characterized in that the B-DNF's of the mixture are preferably present in equal proportions.
9. - Method for the construction of binomial libraries according to claim 7, characterized in that the B-DNF's it contains are deoxynucleoside-phosphoramidites of adenine, guanine, cytokine and thymine, duly protected in the 5 'hydroxyl with a protecting group B.
10. - Method for the construction of binomial libraries according to any of claims 1 or 2, characterized in that the high concentration mixtures containing the DNF's used for the coupling of the 2nd. and 3rd position of the mutant codon of the mutagenesis cycle of part b); it can contain from 1 to 4 protected DNFs in the 5 'hydroxyl, independently of the proportions that they keep among themselves.
11. Method for the construction of binomial libraries according to claim 10, characterized in that the DNF's protected in the hydroxyl 5 'of the mixture are preferably present in equal proportions.
12. - Method for the construction of binomial libraries according to claim 10, characterized in that the DNF's are deoxynucleoside-phosphoramidites of adenine, guanine, cytokine and thymine, duly protected in the 5 'hydroxyl with a protecting group.
13. Method for the construction of binomial libraries according to claim 10, characterized in that the mixture of the DNF's used for the coupling of the 3rd position of the mutant codon of the mutagenesis cycle of part b), preferably contains only guanine and cytokine DNF's .
14. - Method for the construction of binomial libraries according to any of claims 1 or 2, characterized in that preferably the concentration of the mixture of the protected DNFs in the hydroxyl 5 'for the coupling of the Ira. position of the mutant codon, directs and defines the level of mutagenesis.
15. - Method for the construction of binomial libraries according to claim 14, characterized in that the level of mutagenesis is preferably low for the generation of low multiplicity mutants.
16. Method for the construction of binomial libraries according to any of claims 1 or 2, characterized in that the use of a support and the combined use of orthogonal protective groups preferably favors its automation. SUMMARIZES The invention relates to a method of mutagenesis for the construction of binomial libraries of mutagenized oligodeoxyribonucleotides at the codon level comprising the use of two sets of deoxyimieosido-phosphoramidites protected at the 5 'hydroxyl by orthogonal protecting groups with each other, which are combined during the synthesis of oligodeoxyribonucleotides. This method constitutes a valuable tool for the study of the structure-function relationship of proteins and their engineering, since few replacements of amino acids per protein can be generated in a controlled and predictable way, allowing to investigate the individual importance of each one of the proteins. wild amino acids for the function of the protein and at the same time avoid their functional destruction, in order to improve an innumerable amount of proteins with commercial application, as for example the enzymes denominated subtilisins that are used in the biodegradable detergents.
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00980068A EP1262544A2 (en) | 1999-11-15 | 2000-11-15 | Method for constructing oligodesoxyribonucleotide binomial libraries with codon mutagenized using desoxynucleoside-phosphoramitides |
PCT/MX2000/000047 WO2001036439A2 (en) | 1999-11-15 | 2000-11-15 | Method for constructing oligodesoxyribonucleotide binomial libraries with codon mutagenized using desoxynucleoside-phosphoramitides |
AU17371/01A AU1737101A (en) | 1999-11-15 | 2000-11-15 | Method for constructing oligodesoxyribonucleotide binomial libraries with codon mutagenized using desoxynucleoside-phosphoramitides |
CA002391999A CA2391999A1 (en) | 1999-11-15 | 2000-11-15 | Method for constructing oligodesoxyribonucleotide binomial libraries with codon mutagenized using desoxynucleoside-phosphoramitides |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA99010476A true MXPA99010476A (en) | 2001-09-07 |
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