WO1999011818A1 - Procede de detection d'acides nucleiques ou de polypeptides hautement fonctionnels - Google Patents

Procede de detection d'acides nucleiques ou de polypeptides hautement fonctionnels Download PDF

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Publication number
WO1999011818A1
WO1999011818A1 PCT/JP1998/003854 JP9803854W WO9911818A1 WO 1999011818 A1 WO1999011818 A1 WO 1999011818A1 JP 9803854 W JP9803854 W JP 9803854W WO 9911818 A1 WO9911818 A1 WO 9911818A1
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Prior art keywords
shuffling
sequence
fitness
nucleic acids
polypeptides
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PCT/JP1998/003854
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English (en)
Japanese (ja)
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WO1999011818A8 (fr
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Isao Karube
Yoichi Okabe
Koichi Sumikura
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Isao Karube
Yoichi Okabe
Koichi Sumikura
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Application filed by Isao Karube, Yoichi Okabe, Koichi Sumikura filed Critical Isao Karube
Priority to AU88867/98A priority Critical patent/AU8886798A/en
Publication of WO1999011818A1 publication Critical patent/WO1999011818A1/fr
Publication of WO1999011818A8 publication Critical patent/WO1999011818A8/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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
    • C12N15/1027—Mutagenizing nucleic acids by DNA shuffling, e.g. RSR, STEP, RPR
    • 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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1058—Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules

Definitions

  • the present invention relates to the field of biotechnology, and more particularly to the design of polypeptides or nucleic acids.
  • Bio macromolecules such as proteins, DNA, and RNA exhibit various functions such as physiological activity, molecular recognition, and catalytic activity. If it becomes possible to freely design the functions of such polymers, it will be possible to create entirely new functional polymers, which will have a major impact on a wide range of fields such as pharmaceuticals and foods. There is expected.
  • the SELEX method (Ellington, AD & Szostak, JW, 1990, Nature 346, 818-822; Tuerk, C. & Gold, L., 1990, Science 249, 505-510.) Is a method of searching the sequence space.
  • One. Creates a molecular population that contains all sequences of a specific length for DNA or RNA, and has the desired function by repeating selection using specific activity as an index and amplification of the selected molecule It concentrates molecules. Do the same for peptides by associating genetic information with phenotypes
  • the phage display method (Scott, JK & Smith, GP, 1990, Science 249, 386-390.)
  • the polysome display method (Mattheakis, LC et.
  • the enriched sequence is optimized because reactions of many molecules occur in the same solution, and in practice there is competition or inhibition that cannot occur in the case of single molecule reactions. It does not necessarily have the functions provided.
  • Another disadvantage is that when two different sequences have the same activity, the longer sequence is less likely to be detected in the final base sequence decoding stage because of its low percentage in the population. (Sumikura, K. et. Al., Nucleic Acids Symp. Ser., In press.).
  • An object of the present invention is to provide a method for efficiently designing a highly functional biopolymer.
  • a protein has a higher function by connecting a variety of genes encoding polypeptides having a certain length as basic units, and connecting those units. Is believed to have acquired Most eukaryotic genes consist of a region that is translated into amino acids (exons) and an untranslated region between exons (introns). The amino acid sequence of a protein is encoded in multiple exons. In the long term, exons are the basic unit of protein evolution, and introns are thought to be an area for exon shuffling. (De Souza, SJ et. Al., 1996, Genes Cels 1, 493-505.).
  • the present inventors have focused on this exon shuffling mechanism of evolution, and have basically made it possible to artificially rearrange exon shuffling-like genes among multiple individuals using a computer.
  • a molecular design method called “shuffling strategy” was developed. By using this method, it is possible to make the evolution of molecules more dramatic and faster than conventional genetic algorithms, and thus it is possible to search for functional macromolecules more efficiently. .
  • the present invention includes the following.
  • a method for searching for a highly functional polypeptide or nucleic acid comprising the following steps: (a) synthesizing a plurality of polypeptides or nucleic acids having different sequences from each other, (b) measuring the fitness of the synthesized polypeptide or nucleic acid at the laboratory level,
  • step (c) ranking the polypeptides or nucleic acids synthesized in step (a) according to their fitness
  • step (d) a sequence in which a mutation has been introduced into a specific sequence is prepared separately from the polypeptide or nucleic acid having the sequence obtained by shuffling.
  • step (c) The method according to claim 2, wherein the specific sequence is a sequence having a certain rank or more in step (c).
  • step (d) at least one of the sequences obtained by shuffling is subjected to further mutation-introduced sequences and added to the “shuffling library”.
  • step (d) The method according to any one of (1) to (4), wherein in step (d), shuffling is performed only between individuals having a certain rank or higher.
  • a non-naturally occurring polypeptide or nucleic acid obtained by the method according to any one of (1) to (6) and having a certain degree of fitness or more.
  • the term “shuffling” refers to dividing a polypeptide or nucleic acid sequence into a plurality of specific blocks (named “virtual exons”). This refers to the operation of exchanging virtual exons among a number of individuals to create a new polypeptide or nucleic acid sequence.
  • the term “shuffling 'library 1'” refers to a sequence group resulting from performing "shuffling" on a specific polypeptide or nucleic acid sequence.
  • a method known to those skilled in the art can be used to synthesize a polypeptide or a nucleic acid.
  • a polypeptide for a polypeptide, the t-Boc method (Merrifield, B., 1986, Science 232, 34, 347.) or Fmoc method (Gorka, J. et. Al., 1989, Pept. Res. 2, 376-80.).
  • the phosphoramidite method Itakura, K. et. Al., 1984, Ann. Rev. Biochem. 53, 323-356.
  • Etc. for nucleic acids, the phosphoramidite method (Itakura, K. et. Al., 1984, Ann. Rev. Biochem. 53, 323-356.) Etc. can be used.
  • fitness is an index indicating how much a polypeptide or nucleic acid having an individual sequence exhibits a specific biological activity, and is usually determined by measurement at a laboratory level.
  • the fitness of a polypeptide includes binding activity to a target molecule or activity as an antibiotic. The former is based on the EUSA method (Creigh ton, TE (Ed), 1989, Protein Structure. A Practical Approach, IRL). Pres s.) Or BIAcore (Griffiths, DG & Hall, G, 1993, Tibtech 11, 122-130.), And the latter can be measured by examining the ability to inhibit the growth of cells.
  • examples of the fitness of nucleic acid include binding activity to a protein and catalytic activity of cleaving a nucleic acid having a specific base sequence.
  • the former is an ELISA method, a BIAcore, or a gel shift method (Latchman, DS, 1993, Transcription Factors). , IRL pres s.) And the latter using a radioactive label (Santoro, SW & Joyce, G.
  • the cycle of activity measurement and shuffling is usually performed 5 times (generation) or more, preferably 8 times or more, and more preferably 10 times or more.
  • care must be taken if the array to be shuffled is too limited, because it is easy to end up with a local solution instead of the optimal solution.
  • individuals with a certain rank or higher can be divided into a plurality of groups according to the rank, and shuffling can be performed in each group.
  • This allows for global shuffling between a large number of arrays and local shuffling between a small number of arrays in parallel.
  • it is possible to set up two groups within the top 20% and within the top 40%.
  • the optimal solution search can be performed more quickly.
  • Mutations include all common mutations, including point mutations, deletion mutations, insertion mutations, inversions, and translocations.
  • a sequence in which a mutation has been introduced into a specific individual is created, and a “shuffling library” is created.
  • a sequence in which a mutation is introduced into a polypeptide or nucleic acid having a sequence obtained by shuffling is further prepared and added to “Shuffling 'Library 1'”. it can.
  • an array having a high degree of “isolation” in addition to the degree of fitness can be preferentially selected.
  • the “isolation degree” of a sequence is an index indicating the low similarity of a specific sequence to another sequence of the same generation, and is a measure of the similarity of another sequence of the same generation to the specific sequence. It can be defined as being inversely proportional to the sum of "similarity” with the sequence.
  • the term “similarity” of a sequence refers to an index indicating the similarity between two sequences.For example, when two sequences are compared and the same residue or base is present at the same position, 1 Points can be quantified by the total number of points.
  • each sequence of the same generation is ranked by the product of “fitness” and “isolation”, and shuffling can be performed only between individuals having a certain rank or higher.
  • each sequence of the same generation is ranked by the product of “fitness” and “isolation”, and individuals having a certain rank or more are further divided into a plurality of groups according to the rank. Shuffling can also be performed.
  • the initial sequence population (from which the first shuffling is performed) may include naturally occurring sequences. In this case, it is possible to further improve the function of the molecule that exists in nature.
  • a random sequence may be used as the initial sequence. Even if a random sequence is used as the starting sequence, there is no problem in practical use because the fitness is markedly increased by the “shuffling strategy”. Rather, it can be said that using a random sequence as the starting sequence increases the possibility of reaching a highly active biopolymer having a structure significantly different from that of the natural type.
  • FIG. 1 shows the ratio of the binding constant (KA) between the peptide and the antibody.
  • Figure 2 compares the shuffling strategy and the genetic algorithm.
  • the horizontal axis represents the variation of the molecule, and the vertical axis represents the average of the maximum value in 100 trials.
  • FIG. 3 is a diagram showing the structure of a double G quartet.
  • FIG. 4 is a diagram showing the structure of a triple G quartet. BEST MODE FOR CARRYING OUT THE INVENTION
  • Example 1 Simulation of design of a novel peptide that binds to a known protein (Search for topography of fitness based on binding data between a hemagglutinin-derived peptide and a monoclonal antibody)
  • Influenza virus hemagglutinin is a viral membrane protein that is used as an antigen to produce antibodies against the virus in the host body.
  • HA is a trimer consisting of the same subunit of 550 residues, and its monomer is composed of two polypeptide chains, HA1 and HA2 (Wilson, IA. Et. Al., 1981). , Nature 289, 366-73.)
  • the IC50 may be considered to be proportional to the dissociation constant (KD) between the peptide and the antibody (Aita, T. & Husimi, Y., 1996, J. theor. Biol. 182, 469-485.)
  • KD dissociation constant
  • KA binding constant
  • Figure 1 shows the value obtained by dividing the reciprocal of the IC 50 of each analog by the reciprocal of the IC 50 of the control peptide.
  • the letters represent the one-letter code for amino acids). For the above reasons, these values can be considered to be the ratio of the KA values of the analog and the control peptide.
  • the relative value of the equilibrium binding constant (KA) for Fab 17/9 can be determined.
  • the calculation of fitness and the generation of next-generation individuals based on the results were performed for 40 generations.
  • the search for 40 generations was counted as one trial, and 100 trials were performed to examine the directed evolution of the binding ability.
  • the calculation of fitness and the generation of next-generation individuals based on the results were performed for 40 generations.
  • the search for 40 generations was counted as one trial, and 100 trials were performed to examine the directed evolution of the binding ability.
  • the shuffling strategy is also more genetic for the average of the fitness values for each generation, averaged over 100 trials, and for the average of the minimum fitness values for each generation, averaged over 100 trials. It was found that the search for the optimal solution was more efficient than the genetic algorithm. Comparing how many individuals must be synthesized before the fitness exceeds 1 for the first time, the shuffling strategy shows 129 individuals and the genetic algorithm has 155 individuals. Can also see the high search efficiency of shuffling strategies for genetic algorithms.
  • the topography of the fitness was set and the computer simulation was performed.
  • the topography of the fitness was set and the computer simulation was performed.
  • the topography of the fitness was set and the computer simulation was performed.
  • multiple amino acid-substituted / substituted salt substitutions may affect the fitness in some cases.
  • the situation that is different from the simulation occurs, for example, the fitness of all the arrays cannot be measured due to the detection limit of the measuring device. Therefore, we conducted the following experiments, including the process of actually synthesizing molecules and measuring fitness.
  • Thrombin is a type of serine protease and has various functions including blood coagulation.
  • 5'-GGTTGGTGTGGTTGG-3 a single-stranded DNA consisting of 15 residues (SEQ ID NO: 1), called thrombin-abumauma, is known to bind to thrombin and inhibit its biological activity (Bock, LC et. Al., 1992, Nature 355, 564-566.) 0 As shown in Fig. 3, this molecule has a three-dimensional structure stabilized by two G-quartet structures. (Wang, KY et. Al., 19 93, Biochemistry 32, 1899-1904.).
  • N represents A, C, G ⁇ or T
  • the fitness of each sequence was measured using BIAcore2000 (BIAcore AB), an experimental device using the principle of surface plasmon resonance.
  • the synthesized single-stranded DNA was immobilized on a sensor chip for BIAcore2000 via carboxymethyl dextran and streptavidin, and 20 M of thrombin was passed thereto, and the response was examined.
  • BIAcore when a molecule binds to an immobilized substance, the refractive index near the sensor chip changes, and this is detected as a change in the resonance unit.
  • polypeptides or nucleic acids it has become possible to efficiently search for highly functional polypeptides or nucleic acids. For example, it has become possible to dramatically improve the function of naturally occurring polypeptides or nucleic acids. Furthermore, it has become possible to freely search for a polypeptide or nucleic acid having a specific function and a novel structure that does not exist in nature.

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Abstract

Procédé de conception de molécules, qui consiste principalement à reconstruire artificiellement des gènes par déplacement d'exons chez plusieurs individus. Ce procédé consiste plus spécifiquement (a) à synthétiser plusieurs polypeptides ou acides nucléiques de séquence différente; (b) à mesurer en laboratoire la valeur sélective desdits acides nucléiques ou polypeptides synthétisés; (c) à classer les polypeptides ou acides nucléiques synthétisés dans l'étape (a) en fonction de leur valeur sélective; (d) à préparer une bibliothèque de déplacement, des séquences produites par déplacement des structures partielles chez des individus choisis en fonction de leur classement; (e) à synthétiser des polypeptides ou des acides nucléiques faisant partie de ladite bibliothèque; et (f) à répéter les procédures des étapes (b) à (e) un nombre de fois arbitraire, à l'aide des polypeptides ou des acides nucléiques produits dans l'étape (e). Ledit procédé permet d'améliorer sensiblement et rapidement des molécules par rapport à un algorithme génétique classique, ce qui permet de détecter efficacement des polymères fonctionnels, notamment des polypeptides ou des acides nucléiques ayant des fonctions spécifiques ainsi que des nouvelles structures n'existant pas dans la nature.
PCT/JP1998/003854 1997-08-28 1998-08-28 Procede de detection d'acides nucleiques ou de polypeptides hautement fonctionnels WO1999011818A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SG95640A1 (en) * 2001-02-12 2003-04-23 Council Scient Ind Res Safe, eco-friendly, health protective herbal colours and aroma useful for cosmaceutical applications
JP2003521933A (ja) * 2000-02-10 2003-07-22 ゼンコー タンパク質ライブラリーに関するタンパク質設計オートメーション
JP2008118923A (ja) * 2006-11-13 2008-05-29 Nec Soft Ltd 核酸高次構造予測方法、核酸高次構造予測装置及び核酸高次構造予測プログラム
US7462469B2 (en) * 2000-01-11 2008-12-09 Maxygen, Inc. Integrated system for diversity generation and screening
JP2012005458A (ja) * 2010-06-28 2012-01-12 Tdk Corp アプタマーの選別方法

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WO1995022625A1 (fr) * 1994-02-17 1995-08-24 Affymax Technologies N.V. Mutagenese d'adn par fragmentation aleatoire et reassemblage
JPH1014578A (ja) * 1996-07-01 1998-01-20 Mitsubishi Chem Corp 入れ換え変異体遺伝子ｄｎａの構築
WO1998004580A1 (fr) * 1996-07-26 1998-02-05 Yamanouchi Pharmaceutical Co., Ltd. Procede de recherche de peptides et methode de preparation de composes
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7462469B2 (en) * 2000-01-11 2008-12-09 Maxygen, Inc. Integrated system for diversity generation and screening
JP2003521933A (ja) * 2000-02-10 2003-07-22 ゼンコー タンパク質ライブラリーに関するタンパク質設計オートメーション
SG95640A1 (en) * 2001-02-12 2003-04-23 Council Scient Ind Res Safe, eco-friendly, health protective herbal colours and aroma useful for cosmaceutical applications
JP2008118923A (ja) * 2006-11-13 2008-05-29 Nec Soft Ltd 核酸高次構造予測方法、核酸高次構造予測装置及び核酸高次構造予測プログラム
JP2012005458A (ja) * 2010-06-28 2012-01-12 Tdk Corp アプタマーの選別方法

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