DE19646372C1 - Conjugates of polypeptide and encoding nucleic acid - Google Patents

Abstract

Compound comprises a structural unit A (genotype) and a further structural unit B (phenotype), in which the genotype and phenotype are permanently linked to each other, where structural unit A exhibits, besides other regions, \- 1 region coding for \- 1 polymeric molecule constructed from amino acid units and the coding regions are translatable, and furthermore a first structural subunit (terminator) is located in an untranslated section of structural unit A, which permanently links structural unit B as a translation product of structural A to structural unit A.

Description

The present invention relates to a compound of a Structural unit A (genotype) and another structural unit B (phenotype) according to the preamble of claim 1 and a ver drive to molecular genotype / phenotype coupling by Her position of a fusion product according to the invention A compound according to claim 12.

A coupling of genotype and phenotype is particularly important for evolutionary biotechnology desirable since this way the selection of polypeptide molecules with special or improved properties directly on their coding sequence can be used. Procedures are described that enable such a coupling. It is about primarily in the process of selecting the present the molecules in vivo on a surface such. B. on a phage surface (phage display) on a bacteria surface (Bacterial Surface Display) or directly on molecular level (Molecular Display). This in vivo procedure are characterized by serious disadvantages. So is, e.g. B. due to transformation, the repertoire size to about 10⁸ different molecules limited. Even through an in vivo The available repertoire could be diversified only expand to about 10 12 molecules. Would be theoretically possible however a diversity of <10¹⁵ molecules. Beyond that would in vivo systems the expression of cytotoxic genes not ge equip. In addition, such systems only allow that direct access to cytoplasmic or secretory Proteins. These in vivo systems are also with a bio logical security risk in some countries working with such systems is restricted.  

In addition, an in vitro method is described in which DNA molecules, the sequence of a peptide and that of a Contain streptavidin binding peptides, transcribed in vitro will. A streptavidin is chemically attached to the transcripts knotted so that the coding in vitro translation RNA template is linked to the resulting peptide. This However, the process has several disadvantages. So are the changes effects between streptavidin and the streptavidin binding protein not stable under certain assay conditions. In addition can interactions between the streptavidin of an mRNA and the streptavidin binding protein, one not of this mRNA encoded fusion protein occur. In addition, a Influencing the folding structure of the one to be selected Peptides occur through the streptavidin binding protein.

The technical problem underlying the invention exists in a risk-minimized method for genotype-phenotype- Specify coupling, and provide a construct, which the diversity achievable so far exceeds expression cytotoxic genes and equally selection both of cytoplasmic as well as secretory proteins possible and also not dependent on recombinant cells are. It should also be the disadvantages of the above described in vitro procedure can be avoided.

The problem underlying the invention is solved by a compound according to claim 1 and a method according to An Proverb 12 on molecular genotype / phenotype coupling.

The connection according to the invention consists of a structure unit A (genotype) and another structural unit B (pheno type), in which the genotype and phenotype are permanently ver are bound, the structural unit A at least one for at least one polymer composed of amino acid units Has molecule coding region and the one or more encode de (n) area (s) is or are translatable, still in the Structural unit A a first structural subunit (terminator)  is arranged in a non-translated section that the Structural unit B as a translation product of the structural unit A permanently connects to the structural unit A.

The permanent linking of structural units A and B can especially by using ribosomal translation system.

According to the invention, the term terminator denotes a structure subunit, which the structural unit B as translations Product of structural unit A with structural unit A permanent linked. As terminators according to the invention nucleophilic chemical groups, especially organic amino groups, which are a carboxylic acid ester, in particular between a tRNA and the structural unit B, forming a permanent, especially covalent Can split bonds. For example, alipha table, cyclic or aromatic compounds, with a Amino group, natural or unnatural amino acids and Derivatives, or puromycin and derivatives are used. When using a ribosomal translation system the terminator preferably also has a stable molecule group that the 2'-3'-ortho-ester structure of an aminoacyl tRNA imitated in complex with elongation factor EF-Tu and GTP and is therefore bound by the elongation factor.

The coupling reaction can be random, but it is advantageous according to the invention, the linkage of the structural unit A with the structural unit B created by translation a second structure below arranged on the structural unit A. unit such as B. to control a terminator codon.

The terminator codon can be random, even internal half of a coding area of the structural unit A, however preferred to defined at the 3'-terminal end of the coding area lying codons, for example nonsense Codons (UAG, UAA, UGA) or any sense codons, preferred  however rare with regard to the translation system used Sinn codons (e.g. AGA when using an E. coli translation systems) act.

The site-specific coupling to a defined terminator codon can preferably by means of two variants of the invention according to the procedure:

• 1. According to the invention, the site-specific coupling can be found under Use of a sense codon as terminator codon, which is not upstream in the coding area of the structure unit A already exists. For this, preferably rare AGA codon used, in which case the Trans lation approach contain no tRNA specific for the AGA codon should. According to the invention, the peptidyl transfer takes place at the AGA codon on the terminator, which in this case does not match the corresponding tRNA molecules of the translation mixture around the Occupation of the aminoacyl binding site of the ribosome competes.
• 2. According to the invention, the site-specific coupling can also through a codon-anticodon interaction. It is possible according to the invention with the terminator via a spacer to link other areas of the structural unit A. In the event of the site-specific coupling by means of codon-anticodon change effect, a spacer with a tRNA structure is selected, which on Terminator codon the transfer of the nascent structural unit B conveyed to the terminator. This is also preferred the AGA codon as described above, so that the Translation mixture should be pretreated accordingly.

In general, according to the invention, as spacers, for example natural and / or synthetic oligomers or polymers, such as e.g. B. any nucleic acid sequences can be used. The Linking the spacer to other areas of the structural unit A, such as B. the 3'-terminal nucleotide of an mRNA can in done in any way, but preferably via the 3'-position of the terminal nucleotide. It is according to the invention  expedient, this link and other possible ver to choose links within the structural unit A so that these cannot be cleaved under assay conditions. Thus also ensures that after transferring the nascent Another transfer to the polypeptide chain on the terminator a new aminoacyl tRNA is omitted. Such links can be both covalent in nature and molecular Interactions, e.g. B. between streptavidin or avidin and Biotin, nickel-NTA and oligo-histidine, protein and ligand, Antibodies and antigen, or between two hybridizing Nucleic acids. Linking the terminator with a ribonucleic acid spacer can, for example, via the 2 ′ and / or 3'-position.

The compound of the invention and the invention Methods are exemplified below with reference to figures shown.

Fig. 1: General principle of the ribosomally catalyzed coupling of a polypeptide to its own mRNA in vitro.

Fig. 2: Coupling of the nascent polypeptide chain to its own mRNA by an aliphatic amine.

Fig. 3: coupling of the nascent polypeptide to its own mRNA through a puromycin molecule.

Fig. 4: Coupling of the nascent polypeptide chain at their own mRNA by a loaded with phenylalanine, modified tRNA.

Fig. 5: Coupling of the nascent polypeptide to its own mRNA by an anthraniloyl-tRNA.

Fig. 6: Coupling of the nascent polypeptide chain at their own mRNA by an intramolecular laden nonsense suppressor tRNA.

Fig. 7: Coupling of the nascent polypeptide chain at their own mRNA by an intramolecular loaded with puromycin tRNA.

Fig. 8: Coupling of the nascent polypeptide chain to its own mRNA by a loaded tRNA, the amino acid of which is linked to the mRNA.

Fig. 1 shows the general principle of in vitro genotype-phenotype coupling using a ribosomal translation system. The coding, translatable region of the structural unit A is followed by a terminator codon, which is linked to the terminator via a spacer. After synthesis of the construct described, the coding region is translated in vitro. At the terminator codon, the terminator enters the aminoacyl binding site of the translating ribosome. The nascent polypeptide chain (structural unit B) is catalytically transferred to the terminator. The coupling product is a polypeptide that is stably linked to its coding nucleic acid at the C-terminal end via a terminator and a spacer. The stability of the coupling ensures that, after selection of the polypeptide with the desired property, the genetic blueprint is available at the same time. Symbols: A and P, aminoacyl and peptidyl binding site of the ribosome; x terminator.

Fig. 2 shows the use of a primary amine as a terminator. The coding, translatable region of the structural unit A here is an mRNA which, instead of a stop codon, contains a single AGA codon to which a spacer (here ribonucleic acid) is connected. Its 3'-end has a 3'-deoxy-3'-aminoadenosine residue which carries an aliphatic amino group. The translation approach contains no tRNA molecules specific for the arginine codon AGA, so that at the AGA codon the modified 3'-end of the spacer can enter the aminoacyl binding site of the translating ribosome and the nascent polypeptide chain can be transferred to the terminator. A polypeptide is formed as the coupling product, which is covalently linked at the C-terminal end to the terminator, the spacer RNA and mRNA.

FIG. 3 shows the use of puromycin molecule as a terminator. The mRNA contains a single AGA codon instead of a stop codon, followed by a spacer (here ribonucleic acid). Its 3'-end has a puromycin residue coupled via the 5'-OH group. The translation approach does not contain any tRNA molecules specific for the arginine codon AGA, so that at the AGA codon the modified 3'-end of the spacer can enter the aminoacyl binding site of the translating ribosome and the nascent polypeptide chain can be transferred to the terminator. A polypeptide is formed as the coupling product, which is covalently linked at the C-terminal end to the terminator, the spacer RNA and mRNA.

FIG. 4 describes the coupling of the nascent polypeptide chain to its own mRNA by a modified tRNA loaded with phenylalanine. Instead of a stop codon, the mRNA contains a single AGA codon, which is followed by any ribonucleic acid sequence. Their 3'-end has a 3'-deoxy-3'-aminoadenosine residue which is covalently coupled to the X₄₇ base of a tRNA loaded with phenylalanine and modified at the 3'-end. The translation approach does not contain any tRNA molecules specific for the arginine codon AGA, so that at the AGA codon the modified 3'-end of the spacer enters the aminoacyl binding site of the translating ribosome and the nascent polypeptide chain can thus be transferred to the terminator . A polypeptide is formed as the coupling product, which is covalently linked at the C-terminal end to the terminator, the spacer RNA and mRNA.

Fig. 5 illustrates the coupling of the nascent polypeptide chain to its own mRNA by a derivatized anthraniloyl tRNA. Instead of a stop codon, the mRNA contains a single AGA codon, which is followed by any ribonucleic acid sequence. Its 3'-end has a 3'-deoxy-3'-aminoadenosine residue which is covalently coupled to the X₄₇ base of a derivatized anthraniloyl tRNA. The translation approach contains no tRNA molecules specific for the arginine codon, so that at the AGA codon the modified 3'-end of the spacer can enter the aminoacyl binding site of the translating ribosome and the nascent polypeptide chain can be transferred to the terminator. A polypeptide is formed as the coupling product, which is covalently linked at the C-terminal end to the terminator, the spacer RNA and mRNA.

Fig. 6 illustrates the coupling of the nascent polypeptide chain to its own mRNA by an intramolecular, loaded nonsense suppressor tRNA. The mRNA contains the stop codon UAG, which is followed by a spacer (here ribonucleic acid). The spacer contains the nucleotide sequence of the precursor molecule of a nonsense suppressor tRNA and forms a corresponding tertiary structure. The 3'-end is linked to any (natural or unnatural) amino acid by a stable amide bond. At the nonsense codon UAG, the spacer, which acts as a loading suppressor tRNA, can reach the aminoacyl binding site of the translating ribosome and the nascent polypeptide chain can be transferred to the terminator. A polypeptide is formed as the coupling product, which is covalently linked at the C-terminal end to the terminator, the spacer RNA and mRNA.

FIG. 7 shows the coupling of the nascent polypeptide chain to its own mRNA by an intramolecular tRNA loaded with puromycin. The mRNA contains a single AGA codon instead of a stop codon, followed by a spacer (here ribonucleic acid). The spacer contains the nucleotide sequence of the precursor molecule of a tRNA specific for the AGA codon and forms a corresponding tertiary structure. The 3′-terminal nucleotide of the spacer (A₇₆) has a puromycin residue. The translation approach contains no tRNA molecules specific for the arginine codon AGA, so that on the AGA codon the spacer, which acts as a loaded tRNA, can enter the aminoacyl binding site of the translating ribosome and the nascent poly peptide chain can be transferred to the terminator . A polypeptide is formed as the coupling product, which is covalently linked at the C-terminal end to the terminator, the spacer RNA and mRNA.

Fig. 8 illustrates the coupling of the nascent polypeptide chain to its own mRNA by a loaded tRNA, the amino acid of which is linked to the mRNA. The mRNA contains a single AGA codon, which is followed by a stop codon and a spacer (here ribonucleic acid). The spacer is covalently linked at the 3'-end via a phosphodiester bond with the side chain of the termina tors. The terminator in turn is linked to a tRNA via an aminoacyl bond. The translation approach contains no tRNA molecules specific for the arginine codon AGA, so that at the AGA codon the terminator coupled to the mRNA can enter the aminoacyl binding site of the translating ribosome and the nascent polypeptide chain can be transferred to the terminator. The subsequent transfer of the peptidyl residue to water at the stop codon causes the release of the AGA-specific tRNA. A polypeptide is formed as the coupling product, which is covalently linked at the C-terminal end to the terminator, the spacer RNA and mRNA. Neither the tRNA nor the type of amino acid linkage is changed in this embodiment, so that binding by the elongation factor Tu under almost natural conditions and thus an efficient incorporation of the terminator molecule are made possible.

The experimental implementation of the embodiment of the method according to the invention shown in FIG. 8 can be carried out, for example, by the following steps:
Preparatory steps: (final product can be used for any coupling reactions)

• 1. Chemical aminoacylation of the diribonucleotide pC₇₅pA₇₆ with an amino acid, the side chain R with any Diribonucleotide or dideoxyribonucleotide is modified [R = -CH₂-3′-pXpXp-5 ′];
• 2. Ligation of the aminoacylated diribonucleotide with a for the codon AGA specific tRNA (C₇₄ end!) T4 RNA ligase;
• 3. Cleaning the correct and functional ligation products (Aminoacyl tRNAs) by immobilized elongation factor EF-TU in the presence of GTP;

Further steps: (depending on the target to be selected molecule);

• 4. Diversification of the corresponding polypeptide structural gene through chemical resynthesis (codon-based mutagenesis) or by mutagenesis and / or recombination PCR The term "codon-based mutagenesis" is used in the sense the invention understood a chemical synthesis of DNA, in which trimers are used instead of monomer units, which each code for a specific amino acid. By The mixing of trimer building blocks in the synthesis can Installation of different codons in the desired ratio be achieved in any position.
• The term "mutagenesis PCR" is used in the sense of Invention understood an in vitro amplification of DNA, which is faulty due to certain conditions DNA duplication is enforced.
• The term "recombination PCR" is used in the sense of Invention understood an in vitro amplification of DNA, by mixing random DNA subfragments homologous genes new, mosaic-like genes synthesized will. This process is with a natural recom bin comparable;
• 5. Reamplification of the synthesized Structural genes by PCR, the 5'-primer being a promoter  (e.g. T7 promoter) and one Translation initiation region (including ATG start Codon), while the 3'-primer six histidine codons, the Introduces terminator codon AGA and the nonsense codon TAG;
• 6. Preparation of a spacer DNA (e.g. chemical synthesis or by PCR), whose 5'-terminal sequence with the 3'-termina len sequence of the reamplified structural gene overlaps;
• 7. Splicing of both DNA fragments by SOE-PCR (splicing by overlap extension), the spacer being the 3 ′ terminal Fusion partner forms.
• The term "SOE-PCR" is used in the sense of the invention understood an in vitro amplification of DNA in which due to overlapping areas with two DNA fragments be spliced together;
• 8. In vitro transcription of the fusion gene with m⁷GpppG (5′- Cap structure) as a primer (e.g. with T7 DNA polymerase);
• 9. Purification of the run-off transcript using polyacrylamide gel electrophoresis;
• 10. Ligation of the synthesized RNA (free 3'-OH end; 5'-end protected by cap structure) with the amino acid Side chain of aminoacylated tRNA by T4 RNA ligase;
• 11. Cleaning of the product by polyacrylamide gel electro phoresis;
• 12. In vitro translation of the mRNA and coupling of the nascent Polypeptide chain by the specific for the AGA codon, aminoacylated tRNA in the presence of an E. coli trans lationsgemisch, by appropriate pretreatment no tRNA molecules specific for the arginine codon AGA contains more;
• 13. Separation of the translation factors and purification of the Fusion molecules by affinity chromatography on one Nickel -NTA matrix.
• The term "nickel-NTA matrix" is used in the sense of Invention understood a matrix based on a immobilized nickel ions the purification of oligohistidine containing biomolecules;
• 14. In vitro selection of fusion molecules with desired ones  Properties (e.g. through panning);
• 15. Reverse transcription of the enriched fusion molecules and reamplification of the genetic information by PCR, the 5'-primer in turn the promoter and the trans lationsinitiationsregion introduces during the 3'-primer hybridizes with the 3'-terminal sequence of the spacer;
• 16. Repeat the procedure described from step 8.

The experimental implementation of the first preparatory step can in particular also using an amino acid whose Side chain with any oligoribonucleotide or oligo deoxyribonucleotide is modified.

Furthermore, it can be preferred if the side chain of the Amino acid has a free 5'-OH end, which only after Ligation according to step 2 is 5'-phosphorylated. In an advantage The yield of functional ones can thus be obtained Increase ligation products.

Following Fig. 8 is located in a further guide form at least one sense codon between terminator codon and stop codon. In this way, coupling between the terminator and polypeptide chain is no longer restricted to their C-terminal amino acid.

Claims (13)

1. Connection from a structural unit A (genotype) and one further structural unit B (phenotype), in the genotype and Phenotype are permanently linked, the Structure unit A in addition to other areas at least one for at least one composed of amino acid units, has polymeric molecule coding region and the or the coding region (s) is or are translatable, furthermore a first structure in the structural unit A. subunit (terminator) in a non-translated Section is arranged which the structural unit B as Translation product of structural unit A with the structure unit A connects permanently. 2. The compound of claim 1, wherein the linkage of the Structural unit A with the one created by translation Structural unit B via one on structural unit A arranged second structural subunit is controlled. The compound of claim 2, wherein the second structure is under unit is a codon (terminator codon). 4. Connection according to at least one of claims 1 to 3, wherein the structural unit A is a modified mRNA and the structural unit B translated from the mRNA Polypeptide chain. 5. Connection according to at least one of claims 1 to 4, the terminator being connected to other Be range of structural unit A is linked. 6. The compound of claim 5, wherein the spacer with the Interaction of terminator codon of structural unit A. occurs.   7. Connection according to at least one of claims 1 to 6, where the terminator is a nucleophilic chemical group, which a carboxylic acid ester to form a kova lenten bond can split. 8. A compound according to claim 7, wherein the nucleophilic chemical Group is an organic amino group. 9. A compound according to any one of claims 1 to 8, wherein the Terminator a carboxylic acid ester between a tRNA and the structural unit B splits and a permanent Ver bond between the structural units A and B. becomes. 10. A compound according to any one of claims 1 to 9, wherein the Link between the structural units A and B by a covalent connection takes place. 11. A compound according to any one of claims 1 to 10, wherein the Terminator, especially as a nucleophilic group via a Is bound to a spacer with tRNA structure, the one with the terminator codon of structural unit A, the is in particular an mRNA, can interact. 12. Method for molecular genotype / phenotype coupling by means of a compound according to at least one of claims 1 to 11, characterized in that the structural unit A with is implemented in the presence of substances required for translation. 13. The method of claim 12, wherein the coupled genoty pen / phenotypes are isolated.