EP1047706A2 - Procede permettant de faire la synthese de molecules d'acide nucleique - Google Patents

Procede permettant de faire la synthese de molecules d'acide nucleique

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Publication number
EP1047706A2
EP1047706A2 EP99919096A EP99919096A EP1047706A2 EP 1047706 A2 EP1047706 A2 EP 1047706A2 EP 99919096 A EP99919096 A EP 99919096A EP 99919096 A EP99919096 A EP 99919096A EP 1047706 A2 EP1047706 A2 EP 1047706A2
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EP
European Patent Office
Prior art keywords
nucleic acid
acid molecule
synthesis
dna
sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP99919096A
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German (de)
English (en)
Inventor
Annette Bernauer
Hubert Stefan Bernauer
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Individual
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Individual
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Publication of EP1047706A2 publication Critical patent/EP1047706A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the invention relates to a method for the synthesis of nucleic acid molecules.
  • the invention relates to a method of this type that is carried out recursively.
  • the nucleic acid components are preferably of synthetic or semisynthetic origin.
  • the principle of the method according to the invention is based on the fact that a further nucleic acid molecule is attached to or with a nucleic acid molecule provided and / or is linked, the end of the nucleic acid molecule provided is masked, if no further nucleic acid molecule has been attached and / or linked to this or with this, the further nucleic acid molecule is cleaved at a predetermined location, an end again preferably being formed to or with which another nucleic acid molecule can be attached and or linked, and the aforementioned process steps may be repeated until the desired product is synthesized.
  • the invention b also relates to a kit for carrying out the method according to the invention
  • nucleic acid molecules with a precisely defined sequence in the simplest possible manner with little time - and to provide cost
  • the currently most widespread methods for providing such nucleic acid molecules include cloning DNA, for example from cDNA libraries, optionally coupled with subsequent sequencing of the isolated cDNA.
  • DNA with the desired sequence can be produced synthetically, for example using the conventional phoshoamidite method become
  • DNA molecules of interest must be isolated, for example, by cDNA or positioning cloning and cloned into suitable vectors.
  • the resulting vectors and thus the DNA molecules of interest must be multiplied "In vivo"
  • the vectors have to be introduced into suitable host cells, for example bacteria or yeasts.
  • suitable host cells for example bacteria or yeasts.
  • the DNA has to be isolated again from the host organisms they are again available for manipulation purposes.
  • they must again be introduced into suitable host organisms.
  • process steps and / or complicated manipulations are often necessary in order to obtain a certain to generate DNA WANTED It is also, unfortunately, to imagine and well known to the skilled person that still multiply this effort unless a larger number of various DNAs are prepared to
  • the object of the present invention was therefore to provide a method which enables the synthesis of nucleic acid molecules of the desired sequence and length in a simple and time-saving manner. This object is achieved by the embodiments characterized in the claims
  • the invention thus relates to a method for the synthesis of nucleic acid molecules, the following
  • nucleic acid molecule which has at least one end which permits attachment and / or linkage of or with a further nucleic acid molecule
  • 2 Attachment and / or linkage of at least one further nucleic acid molecule to or with the nucleic acid molecule
  • the one end of the at least one another nucleic acid molecule is attached to and / or linked to the at least one end of the nucleic acid molecule and / or linked
  • the other end of the at least one further nucleic acid molecule is masked in the event of a link
  • 3 optionally masking the at least one end of the nucleic acid molecule to the or with which no other nucleic acid molecule has been attached and / or linked
  • step (2) Repeat steps (2) to (4) at least once, possibly several times, with suitable nucleic acid molecules being used in step (2)
  • the further nucleic acid molecule is a single-stranded nucleic acid molecule
  • the method according to the invention comprises the following step after step (2)
  • the method according to the invention comprises the following step after step (4) or (5)
  • the method according to the invention is suitable for the synthesis of single-stranded (dsDNA) or partially double-stranded DNA
  • FIG. 1 The principle of the method according to the invention is shown in FIG. 1. Further embodiments are shown in FIGS. 2 to 7
  • a single-stranded nucleic acid molecule a partially double-stranded nucleic acid molecule with an overhanging 5 'or 3' end or a double-stranded nucleic acid molecule with a smooth end is provided.
  • the next step is by means of a ligase activity, for example a T4-RNA ligase, a single-stranded nucleic acid molecule covalently bound
  • the single-stranded nucleic acid molecule can be bound with its 5 '- phosphate or 3' - hydroxy end to the provided nucleic acid molecule.
  • the method according to the invention comprises nucleic acid synthesis in 3 '-5 '- or in the 5' -3 'direction (starting from the orientation of the precursor molecules)
  • masked means that masked means that this end in this ligation approach cannot be linked to another single-stranded nucleic acid molecule of the same type, and this results in single-stranded molecules which consist of several copies of the same nucleic acid molecule and also can be linked to the nucleic acid molecule provided.
  • a masking is a chemical, enzymatic or other modification of the end which prevents the above-mentioned linkage.
  • Masking in the sense of this invention is described in more detail below.
  • the ends of the nucleic acid molecules provided are masked, which have not been linked to a single-stranded nucleic acid molecule
  • the single-stranded nucleic acid molecule which has been ligated to the provided nucleic acid molecule is attached to a predetermined one Cleaved site, removing the mask and creating an end that allows a link to a next single-stranded nucleic acid molecule.
  • the method according to the invention advantageously ensures that only those nucleic acid molecules to those in the previous one are extended in further ligation sections Step a single-stranded nucleic acid molecule was ligated
  • the ligation, masking and cleavage step can now be repeated any number of times in this order with new molecules to be added, in each case using suitable single-stranded nucleic acid molecules
  • the complementary counter strand with a polymerase activity is synthesized, the single strand was synthesized in the 3 '- 5' direction and became a double-stranded nucleic acid molecule with a smooth end or a partial one provided double-stranded nucleic acid molecule, the complementary nucleic acid strand can be synthesized directly from the free 3 'end of the nucleic acid molecule provided.
  • the corresponding complementary nucleic acid strand is synthesized by means of a polymerase activity directly after (each) cleavage of the ligated single-stranded nucleic acid molecule before the ligation of the next single-stranded nucleic acid molecule.
  • the procedure is essentially as described above for the synthesis of the complete complementary nuclein
  • each single-stranded nucleic acid molecule is selected such that it advantageously forms a hairpin structure at the 3 'end.
  • the masking is carried out before the polymerase reaction which occurs before the ends of the nucleic acid molecules provided are masked removed at the 3 'end of the ligated nucleic acid molecule
  • the further nucleic acid molecules which are attached to and / or linked to the provided nucleic acid molecule are double-stranded.
  • the nucleic acid molecule provided is single-stranded or partially double-stranded with an overhanging 3 'or 5' end another nucleic acid molecule a corresponding, in its sequence complementary, 3'- or 5'-overhanging end, an attachment takes place by hybridization of the single-stranded overhanging ends.
  • the other end of the further nucleic acid molecule is preferably smooth. The masking described above ensures that this is attached to it There is no addition via hybridization of cohesive ends of further nucleic acid molecules of the same type
  • the further nucleic acid molecule is single-stranded and there is an attachment to the nucleic acid molecule provided via hybridization of complementary terminal nucleotides.
  • the nucleic acid molecule provided is single-stranded or partially double-stranded with an overhanging 3 'or 5' End
  • the single-stranded further nucleic acid molecule can additionally covalently with the nucleic acid molecule provided by means of a Ligase activity linked If the hybridization takes place via 3 '- terminal nucleotides, the complementary strand can be synthesized in the next step by means of a polymerase activity.
  • the 3 ' end of the further single-stranded nucleic acid molecule is chosen such that it forms a hairpin structure, so that a 3 'end is provided for the subsequent polymerization reaction for the synthesis of the complementary nucleic acid strand.
  • the synthesized nucleic acid duplex is cleaved at a predetermined location, the recognition sequence necessary for the cleavage and the smooth end or the hairpin structure is removed and a preferably cohesive end is formed which allows attachment via hybridization and, if appropriate, covalent linkage of the nucleic acid molecule to a further single-stranded nucleic acid molecule
  • the present invention also encompasses processes whose addition, masking and / or cleavage combinations represent the corresponding steps of the abovementioned embodiments.
  • a single-stranded nucleic acid molecule can be covalently linked to a nucleic acid molecule provided, and then the complementary nucleic acid strand can be synthesized , the double strand is cleaved as described above, and another single-stranded nucleic acid molecule is attached via hybridization in the next synthesis cycle
  • the synthesis of the complementary nucleic acid strand does not have to take place after every addition, masking and / or cleavage step or at the end of the synthesis of the complete nucleic acid single strand.
  • the time of filling up the complementary strand can be chosen arbitrarily , in the sense that it is selected, for example, after any permuting addition, masking and / or cleavage step
  • Masked single-stranded 3-ends can be, for example, by the incorporation of an amino block, a dideoxynucleotide, a 3′-phosphate or an artificially incorporated 5-end
  • masked single-stranded 5 ' ends are distinguished, for example, by a missing phosphate group or by the incorporation of a 5-modified nucleotide (eg biotin-dNTP, digoxygenin-dNTP).
  • a partially double-stranded nucleus Cleic acid molecule with overhanging single-stranded ends can thus be masked by removing an overhanging 3 ' end by means of exonuclease activity, or by synthesizing the complementary strand to an overhanging 5-end by means of polymerase activity, so that in both cases a double-stranded nucleic acid molecule with smooth ends arises
  • the term “providing a nucleic acid molecule” encompasses any form of providing, for example the cloning of a gene with subsequent restriction cleavage and isolation of a fragment with, for example, an overhanging or smooth end which serves as starting material for the process according to the invention.
  • the nucleic acid molecule is by attaching two at least partially complementary synthetic oligonucleotides to each other, whereby an overhang can result from the attachment to one another.
  • single-stranded oligonucleotides are provided
  • the "attachment" of the nucleic acid single-strand molecules is preferably carried out by hybridization.
  • the necessary hybridization conditions can, if necessary, be modified by the person skilled in the art for each step of the addition of a new single strand based on his specialist knowledge
  • the nucleic acid single-stranded molecules used in the process according to the invention have a maximum length of approximately 150 nucleotides. A length of between 15 and 130 nucleotides is preferred.
  • the yield of intact oligonucleotides in the chemical synthesis of single-stranded precursor molecules with increasing length decreases because of incorrect incorporation of nucleotides.Therefore, a compromise must be made between the length of the oligonucleotides and their yield.
  • the quality of the single-stranded molecules used for the synthesis also has an influence on the yield of the desired nucleic acid with the gonucleotide purification with the help of HPLC, the individual nucleic acid single-stranded molecules for further syntheses are intact.
  • the length of the oligonucleotides used in further syntheses will vary according to the amount required for a synthesis orient tt and the yield in chemical synthesis
  • predetermined position means that this sequence is defined either by its primitive sequence or by its relative positioning to the actual cleavage point
  • a predetermined site for cleavage of a single nucleic acid strand can be created, for example, by incorporation of one or more artificial or modified nucleotides, base analogs or a chemical group, internal or terminal, which can be cleaved by means of a physical, chemical or enzymatic process so that a 3'-OH and / or a 5 - phosphate end is formed (eg Maxam-Gilbert reaction etc).
  • Nucleotides which are suitable for the process according to the invention are, for example, 5-hydroxy-2-deoxycytide, 5-hydroxy- 2- deoxyu ⁇ dm or 5-hydroxy-2-deoxyu ⁇ dm While the first two nucleotides are substrates for E co // - endonuclease III and formamidopy ⁇ midine DNA glycosylase, the latter nucleotide can be cleaved using uracil DNA glycosylase and apy ⁇ midase or alkali treatment
  • phosphoborane nucleotides or thioate nucleotides in the DNA sequence can bring a terminal digestion of exonuclease II or T7 (gene 6) nuclease to a standstill, the sequence being predetermined up to the modified nucleotide for cleavage
  • a further possibility for cleaving the nucleic acid single strand at a predetermined point is to introduce a "mismatch" in an artificial hairpin structure.
  • This structure can be cleaved efficiently and precisely by means of a "mismatch repa" enzyme
  • which (molecular) agent is ultimately used as residual activity for cleaving one or more predetermined positions in a nucleic acid double strand in the process according to the invention is not essential to the invention.
  • the recognition sequence is usually removed by cleavage from the growing nucleic acid double-stranded molecule.
  • class II S residual endonucleases have properties which meet the requirements for such an agent Depending on the embodiment of the method according to the invention, representatives of this class, which generate a free, cohesive 3 ' end or a protruding, cohasive 5' end, are suitable
  • the restricting agent can be of diverse nature, including all nucleic acids specifically cleaving synthetic agents such as synthetic peptides, PNA (peptide nucleic acid), triple kale DNA binding oligonucleotides, which are necessary for the specific processing of the
  • Nucleic acid term (s) are suitable for the purposes of this invention, as are naturally occurring DNA-cleaving enzymes.
  • the person skilled in the art is able to use suitable (exo) nuclease or residual functional activities for his particular purposes,
  • these can be, for example, residual II endionucleases, (c) asymmetric recognition sequences (residual endonucleases of Class II S), as well as symmetrical recognition sequences can be used, (d) as already mentioned above, the cleavage sites may be caused by the restriction activity generated, do not lie within the specific recognition sequence, but must be located 5 or 3 ' distally thereof, (e) the distance of the interface from the recognition sequence must be precisely and clearly defined,
  • Agent preferably cohesive ends This also eliminates the need for masking the single strands discussed above, which, for example, are attached to the smooth ends
  • a suitable selection of restricting agents can be found in the accompanying literature
  • step (5) or the frequency of its implementation ultimately depends on the length of the desired end product and also on the length of the strand of the starting material available
  • a generally synthetic, single-stranded DNA molecule (+) is made available, the 5 'templates of which are brought to hybridization with the 3' end of the previously synthesized single-stranded DNA molecule (-)
  • a double stranded strand is formed 3'-Term ⁇ nus which terminally masks the double strand
  • the complementary template strand can be completely or partially (FIG. 8A) degraded to facilitate the subsequent hybridization reaction by means of, for example, 5'-3 'exonuclease activity (FIG. 8B).
  • nucleic acids for example genes
  • nucleotide sequences are available at any time, anywhere and quickly (within days) if the nucleotide sequences are known from databases, for example. Knowing these sequences and the teaching according to the invention, the person skilled in the art can synthesize any desired nucleotide molecule
  • Nucleic acid molecules in the size of genes no longer have to be physically stored in refrigerators with high energy consumption. Your sequences can be managed in EDP systems and, if necessary, easily synthesized in a biological system. This eliminates the need for transport. DNA sequences can also be sent by email become
  • nucleotide sequences such as DNA sequences and gene sequences are mutated in vitro to achieve certain properties, such as stability to heat, pH changes or solubility in certain solvents, as well as the optimization or change in the biological behavior of their gene products.
  • fro mutagenesis is a complex undertaking. Due to the synthetic possibility, it is possible to insert any number of mutations even at widely spaced sequence positions, since the sequence is freely definable and predeterminable. Any number of variants can be generated The free synthesizability of the DNA sequences will shift many of the currently common methods of time-consuming DNA manipulations from the laboratory to the synthesis machine, which results in a large saving in costs and, consequently, in a large amount of time.
  • the experiment to be carried out by the person skilled in the art then consists of the design of a nucleic acid sequence on a computer editor and checking whether the properties desired by the sequence manipulations are set on the biological model or in vitro.
  • the method according to the invention is thus also a contribution to the further development of techniques of reverse genetics
  • nucleic acid molecules, fragments thereof and / or nucleotides after step (2), (2a), (3), (4), (4a), (5) and / or are not incorporated into the nucleic acid molecule provided. or (5a) separated
  • the separation of the non-incorporated single-stranded nucleic acid molecules is preferred, but not absolutely necessary, and can be accomplished by a person skilled in the art by standard methods, for example by column chromatography methods.
  • concentration of free nucleotide phosphates could be limiting, in particular for the overall yield of desired nucleic acid, and the nucleotides consumed in the synthesis and interfere with the ligation reaction, which is necessary, for example, in the case of the generation of blunt ends or the attachment of single nucleic acid strands to blunt ends and the subsequent generation of the complementary strand when carrying out the method according to the invention, as described above.
  • each individual synthesis step can be carried out under optimal conditions. It is therefore advisable to separate the one that is not required n Single strands in each case before the next synthesis step, for example in a matrix-coupled reaction, after the expiration of which used nucleotides and excess single-strand nucleic acids are eluted. Of course, the separation can also take place after or during the implementation of step (5)
  • step (5) An example of the ligation after step (5) is provided by the case that bacteria, for example BE coli, are transformed with the non-ligated synthesis product and the ligation is carried out by endogenous ligases. It is known that with increasing size the complementary overlap of cohesive ends results in a Transformation of, for example, £ coli with suitable DNA using the endogenous ligase activity for circulatory purposes is possible.
  • Gaps and protruding single-stranded DNAs are entirely tolerated, since repair mechanisms restore the integrity of the circular double strand Filled and repaired if at least one phosphodiester backbone is intact. Ligation is preferably carried out when the overhangs are only a few nucleotides long.
  • the predetermined position of the nucleic acid molecule is generated by incorporation of an artificial or modified nucleotide, a base analog, a chemical group or a “mismatch” in an artificial hairpin structure, which means a physical, chemical or enzymatic process can be split
  • the artificial or modified nucleotide is 5-hydroxy-2-deoxy-cyt ⁇ d ⁇ n, 5-hydroxy-2-deoxyur ⁇ d ⁇ n, or 5-hydroxy-2 ' -deoxyu ⁇ d ⁇ n
  • the present invention relates in a further preferred embodiment to a method in which the linkage of two terminal nucleotides via a 3 ' hydroxy and a 5 ' phosphate end with the aid of a ligase activity, and the attachment via the hybridization of complementary sequences take place
  • the nucleic acid is DNA. In another preferred embodiment of the method according to the invention, the nucleic acid is RNA.
  • the method according to the invention also includes the generation of DNA / RNA hybrids
  • the masking in step (3) is carried out additively and subtractively by adding or removing a chemical group or a chemical molecule.
  • a 5 "end is masked by removing the phosphate Group (s) or the incorporation of a 5-modified nucleotide (eg Biotm-dNTP, digoxygenin-dNTP etc.)
  • a 5-modified nucleotide eg Biotm-dNTP, digoxygenin-dNTP etc.
  • the masking is effected at least by the incorporation of a 5 'nucleotide -mod ⁇ f ⁇ z ⁇ erten
  • a masked 3 ' end is distinguished by the presence of an amino block, a dideoxynucleotide, a 3 ' phosphate, or an artificial 5 ' end
  • the further nucleic acid molecule forms a hairpin loop on the nucleic acid molecule provided after attachment and / or linkage at the distal end, which serves as a primer for the polymerase activity
  • the invention relates to a method, wherein the cleavage at a predetermined point in step (4) is carried out by a sequence-specific cleaving helical DNA
  • a triple-helical DNA is formed, for example, when a single-stranded DNA, at the end of which a heavy metal (SM) is coupled, attaches to a double-stranded DNA and, if the sequence conditions are suitable, a triple-helical structure with a double-stranded DNA forms The nucleic acid duplex is cleaved by the heavy metal at a defined position
  • any specific physical, chemical and enzymatic nucleic acid cleavage can be used according to the invention, which is necessary for the attachment of a single-stranded nucleic acid molecule for subsequent ligation with the double-stranded nucleic acid molecule.
  • Further examples of this are methodological approaches based on designed peptides or PNA ( peptide nucleic acid)
  • Another preferred embodiment of the invention relates to a method in which the cleavage at a predetermined point in step (4) is carried out by a type II S residual endonuclease.
  • Type or class II S enzymes have an asymmetric, that is to say non-parametric, recognition sequence.
  • the cleavage sites are either 5 ' - or 3 ' -d ⁇ stal to the recognition sequence Either 5 - (eg BspMI) or 3 - (eg RleAl) projecting ends or smooth ends (eg BsmFI) are generated
  • the type II S residual endonuclease is the RleAl enzyme from Rh zobium leguminosarum (Vesely Z, Muller A, Schmitz G, Kaluza K, Jarsch M, Kessler C (1990) RleAl a novel class-IIS restnction endonuclease from Rhrzobium leguminosarum recognizing 5 ' -CCCACA (N12 / 9) - 3 ' , Gene 95 129-131)
  • the nucleic acid double-stranded molecules and / or the nucleic acid single-stranded molecules are of synthetic or semisynthetic origin, the use of synthetic single-stranded molecules being particularly preferred for the synthesis
  • Semisynthetic molecules can be produced by incorporating nucleic acid fragments from "in vivo" (bacteria, yeast) amplified DNA (dsDNA, ssDNA) or RNA in one or more intermediate steps of the synthesis according to the invention at defined sites by ligation reactions. This strategy can be costly in individual cases help reduce
  • the nucleic acid molecule as a starter molecule can also be an “in vivo” DNA molecule to which any DNA sequences are attached by recursive DNA synthesis
  • the synthesis is at least partially automated.
  • a nucleic acid (gene) synthesizer for nucleic acid double strands from single nucleic acid strands
  • a battery of automated chemical oonucleotide syntheses (a technology already practiced to a large extent) can be used Providing raw material for the synthesis of biologically active, double-stranded DNA molecules (eg entire genes) These are produced from the chemically synthesized oligonucleotides in a likewise automated process
  • the double-stranded nucleic acids to be extended are bound to the synthesis matrix in a synthesis chamber.
  • this synthesis chamber the same steps described above are repeated in a cyclical reaction sequence.
  • the reaction by-products of the previous reaction are washed out of the synthesis chamber before the start of a new reaction.
  • the starter molecule is extended by one nucleic acid molecule remains bound to the synthesis matrix
  • a nucleic acid with a different sequence sequence is incorporated, so that ultimately a possibly double-stranded nucleic acid with the desired nucleotide sequence is formed
  • the invention relates to a method in which the synthesis is carried out in a matrix-bound manner
  • All carrier materials to which a nucleic acid can be bound and whose properties are compatible with the desired recursive nucleic acid synthesis are suitable as a synthesis matrix, for example streptavidin-coated surfaces, the nucleic acid double-strand molecule used as the starter molecule being incorporated via a built-in biochemical synthesis of the nucleotide
  • Further preferred synthesis matrices include nylon surfaces to which polydT-containing sequences are coupled by UV radiation, and tosyl-, active ester- or epoxy-activated surfaces (eg GOPS), the binding preferably taking place via an “amino link”, such as glass (CPG, glass wool etc), silicate, latex, polystyrene, epoxy or silicon
  • the synthesized nucleic acid molecule is isolated after the synthesis
  • a plasmid is linked to the synthesis product and the resulting nucleic acid molecule, optionally after its recirculation, is introduced and reproduced in bacteria.
  • defined sequences are incorporated into the primer in the nucleic acid molecule provided and in the nucleic acid molecule used in the last synthesis step Can bind specifically With the aid of these polymers, the completely synthesized nucleic acid molecule can be amplified in a PCR reaction, whereby the synthesis product is isolated from the affinity matrix.
  • Sequence motifs for example, type III recognition sites
  • single-stranded nucleic acid molecules are isolated by denaturing the double-stranded nucleic acid molecule
  • This embodiment of the method according to the invention is suitable for producing single-stranded nucleic acid molecules of any composition.
  • the invention relates to a kit comprising
  • the manufacturer of the kit according to the invention knows how to produce and formulate the individual components of the kit, for example the buffers contain bound starter molecule and / or a set of suitable single-stranded molecules Description of the figures
  • FIG. 1 In vitro" -ssDNA synthesis in the 3 '-5' direction (1) A starter molecule (s) coupled to a matrix is linked by means of a ligase activity around an n + lth single-stranded molecule through a 3'- 5 ' phosphodiester bond The n + lth ssDNA has a uracil deoxynucleotide at the terminal.
  • the glycosidic linkage of the base uracil is cleaved by the DNA uracil glycosylase, which results in an apyrimidine position - this in turn is cleaved by an apyrimidine endonuclease activity (exonuclease cell) so that a 5 'phosphate and a 3 'OH end is formed.
  • the n + 2nd ssDNA molecule is linked to the released 5' phosphate end in the n + 2nd ligation reaction - a subsequent phosphate reaction (not shown, see FIG.
  • Figure 2 In vitro - dsDNA synthesis in the 3 '-5' direction. All steps are carried out as in Figure 1, but then in the last step, starting from the 3 'end of the starter molecule, a polymerization reaction is started, which the new Synthesized single strand to double strand - Alternatively, in the first and in the last step, a primer of a pair of primers can be installed and thus a double strand molecule can be generated by amplification by means of the PCR reaction -
  • Figure 3 "In vitro" - dsDNA synthesis in the 3 '-5' direction. All steps are carried out as in Figure 1 .. Within each synthesis cycle, a polymerization step is initiated after the phosphatase treatment, which converts the ligated ssDNA into a dsDNA. The processing can also be carried out using a restriction endunuclease of the type HS, provided that a recognition sequence is built into each of the ssDNA fragments.
  • a polymerization step is initiated after the phosphatase treatment, which converts the ligated ssDNA into a dsDNA.
  • the processing can also be carried out using a restriction endunuclease of the type HS, provided that a recognition sequence is built into each of the ssDNA fragments.
  • Steps take place as in Figure 1 - one by one
  • Ligation step carried out phosphate reaction inactivates all DNA molecules for the next ligation step and for all subsequent ligation steps, provided that no ssDNA molecule in the n-lth
  • a 5 'phosphate can again be made available for the next reaction sequence by processing -
  • n + 2nd ssDNA molecule is linked to the released 5 'phosphate end in the n + 2nd ligation reaction.
  • a subsequent terminal transferase reaction with a dideoxytrinucleotide (not shown, see FIG. 7) inactivates all DNA chains for the n + 3rd ligation step for all subsequent ligation steps, unless there is an n + 2nd ssDNA mole was installed in the n + 2nd step.
  • the 3-OH for the next reaction sequence (n + 3) can be made available again through processing by the DNA uraclyglycosylase and the apyrimidine endonuclease activity. All steps are repeated k times until the last ssDNA molecule in the mth step was installed.
  • a 3 'end can be provided for a DNA polymerization reaction or dsDNA polymerization can be carried out as described in FIG.
  • FIG. 7 "In vitro" SSDNA synthesis in the 5 '-3' direction (1) /
  • FIG. 8A shows a complete degradation of the template molecule with T7 (Gen6), while FIG. 8B shows a partial degradation with exonuclease III.
  • the recursive DNA synthesis "in vitro" can be used for manipulation of DNA sequences "in vitro".
  • gene mutations such as deletion mutagenesis, also several deletions in one gene at the same time, gene fusions with generation of new properties, insertion mutagenesis, substitution mutagenesis and also sequence inversions
  • one to any number of point mutations can be introduced into a sequence. All DNA sequences can be generated directly in parallel syntheses without intermediate cloning steps
  • the functional changes in biological activity “in vivo” resulting from the sequence manipulations can have an effect on the protein level, provided that the coding sequences can be translated into functional proteins.
  • the method can thus be used to implement considerations in enzyme or protein design
  • RNA sequences of regulatory cis elements in order to change the binding activity of transactivators and suppressors, to investigate their behavior or even to create completely new combinations of cis elements.
  • activity of RNA was also able to -Molecules are manipulated (e.g. ribozymes), provided the manipulated DNA is transcribed
  • the following example of the application of the recursive DNA “in vitro” synthesis method is the manipulation of DNA sequences for the analysis of the binding activity of a transactive regulatory protein on a bacterial promoter region.
  • the “in vitro” mutagenesis of the binding sites has an impact on the DNA -binding protein examined
  • the DNA wild-type sequence and the mutants of the cis-active element are shown in FIG. 7, the sequence manipulations are explained in the text.
  • the aim of the experiments was to test the functionality of the binding of the regulator in a different sequence context “in vitro” and possibly also to be able to be examined “in vivo”
  • the WT-6-HDNO promoter fragment from the 5 control region of the 6-HDNO gene has some very interesting sequence features (see FIG. 7). It is characterized by extended inverted sequence repetitions (IR) and other striking sequence motifs. Characteristic sequence arrangements within the 6-HDNO -Gen-promoter region are shown in FIG. 7. This shows two inverted repeats, IR1 and IR2, which have extensive homologies with one another (FIG. 7). The right one Palmdromic half-side of IR2 repeats itself in the 5 range. Such palindromes are structural features that can be found in many bacterial cis-active regulator regions
  • IR1 and IR2 are separated by a 50 bp interpalindromic sequence.
  • the palindromic half-sides of IR1 are over 17 bp homologous, those of IR2 over 9 bp.
  • the palindrome of IR1 is twelve base pairs larger, but shows two insertions in this area of two and one base pair (AT, A)
  • Ten of twelve base pairs of IR1 in the 5 half and 9 of 12 base pairs in the 3 half of the sequence are homologous to IR2 (FIG. 7A, sequences of IR1 and IR2).
  • IR1 represents an almost perfect S 70 -like promoter sequence, with a remarkable modification.
  • the -10 region differs from the consensus sequence TAT AAT by inserting a C, which results in the sequence TAT-CAAT.
  • sequence of IR2 one finds ei ne -30 region, but no similarity to the known -10 region of an S 70 -like promoter sequence.
  • the spacing between the -10 and -30 region corresponds to the S 70 ideal of 17 at 16 bp.
  • the -35 region of an S 70 -like promoter lacks a consensus-like -10 region.
  • IR1 and IR2 there are three channels - (CATG) recognition pal ⁇ ndrome at homologous position
  • CAG channel - recognition pal ⁇ ndrome
  • the question of chance or necessity of such a structure arises outside the palindromic sequences of IR1 and IR2 there are also striking sequence motifs GC and AT-rich sequences are arranged alternately
  • An interesting Seq The feature of this domain is the presence of GC-rich sequence segments which are interrupted by an AT-rich sequence segment in the 6-HDNO-5 sequence.
  • the GC sequence blocks are located above the 5 region of the S 70 -like promoter.
  • the AT-rich positions turn into the protein with their small DNA groove, the GC-rich sequence blocks show GC-rich promoters on the outside, which are linked to the known S 70 -like promoters no longer have sequence similarity can be found in Streptomyces species
  • the plasmid pUC19 (Yanish-Perron et al, 1985) was double-digested with BamHI and Kpnl and purified via an agarose gel.
  • Kpnl has the recognition sequence 5 ' -GGTAC ' C- 3 '
  • An oligonucleotide of complementary sequence in the presence of a T4 ligase, T4 DNA polymerase and 0.2 mM dNTP was attached to the 3' protruding Kpnl end under standard conditions (Sambrook et al, (1989)), ligated and to Double strand filled in.
  • the oligonucleotide has the recognition sequence of the restriction endonuclease RleAl plus a few additional bases (see FIG. 7A (1)).
  • the now double-stranded DNA molecule (filled-in supernatant synthetic oligonucleotide) was made up of an enrichment fraction of the restriction enduminosolone leonuclum leone nuclobium leonuclease leonuclease (each Fig. 7 (2) and (3))
  • the reaction conditions were taken from Veseley et al, (1990).
  • This enzyme produces 3 - protruding ends outside se in an asymmetric binding site This specificity is unique so far and allows the repeated attachment of an O gonucleotides and the Primmg for a DNA polymerisation.
  • the short DNA fragment with the RleAl recognition sequence was cleaned away from the plasmid over an agarose gel.
  • FIG. 7B The changes which were introduced by “recursive DNA synthesis in vitro” in the sequence of the promoter-containing IR1 palindrome and in the interpalindromic sequence are shown in FIG. 7B.
  • FIG. 7B-4 shows retention only by the binding of N ⁇ cR1 to IR2 since the size of the complex to IR2 is the same size like the complex at IR1, this is an indication of the binding of the same protein to both palindromes Contrary to the pronounced effect created by the changes in both the length and the symmetry of the palindrome IR1 on the N ⁇ cR1 bond, changes in the number of helical turns in the interpalindromic sequence did not result in any difference in the N ⁇ cR1 bond to both palindromes Die Lange der The interpalindromic sequence was changed by deletions as well as insertions of 5 bp each (FIGS.
  • IR1 contains the -10 region of the promoter of the 6-HDNO gene, which differs from the consensus sequence of the promoter of the S 70 _ RNA polymerases by the insertion of an additional base position in the TATAAT sequence containing cytosine (FIG. 7B -1)
  • the question arose whether this unusual S 70 -10 region has a share in the specificity of the N ⁇ cR1 binding in IR1.
  • Fig. 7B-2 with N ⁇ cR1, the deletion of the cytosine residue at the corresponding position (Fig 7B-2) no change in the protein binding pattern compared to the pattern obtained with the unchanged DNA fragment, but in the binding of the S 70- like RNA polymerase from £ coli
  • PLASMID ⁇ - B - N 7885 bp Huang, Little, Seed (1985) in Vectors: A survey of molecular cloning and their applications "Rodriguez, R., ed, Butterworth Publishers, Stoneham, MA, United States. STARTER MOLECULE:
  • biotinylated starter molecule was bound to streptavidingecoatete DynalBeads. Then 0-2 rnM Biotin-deoxyUracil was set (0.5 h incubation at RT each) to block all biotin binding sites.
  • the ssDNA was filled in to the dsDNA using the T4 DNA polymerase. If need be, the dsDNA was released from its attachment to the DynalBeads. The same amount of fresh DynalBeads were added. Only the molecules with biotin were bound to the column. Molecules without biotin from the last ligation reaction were washed away.
  • the restriction endonuclease Pacl was subsequently cleaved.
  • the Dynabeads were pelletized with a magnet.
  • the molecules were precipitated from the supernatant using ethanol and the molecule was circularized using the T4 DNA ligase. " ⁇ " The circularized synthetic plasmid molecules were then in E. coli DHlO / P3 transformed according to standard protocol and selected against Tet / Amp on LB plates.

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Abstract

L'invention concerne un procédé permettant de faire la synthèse de molécules d'acide nucléique. L'invention concerne plus particulièrement un procédé de ce type, appliqué de manière récursive. Les constituants de l'acide nucléique sont de préférence d'origine synthétique ou semi-synthétique. Ce procédé repose sur le principe selon lequel une autre molécule d'acide nucléique est déposée et/ou combinée sur/avec une molécule d'acide nucléique préparée. L'extrémité de la molécule d'acide nucléique préparée est masquée, si aucune autre molécule d'acide nucléique n'a été déposée dessus et/ou combinée avec elle. L'autre molécule d'acide nucléique est séparée en un point prédéterminé et on obtient à nouveau une extrémité sur laquelle peut être appliquée une autre molécule d'acide nucléique et/ou bien être combinée avec. Les étapes précitées peuvent, le cas échéant, être répétées jusqu'à ce que le produit voulu soit synthétisé. L'invention concerne en outre un kit permettant de mettre ledit procédé en oeuvre.
EP99919096A 1998-03-19 1999-03-18 Procede permettant de faire la synthese de molecules d'acide nucleique Withdrawn EP1047706A2 (fr)

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DE1998112103 DE19812103A1 (de) 1998-03-19 1998-03-19 Verfahren zur Synthese von Nucleinsäuremolekülen
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