EP1230355A2 - Banque d'adn et son utilisation dans des procedes de selection et conception des polypeptides - Google Patents

Banque d'adn et son utilisation dans des procedes de selection et conception des polypeptides

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Publication number
EP1230355A2
EP1230355A2 EP00964464A EP00964464A EP1230355A2 EP 1230355 A2 EP1230355 A2 EP 1230355A2 EP 00964464 A EP00964464 A EP 00964464A EP 00964464 A EP00964464 A EP 00964464A EP 1230355 A2 EP1230355 A2 EP 1230355A2
Authority
EP
European Patent Office
Prior art keywords
library
dna
sequences
sequence
polypeptide
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.)
Withdrawn
Application number
EP00964464A
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German (de)
English (en)
Inventor
Yen MRC Lab. of Molecular Biology CHOO
Aaron Klug
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sangamo Therapeutics Inc
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Sangamo Biosciences Inc
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Filing date
Publication date
Priority claimed from GBGB9923327.2A external-priority patent/GB9923327D0/en
Priority claimed from GB0011068A external-priority patent/GB0011068D0/en
Priority claimed from GB0013106A external-priority patent/GB0013106D0/en
Application filed by Sangamo Biosciences Inc filed Critical Sangamo Biosciences Inc
Publication of EP1230355A2 publication Critical patent/EP1230355A2/fr
Withdrawn legal-status Critical Current

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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
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
    • 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
    • C12N15/1048—SELEX

Definitions

  • the present invention relates to a library of DNA sequences immobilised onto a solid support and its use in methods of selecting and designing polypeptides comprising nucleic acid binding motifs, in particular zinc fmger polypeptides.
  • Zf zinc finger
  • DNA-binding protein domains are able to discriminate between different DNA sequences.
  • the zinc fmger motif has been studied extensively, with a view to providing some insight into this problem, owing to its remarkable prevalence in the eukaryotic genome, and its important role in proteins which control gene expression in Drosophila, mice and humans (Kinzler et al., 1988 Nature (London) 332, 371).
  • sequence-specific DNA-binding proteins bind to the DNA double helix by inserting an ⁇ -helix into the major groove. Sequence specificity results from the geometrical and chemical complementarity between the amino acid side chains of the and the accessible groups exposed on the edges of base-pairs. In addition to this direct reading of the DNA .1-
  • Randomised mutagenesis (at the same postions as those selected by Rebar & Pabo) of finger 1 of Zif 268 with phage display has also been used by Jamieson et al, (1994 Biochemistry 33, 5689-5695) to create novel binding specificity and affinity.
  • Zf protein motifs are widespread in DNA binding proteins and that binding is via three key amino acids, each one contacting a single base pair in the target DNA sequence.
  • Motifs are modular and may be linked together to form a set of fingers which recognise a contiguous DNA sequence (e.g. a three fingered protein will recognise a 9mer etc).
  • the key residues involved in DNA binding have been identified through sequence data and from structural information. Directed and random mutagenesis has confirmed the role of these amino acids in determining specificity and affinity.
  • Phage display has been used to screen for new binding specificities of random mutants of fingers.
  • a recognition code, to aid design of new finger specificities, has been worked towards although it has been suggested that specificity may be difficult to predict.
  • the assay should include all possible DNA sequences, of given length, to establish the preferred specificity of the protein motif to rank other acceptable DNA sequences in terms of affinity. Therefore, wherever possible, an idea of the absolute affinity should emerge in parallel, i.e. the assay should not be simply comparative. This is possible by, for example, determining the apparent Kd of a protein for a series of related binding sites.
  • the present invention provides a library of DNA sequences consisting of 4 1 sequences, where N is greater than or equal to three, each sequence varying from the other sequences by comprising a different one of the 4 N possible permutations of a DNA sequence of length N, wherein the library of DNA sequences is immobilised on a solid substrate.
  • the present invention also provides a method for designing a zinc finger polypeptide having specificity for a particular DNA sequence comprising a contiguous sequence of N nucleotides. where N is greater than or equal to three, which method comprises: (i) providing a zinc finger polypeptide, preferably by designing using a rational design method or by selection from a library; (ii) producing the polypeptide;
  • step (iii) determining the sequence specificity for the polypeptide by contacting a library of DNA sequences with the polypeptide and identifying the sequence or sequences with which the polypeptide binds to with greatest affinity; (iv) if the sequence or sequences identified in step (iii) are not the desired sequences, making modifications to the amino acid sequence of the polypeptide, preferably based on rational design or by selection from a library, and repeating steps (ii) and (iii).
  • the library of DNA sequences consist of 4' sequences, each sequence varying from the other sequences by comprising a different one of the 4 possible permutations of the DNA sequence of length N, wherein the library of DNA sequences is immobilised on a solid substrate.
  • the present invention also provides a method for isolating a zinc finger polypeptide having specificity for a particular DNA sequence comprising a contiguous sequence of N nucleotides, where N is greater than or equal to three, which method comprises:
  • step (iii) optionally repeating selection steps (i) and (ii) with those carrier organisms selected in step (ii), wherein the library of DNA sequences consist of 4 N sequences, each sequence varying from the other sequences by comprising a different one of the 4 ' possible permutations of the DNA sequence of length N, wherein the library of DNA sequences is immobilised on a solid substrate.
  • the present invention provides a method for determining the preferred base recognition specificity of a zinc finger polypeptide, which method comprises contacting a library of DNA sequences with the polypeptide, measuring the affinity with which the polypeptide binds to each of the sequences, and optionally ranking the sequences in order of the affinity with which the polypeptide binds, wherein the library of DNA sequences consist of 4 sequences, each sequence varying from the other sequences by comprising a different one of the 4 possible permutations of the DNA sequence of length N, wherein the library of DNA sequences is immobilised on a solid substrate.
  • each of the DNA sequences within the library occupies a discrete position on the solid substrate.
  • the present invention also provides the use of a library of the invention in a method for designing a zinc finger polypeptide having specificity for a particular DNA sequence.
  • the present invention further provides the use of a library of the invention in a method for isolating a zinc finger polypeptide having specificity for a particular DNA sequence.
  • the present invention additionally provides the use of a library of the invention in a method for determining the preferred base recognition specificity of a zinc finger polypeptide.
  • the DNA library may be arranged into two or more sub-libraries. Each sub-library may occupy a discrete position on the solid substrate. Preferably, each sub-library comprises a subset of the 4 N sequences. In a preferred embodiment of the invention, the library is arranged in 4N sub-libraries, wherein for any one sub-library one base in the DNA sequence of length N is defined and the other N-l bases are randomised. According to a further aspect of the invention, we provide such a sub-library. Brief Description of the Drawings
  • Step 1 Two pre-made zinc finger phage-display libraries, Lib 12 and Lib23, contain randomised DNA-binding amino acid positions in fingers 1 and 2 (black) or fingers 2 and 3
  • Zinc finger genes are amplified from the recovered phage using PCR and sets of 'one-and-a-half fingers are paired to yield recombinant three-finger DNA-binding domains.
  • Step 3 The recombinant DNA-binding domains are cloned back into phage and subjected to further rounds of selection, or immediately validated for binding to a composite 10 bp DNA of pre-defined sequence.
  • the DNA-binding positions of each zinc finger are numbered and randomised residues in the two libraries are circled.
  • Broken arrows denote possible DNA contacts from Lib 12 to bases H'UKLM and from Lib23 to bases MNOPQ.
  • Solid arrows show DNA contacts from those regions of the two libraries that carry the wild-type Zif268 amino acid sequence, as observed in the crystal structure.
  • the wild-type portion of each library target site (white boxes) determines the register of the zinc finger-DNA interactions, such that the selected portions of the two libraries can be recombined to recognise the composite site H'lJKLMNOPQ.
  • Amino acid composition of the randomised DNA-binding positions on the ⁇ - helix of each zinc finger are numbered and randomised residues in the two libraries are circled.
  • positions 4 and 5 of F2 are speciiied by the codons CTG AGC, which contain the recognition site of the restriction enzyme Ddel (underlined), used as a breakpoint to recombine the products of the two libraries.
  • Figure 3 shows a matrix specificity assay for seven zinc finger DNA-binding domains designed to bind sequences in the HIV-1 promoter.
  • the seven constructs and their respective binding sites are labelled A-G. Binding of zinc fingers to 0.4 pmol DNA per 50 ⁇ l well is plotted vertically from phage ELISA absorbance readings (A 4 5o-A 6 so). Each clone is tested using all seven DNA sequences but strong binding is only observed to those sequences against which they have been designed.
  • nucleic acid binding proteins or polypeptides e.g., helix-turn-helix proteins
  • other nucleic acids such as DNA, RNA, or PNA (protein-nucleic acid)
  • small molecules such as drug, an intercalating molecule, a major or minor groove binding molecule (such as distamycin), etc.
  • our invention encompasses libraries and methods for designing, isolating, and determining the preferred base recognition specificity of any nucleic acid binding molecule.
  • a DNA library of the invention is used to test the selectivity of a zinc finger for a nucleotide sequences of length N. Consequently, since there are four different nucleotides that occur naturally in genomic DNA, the total number of sequences required to represent all possible base permutations for a sequence of length N is 4 N . However, uracil, which occurs in RNA, or other natural or non natural bases, may also be included, either in substitution for thymidine, or in addition. Thus, the DNA library of the invention may have 5 N sequences.
  • N is an integer having a value of at least three. That it to say that the smallest library envisaged for testing binding to a nucleotide sequence where only one DNA triplet is varied, consists of 64 different sequences. However, N may be any integer greater than or equal to 3 such as 4, 5, 6, 7, 8 or 9. Typically, N only needs to be three times the number of zinc fingers being tested, optionally including a few additional residues outside of the binding site that may influence specificity. Thus, by way of example, to test the specificity of a protein comprising three zinc fingers, where all three fingers have been engineered, it may be desirable to use a library where N is at least 9.
  • the DNA sequences in the library are typically immobilised at discrete positions on a solid substrate, such as a DNA chip, such that each different sequence is separated from other sequences on the solid substrate.
  • the 4 N possible permutations of the DNA sequence of length N sequence are typically (but need not be) arranged in sub-libraries.
  • the library is sub-divided into 4N sub-libraries, wherein for any one sub-library one base in the DNA sequence of length N is defined and the other N-l bases are randomised.
  • the nucleotide sequence of length N may be generally, but need not be, part of a longer DNA molecule.
  • the DNA sequences within the library may consist of sequences against which the binding of a binding molecule is tested (i.e., every base position in the DNA sequence is potentially involved binding to the binding molecule).
  • An example is a library of 64 sequences of length 3 representing all possible targets for a zinc fmger motif.
  • the DNA sequences comprise other flanking sequences which are not directly relevant to or involved in binding.
  • flanking sequences include vector sequences, dimerisation sequences, or nucleic acid sequences which are capable of hybridising to other nucleic acid sequences to form double stranded regions, other binding targets, etc.
  • the sequence and (where applicable, the binding specificity) of such flanking regions may be known or unknown.
  • the DNA sequences may comprise one or more binding targets for another binding domain, whether this is a zinc finger domain or otherwise.
  • Such libraries are useful in designing, isolating, and determining the binding affinity and preferred base recognition specificity of a hybrid binder such as a zinc finger-homeodomain fusion protein.
  • the nucleotide sequence of length N typically occupies the same position within the longer molecule in each of the varied sequences even though the sequence of N itself may vary.
  • the other sequences within the DNA molecule are generally the same throughout the library.
  • the library can be said to consist of a library of 4' DNA molecules of the formula R -[A/C/G/T] N -R", wherein R 1 and R 2 may be any nucleotide sequence.
  • each sequence is also represented as a dilution concentration series.
  • the immobilised DNA library may occupy Z4 N discrete positions on the chip where Z is the number of different dilutions in the series and is an integer having a value of at least 2.
  • the range of DNA concentrations for the dilution series is typically in the order of 0.01 to 100 pmol cm "' , preferably from 0.05 to 5 pmol cm "2 .
  • the concentrations typically vary 10- fold, i.e. a series may consist of 0.01, 0.1, 1, 10 and 100 pmol cm "2 , but may vary, for example, by 2- or 5-fold.
  • the DNA molecules in the library are at least partially double-stranded, in particular at least the nucleotide sequence of length N is double-stranded.
  • Single stranded regions may be included, for example to assist in attaching the DNA library to the solid substrate.
  • 5,837,832 also provides references for earlier techniques that may also be used.
  • an important aspect of the present invention is that it relates to DNA binding proteins, zinc fingers, that bind double-stranded DNA.
  • single-stranded nucleic acid molecule libraries using the prior art techniques referred to above will then need to be converted to double-stranded DNA libraries by synthesising a complementary strand.
  • An example of the conversion of single-stranded nucleic acid molecule libraries to double- stranded DNA libraries is given in Bulyk et al, 1999, Nature Biotechnology 17, 573-577, the contents of which are incorporated herein by reference.
  • the technique described in Bulyk et al, 1999 typically requires the inclusion of a constant sequence in every member of the library (i.e.
  • a nucleotide primer is bound to act as a primer for second strand synthesis using a DNA polymerase and other appropriate reagents.
  • deoxynucleotide triphosphates dNTPs
  • the detectable label may assist in detecting binding of zinc fingers when the immobilised DNA library is in use.
  • double-stranded molecules may be synthesised off the solid substrate and each pre-formed sequence applied to a discrete position on the solid substrate.
  • An example of such a method is to synthesis palindromic single-stranded nucleic acids - see U.S. Patent No. 5556752. the contents of which are incorporated herein by reference.
  • DNA may typically be synthesised in situ on the surface of the substrate.
  • DNA may also be printed directly onto the substrate using for example robotic devices equipped with either pins or pizo electric devices.
  • the library sequences are typically immobilised onto or in discrete regions of a solid substrate.
  • the substrate may be porous to allow immobilisation within the substrate or substantially non-porous, in which case the library sequences are typically immobilised on the surface of the substrate.
  • the solid substrate may be made of any material to which polypeptides can bind, either directly or indirectly.
  • suitable solid substrates include flat glass, silicon wafers, mica, ceramics and organic polymers such as plastics, including polystyrene and polymethacrylate. It may also be possible to use semi-permeable membranes such as nitrocellulose or nylon membranes, which are widely available.
  • the semi-permeable membranes may be mounted on a more robust solid surface such as glass.
  • the surfaces may optionally be coated with a layer of metal, such as gold, platinum or other transition metal.
  • a particular example of a suitable solid substrate is the commercially available BiaCoreTM chip (Pharmacia Biosensors).
  • the solid substrate is generally a material having a rigid or semi-rigid surface.
  • at least one surface of the substrate will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different polymers with, for example, raised regions or etched trenches.
  • the solid substrate may be a microtitre plate or bead. It is also preferred that the solid substrate is suitable for the high density application of DNA sequences in discrete areas of typically from 50 to 100 ⁇ m, giving a density of 10000 to 40000 cm "2 .
  • the solid substrate is conveniently divided up into sections. This may be achieved by techniques such as photoetching, or by the application of hydrophobic inks, for example teflon-based inks (Cel-line. USA).
  • the sections may conveniently comprise the wells of the microtitre plate. Each well may comprise a discrete DNA sequence of the library, or, in the case where the library is sub- divided into sub-libraries, each well may comprise one or more sub-libraries.
  • Discrete positions, in which each different member of the library is located may have any convenient shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
  • a discrete position is commonly referred to as a "spot".
  • Each discrete position may comprise, preferably consist of, one DNA sequence of the library.
  • the discrete position may comprise a single molecule, or a number of DNA molecules of homogenous composition. The latter arrangement is advantageous in that the signal strength is likely to be higher.
  • each discrete position comprises a number of DNA molecules of heterogenous composition.
  • a number of different DNA sequences are immobilised at a discrete spot.
  • each discrete spot comprises the sequences within the sub-library.
  • each of the sub-libraries is immobilised in a discrete position on the solid substrate. This embodiment is referred to as "multiplexing".
  • Attachment of the library sequences to the substrate may be by covalent or non-covalent means.
  • the library sequences may be attached to the substrate via a layer of molecules to which the library sequences bind.
  • the library sequences may be labelled with biotin and the substrate coated with avidin and/or streptavidin.
  • biotinylated library sequences A convenient feature of using biotinylated library sequences is that the efficiency of coupling to the solid substrate can be determined easily. Since the library sequences may bind only poorly to some solid substrates, it is often necessary to provide a chemical interface between the solid substrate (such as in the case of glass) and the library sequences. Examples of suitable chemical interfaces include hexaethylene glycol.
  • polylysine coated glass Another example is the use of polylysine coated glass, the polylysine then being chemically modified using standard procedures to introduce an affinity ligand.
  • Other methods for attaching molecules to the surfaces of solid substrate by the use of coupling agents are known in the art. see for example W098/49557.
  • Binding of zinc fingers to the immobilised DNA library may be determined by a variety of means such as changes in the optical characteristics of the bound DNA (i.e. by the use of ethidium bromide) or by the use of labelled zinc fmger polypeptides, such as epitope tagged zinc finger polypeptides or zinc finger polypeptides labelled with fluorophores such as green fluorescent protein.
  • Other detection techniques that do not require the use of labels include optical techniques such as optoacoustics, reflectometry, ellipsometry and surface plasmon resonance (SPR) - see W097/49989, incorporated herein by reference.
  • Binding of epitope tagged zinc finger polypeptides is typically assessed by immunological detection techniques where the primary or secondary antibody comprises a detectable label.
  • a preferred detectable label is one that emits light, such as a fiuorophore, for example phycoerythrin.
  • the complete DNA library is typically read at the same time by charged coupled device (CCD) camera or confocal imaging system.
  • the DNA library may be placed for detection in a suitable apparatus that can move in an x-y direction, such as a plate reader. In this way, the change in characteristics for each discrete position can be measured automatically by computer controlled movement of the array to place each discrete element in turn in line with the detection means.
  • the detection means are capable of interrogating each position in the library array optically or electrically.
  • suitable detection means include CCD cameras or confocal imaging systems.
  • any of the immobilised DNA sequences of the library may be removed from the solid substrate for further manipulation.
  • it may be desired to remove a particular DNA sequence which shows binding to a particular zinc finger, for example.
  • Removal from the solid substrate may be achieved by various means, for example, by elution using an appropriate solvent, by chemical or enzymatic cleavage, photochemical lysis (e.g., by application of laser energy), etc.
  • the removed sequence may be amplified by PCR, for example.
  • a zinc finger binding motif is the ⁇ -helical structural motif found in zinc finger binding proteins, well known to those skilled in the art.
  • the amino acid numbering used throughout is based on the first amino acid in the ⁇ -helix of the zinc finger binding motif being position +1. It will be apparent to those skilled in the art that the amino acid residue at position -1 does not, strictly speaking, form part of the ⁇ -helix of the zinc binding finger motif. Nevertheless, the residue at -1 is shown to be very important functionally and is therefore considered as part of the binding motif ⁇ -helix for the purposes of the present invention.
  • zinc finger polypeptide sequences to be tested and/or selected using the methods of the invention are typically obtained by modifying one or more amino acids residues known to be important in binding specificity.
  • zinc fmger polypeptide sequences may comprise a substitution at one or more of the following positions: -1, +1, +2, +3, +5 +6 and +8.
  • Zinger finger polypeptides may in one embodiment be tested individually using the library and methods of the invention. For example, it may be desired to determine the preferred base recognition specificity of a zinc finger polypeptide designed using rational design techniques.
  • a zinc finger may be designed to bind to a nucleic acid quadruplet in a target nucleic acid sequence, wherein binding to each base of the quadruplet by an ⁇ - helical zinc finger nucleic acid binding motif in the protein is determined as follows: if base 4 in the quadruplet is G, then position +6 in the ⁇ -helix is Arg or Lys; if base 4 in the quadruplet is A, then position +6 in the ⁇ -helix is Glu.
  • position +2 Arg or Gin if base 1 in the quadruplet is C, then position +2 is Asn, Gin, Arg. His or Lys; if base 1 in the quadruplet is T, then position +2 is Ser or Thr.
  • position +2 in the helix is responsible for determining the binding to base 1 of the quadruplet. In doing so. it cooperates synergistically with position +6. which determines binding at base 4 in the quadruplet, bases 1 and 4 being overlapping in adjacent quadruplets.
  • zinc finger polypeptides are considered to bind to overlapping quadruplet sequences
  • rational design rules such as the rules set out above allow polypeptides to be designed to bind to target sequences which are not multiples of overlapping quadruplets.
  • a zinc finger polypeptide may be designed to bind to a palindromic target sequence. Such sequences are commonly found as, for example, restriction enzyme target sequences.
  • creation of zinc fingers which bind to fewer than three nucleotides may be achieved by specify ing, in the zinc finger, amino acids which are unable to support H-bonding with the nucleic acid in the relevant position.
  • this is achieved by substituting Gly at position -1 (to eliminate a contact with base 2) and/or Ala at positions +3 and/or +6 (to eliminate contacts at the 3rd or 4th base respectively).
  • the contact with the final (3') base in the target sequence may be strengthened, if necessary, by substituting a residue at the relevant position which is capable of making a direct contact with the phosphate backbone of the nucleic acid.
  • a library of zinc finger polypeptides having different amino acids at one or more positions involved in binding specificity may be screened ("empirical selection") using the library and methods of the present invention and zinc finger polypeptides selected that bind to a target nucleotide sequence.
  • a library of sequences may conveniently be obtained by random mutagenesis at particular positions to produce a phage display library using standard techniques (see WO96/06166 for construction of a randomised Zif268 library).
  • the zinc fingers are randomised at one or more of, or may have a random allocation at some or all, preferably all, of positions -1, +1, +2, +3, +5 +6, +8 and +9. More preferably, the zinc fingers are randomised at positions -1, +2, +3 and +6, and at least one of +1, +5 and +8.
  • sequences may also be randomised at other positions (e.g. at position +9, although it is generally preferred to retain an arginine or a lysine residue at this position).
  • allocation of amino acids at the designated "random" positions may be genuinely random, it is preferred to avoid a hydrophobic residue (Phe, Tip or Tyr) or a cysteine residue at such positions.
  • the zinc finger binding motif is present within the context of other amino acids (which may be present in zinc finger proteins), so as to form a zinc finger (which includes an antiparallel ⁇ -sheet).
  • the zinc finger is preferably displayed as part of a zinc finger polypeptide, which polypeptide comprises a plurality of zinc fingers joined by an intervening linker peptide.
  • the library of sequences is such that the zinc fmger polypeptide will comprise two or more zinc fingers of defined amino acid sequence (generally the wild type sequence) and one zinc finger having a zinc finger binding motif randomised in the manner defined above. It is preferred that the randomised finger of the polypeptide is positioned between the two or more fingers having defined sequence. The defined fingers will establish the "phase" of binding of the polypeptide to DNA, which helps to increase the binding specificity of the randomised finger.
  • sequences encode the randomised binding motif of the middle finger of the Zif268 polypeptide.
  • sequences also encode those amino acids N-terminal and C-terminal of the middle finger in wild type Zif268, which encode the first and third zinc fingers respectively.
  • the sequence encodes the whole of the Zif268 polypeptide.
  • the randomised sequence encoding zinc finger polypeptides are such that the zinc finger binding domain can be cloned as a fusion with the minor coat protein (pill) of bacteriophage fd.
  • the encoded polypeptide includes the tripeptide sequence Met-Ala-Glu as the N terminal of the zinc finger domain, which is known to allow expression and display using the bacteriophage fd system.
  • the polypeptide library comprises 10 or more different sequences (ideally, as many as is practicable).
  • the immobilised DNA library of the present invention may conveniently be used to verify the results of rationally designing zinc fingers with desired specificity.
  • a zinc finger motif is designed as described above and then produced by recombinant or synthetic means.
  • the zinc finger polypeptide is contacted with the immobilised DNA library and binding detected as described above.
  • the specificity and affinity of the zinc finger for the various sequences in the library can then be determined. If the desired binding is not seen then further modifications may be made to the zinc finger motif and the screening process repeated.
  • a library of zinc finger polypeptides is contacted with the DNA library and the zinc fingers that bind to the target sequence(s) selected.
  • the zinc finger library is in the form of a library of carrier organisms that express on their surface a zinc finger polypeptide. Typical carrier organisms include phage and bacteria.
  • the libraries may be segregated into compartments or microcapsules, as described in WO99/02671.
  • This document discloses a method for isolating one or more genetic elements encoding a gene product having a desired activity. Genetic elements are first compartmentalised into microcapsules, and then transcribed and/or translated to produce their respective gene products (RNA or protein) within the microcapsules. Alternatively, the genetic elements are contained within a host cell in which transcription and/or translation (expression) of the gene product takes place and the host cells are first compartmentalised into microcapsules. Genetic elements which produce gene product having desired activity are subsequently sorted. Polysome display techniques, such as those disclosed in WO00/27878, may also be applied to the libraries and methods of our invention.
  • More than one round of selection may take place, for example to confirm that specificity of zinc finger polypeptides selected in any particular round. Desirably at least two, preferably three or more, rounds of screening are performed.
  • the library of zinc fmger polypeptides need not necessarily be completely random but may be partially random, for example at certain positions only.
  • the positions chosen and the range of different amino acids at any given position may be based on rational design principles.
  • the two methods are not mutually exclusive and may both be used as part of a design and selection strategy.
  • the first step comprising screening each of a plurality of zinc finger binding motifs (typically in the form of a display library), mainly or wholly on the basis of affinity for the target sequence; the second step comprising comparing binding characteristics of those motifs selected by the initial screening step, and selecting those having preferable binding characteristics for a particular DNA triplet.
  • the non-specific component of all protein-DNA interactions which includes contacts to the sugar-phosphate backbone as well as ambiguous contacts to base-pairs, is a considerable driving force towards complex formation and can result in the selection of DNA-binding -II.
  • selections should preferably be performed with low concentrations of specific binding site in a background of competitor DNA. and binding should desirably take place in solution to avoid local concentration effects and the avidity of multivalent phage for ligands immobilised on solid surfaces.
  • the immobilised DNA library of the present invention may be used in a general sense to determine the preferred base recognition specificity of a zinc finger polypeptide, whether the zinc finger polypeptide be a naturally occurring zinc finger polypeptide, or a fragment thereof comprising a zinc finger motif, a zinc finger polypeptide identified by a screening procedure, such as the screening method of the invention, or a zinc finger obtained by rational design methods.
  • the zinc fmger polypeptide of interest in contacted with the DNA library as described above and the extent of binding at each position on the immobilised DNA library determined.
  • the results for each different sequence in the library may then be placed in order of the affinity with which the zinc fmger polypeptide binds.
  • the resulting ranking will provide a clear indication of the preferred base recognition specificity of the zinc finger polypeptide and may even be used to determine an optimal consensus binding sequence.
  • zinc finger binding motifs Once suitable zinc finger binding motifs have been identified and obtained, they will advantageously be combined in a single zinc finger polypeptide. Typically this will be accomplished by use of recombinant DNA technology; conveniently a phage display system may be used.
  • the invention provides a zinc finger polypeptide designed and/or selected by one or both of the methods defined above.
  • the zinc finger polypeptide designed by the method comprises a combination of a plurality of zinc fingers (adjacent zinc fingers being joined by an intervening linker peptide), each finger comprising a zinc finger binding motif.
  • each zinc finger binding motif in the zinc finger polypeptide has been selected for preferable binding characteristics by the method defined above.
  • the intervening linker peptide may be the same between each adjacent zinc finger or, alternatively, the same zinc finger polypeptide may contain a number of different linker peptides.
  • the intervening linker peptide may be one that is present in na ⁇ jjally-occurring zinc fmger polypeptides or may be an artificial sequence.
  • the sequence of the intervening linker peptide may be varied, for example, to optimise binding of the zinc finger polypeptide to the target sequence.
  • each motif binds to those DNA triplets which represent contiguous or substantially contiguous DNA in the sequence of interest.
  • candidate binding motifs or candidate combinations of motifs may be screened against the actual target sequence to determine the optimum composition of the polypeptide. Competitor DNA may be included in the screening assay for comparison, as described above.
  • those motifs selected for inclusion in the polypeptide could be artificially modified (e.g. by directed mutagenesis) in order to optimise further their binding characteristics.
  • the length and amino acid sequence of the linker peptide joining adjacent zinc binding fingers could be varied, as outlined above. This may have the effect of altering the position of the zinc finger binding motif relative to the DNA sequence of interest, and thereby exert a further influence on binding characteristics.
  • Zinc) Zincogenase-containing zinc finger polypeptides Possible uses of suitably designed zinc finger polypeptides are: a) Therapy (e.g. targeting to double stranded DNA) b) Diagnosis (e.g. detecting mutations in gene sequences: the present work has shown that "tailor made” zinc finger polypeptides can distinguish DNA sequences differing by one base pair). c) DNA purification (the zinc finger polypeptide could be used to purify restriction fragments from solution, or to visualise DNA fragments on a gel - for example, where the polypeptide is linked to an appropriate fusion partner, or is detected by probing with an antibody).
  • Therapy e.g. targeting to double stranded DNA
  • Diagnosis e.g. detecting mutations in gene sequences: the present work has shown that "tailor made” zinc finger polypeptides can distinguish DNA sequences differing by one base pair.
  • DNA purification the zinc finger polypeptide could be used to purify restriction fragments from solution, or
  • zinc finger polypeptides could even be targeted to other nucleic acids such as single-stranded or double-stranded RNA (e.g. self-complementary RNA such as is present in many RNA molecules) or to RNA-DNA hybrids, which would present another possible mechanism of affecting cellular events at the molecular level.
  • RNA single-stranded or double-stranded RNA
  • RNA-DNA hybrids which would present another possible mechanism of affecting cellular events at the molecular level.
  • Example 1 Use of a DNA chip to study a phage display library of the pZif268 middle finger.
  • the DNA chip used in this protocol has 64 different features which correspond to the 64 possible middle triplets of the Zif268 binding site. Each DNA binding site is applied to the chip at various densities, covering a roughly 100-fold range, from 0.04 to 4 pmol/cm .
  • the DNA sequence synthesised on the chip is: 3'-cctggctaactgaactATATATGCG-NNN- GCGATATAT-5'.
  • Every member of the Zif268 phage library (as described in Choo and Klug, 1994, Proc Natl Acad Sci U S A 91, 1 1 163-11167) can be screened against every possible binding site to establish whether the phage display library has any limitations on DNA recognition. This helps ascertain the quality of a library. For instance we now know that the Choo and Klug library has certain sequence-restrictions which arise from the synergy of fingers 2 and 3. The overlap restricts binding to middle triplets with 5' G or T - this is discussed fully in Isalan et al. 1998, Biochemistry 37: 12026-33 and Isalan et al, 1997, Proc Natl Acad Sci U S A. 94: 5617-21.
  • the frameshift means that finger 1 is forced to recognise the triplet GCT rather than GCG, which is suboptimal.
  • the triplet GAC is offered to these fingers in the context of the correct binding site for fingers 1 and 3, binding is optimal and a higher signal is obtained.
  • the amino acid sequences of zinc fingers isolated from separate selections using the triplets TCC and GAC are compared it is seen that the same fingers have been isolated, thus confirming the above hypothesis.
  • Clone A is seen to bind specifically to the feature containing GCG - it is concluded that this clone is highly sequence-specific.
  • Clone B lights up features with both GCG and GTG - this clone is bispecific. From the relative intensities of binding to the gradients of DNA on the chip it is concluded that the two clones have roughly equal affinity for the GCG site, and it is deduced that this affinity is in the nanomolar range.
  • a 6-f ⁇ nger protein is constructed comprising a fusion between wild-type Zif268 three-fingers and a three- finger protein selected from the Choo and Klug library to bind GAC, linked by the linker H (zinc chelating)-LRQKDGERP-Y (hydrophobic core) where H and Y are the last and first structural elements of two adjacent fingers.
  • the protein is applied to the chip and appreciable binding is seen to those features in which the spacing (Nx) is from 0 to 3 nucleotides, but no binding is observed to features where the spacing is greater than 7. It is concluded that the linker design restricts binding to short spacings between adjacent binding sites.
  • a bipartite-complementary' system for the construction of DNA-binding domains by phage display may be used (Fig. 1).
  • This system comprises two master libraries, Libl2 and Lib23, each of which encodes variants of a three-finger DNA-binding domain based on that of the transcription factor Zif268 (Pavletich, N. P. & Pabo, C. 0.
  • Zinc finger-DNA recognition Crystal structure of a Zif268-DNA complex at 2.1 A. Science 252, 809-817 (1991); Christy, B. A., Lau. L. F. & Nathans, D.
  • a gene activated in mouse 3T3 cells by serum growth factors encodes a protein with "zinc finger" sequences. Proc. Natl.
  • the two libraries are complementary because Libl2 contains randomisations in all the base-contacting positions of FI and certain base-contacting positions of F2, while Lib23 contains randomisations in the remaining base-contacting positions of F2 and all the base-contacting positions of F3 (Fig. 2a).
  • the non-randomised DNA-contacting residues carry the nucleotide specificity of the parental Zif268 DNA- binding domain.
  • each library contains members that are randomised in the ⁇ -helical DNA-contacting residues from more than one zinc fmger.
  • the proteins produced by these libraries are therefore not limited to binding DNA sequences of the form GNNGNN..., as is the case with many prior art libraries (eg. 9. 13, 20).
  • the repertoire of randomisations does not encode all 20 amino acids, rather representing only those residues that most frequently function in sequence-specific DNA binding from the respective ⁇ -helical positions (Fig 2b). Excluding the residues that do not frequently function in DNA recognition advantageously helps to reduce the library size and/or the 'noise' associated with non-specific binding members of the library.
  • Phage libraries for use in the present invention are prepared as follows.
  • Genes for the two zinc finger phage display libraries are assembled from synthetic DNA oligonucleotides by directional end-to-end ligation using short complementary DNA linkers.
  • a large number of appropriately randomised oligonucleotides are used in combinations to assemble the gene cassettes. These are amplified by PCR, digested with S ⁇ l and Notl endonucleases, and ligated into the phage vector Fd-Tet-S ⁇ (Pavletich, ⁇ . P. & Pabo, C. 0.
  • Zinc finger-D ⁇ A recognition Crystal structure of a Zif268-D ⁇ A complex at 2.1 A. Science 252, 809-817 (1991)).
  • E. coli TGI cells are transformed with the recombinant vector by electroporation and plated onto TY ⁇ medium (1.5 % (w/v) agar, 1 % (w/v) Bactotryptone, 0.5 % (w/v) Bactoyeast extract, 0.8 % (w/v) NaCl) containing 15 ⁇ g/ml tetracycline.
  • TY ⁇ medium 1.5 % (w/v) agar, 1 % (w/v) Bactotryptone, 0.5 % (w/v) Bactoyeast extract, 0.8 % (w/v) NaCl
  • Phage selections from the two master libraries described in Example 2 are performed using the generic DNA sequence 3'-HIJKLMGGCG-5' for Lib 12, and 3'- GGCGMNOPQ-5' for Lib23, where the underlined bases are bound by the wild-type portion of the DNA-binding domain and each of the other letters represents any given nucleotide (Fig. 2a). A number of sites in the well-characterised promoter of HIV-1 are targeted.
  • the two zinc finger libraries (Lib 12 and Lib23) are subjected to selection in parallel, the nucleotide sequences used (ie. HIJKL/MNOPQ) being from HIV-1 between positions -80 and +60 (see Table 1/Fig. 3).
  • Tetracycline resistant bacterial colonies are transferred to 2 x TY liquid medium (16 g/litre Bactotryptone, 10 g/litre Bactoyeast extract, 5 g/litre NaCl) containing 50 ⁇ M ZnCb and 15 ⁇ g/ml tetracycline, and cultured overnight at 30°C in a shaking incubator.
  • 2 x TY liquid medium (16 g/litre Bactotryptone, 10 g/litre Bactoyeast extract, 5 g/litre NaCl) containing 50 ⁇ M ZnCb and 15 ⁇ g/ml tetracycline
  • Cleared culture supernatant containing phage particles is obtained by centrifuging at 300 g for 5 minutes.
  • biotinylated DNA target site is bound to streptavidin-coated tubes (Roche), in 50 ⁇ l PBS containing 50 ⁇ M ZnCb.
  • Bacterial culture supernatant containing phage is diluted 1 :10 in selection buffer (PBS containing 50 ⁇ M ZnCb, 2 % (w/v) fat-free dried milk (Marvel), 1 % (v/v) Tween, 20 mg/ml sonicated salmon sperm DNA), and 1 ml is applied to each tube.
  • Binding reactions are incubated for 1 hour at 20 ° C, after which the tubes are emptied and washed 20 times with PBS containing 50 ⁇ M ZnCb, 2 % (w/v) fat- free dried milk (Marvel) and 1 % (v/v) Tween.
  • Retained phage are eluted in 0.1 M triethylamine and neutralised with an equal volume of 1 M Tris-HCl (pH 7.4).
  • Logarithmic-phase E. coli TGI are infected with eluted phage, and cultured overnight at 30°C in 2 ⁇ TY medium containing 50 ⁇ M ZnCb and 15 ⁇ g/ml tetracycline, to amplify phage for further rounds of selection.
  • E. coli TGI infected with selected phage are plated and individual colonies are picked and cultured in liquid medium to prepare phage for ELISA DNA-binding assays (Choo, Y. & Klug, A. Selection of DNA binding sites for zinc fingers using rationally randomised DNA reveals coded interactions. Proc. Natl. Acad. Sci. U.S.A. 91, 1 1 168-1 1 172 (1994); Example 4).
  • Clones which recognise their target site may be retained for subsequent recombination of the two complementary halves recovered from Lib 12 and Lib23 to produce molecules having high affinity for the HIV-1 promoter.
  • clones B-G Six (clones B-G) are engineered according to the full 'bipartite' protocol, while one protein (clone A) is derived directly by selection from Lib23. This illustrates a further use of the master libraries, namely to select zinc finger domains that bind DNA sequences containing the motif 5'-GCGG-3' or 5'-GGCG-3'.
  • clones A-D Four proteins have binding sites that are dispersed upstream of the transcription initiation site (clones A-D), including two that flank the TATA box (clones C-D). Another three proteins bind to a cluster of sites at the beginning of the ORF, within the coding region for TAR (clones E-G). Clone H (HIV A') binds between the sites for HIV A and HIV B.
  • the immobilised DNA library of the present invention may be used to verify the binding ability of rationally designed zinc fingers, or they may be used to screen for zinc fingers having specificity for one or more DNA sequences, or to determine the preferred base recognition specificity of a zinc fmger.
  • the binding specificity of the zinc finger sequences to a particular sequence or sequences within the immobilised library may be determined by any suitable binding assay as known in the art, for example, an ELISA assay as follows:
  • PBS (10 x stock solution: NaCl, 80 g/l;KCl, 2g/l; Na 2 HP0 .7H 2 0 1 1.5 g/l;KH 2 P0 4 , 2 g 1) • Fat-free freeze-dried milk (Marvel; Premier Brands UK Ltd.)
  • ELISA developer solution [0.1 M Na (CH 3 .COO), pH 5.5; 3', 3', 5' 5'- tetramethylbenzidine (TMB; Sigma), 0.5 mg/ml; dimethyl sulphoxide (DMSO), 1%
  • Certain nucleic acid-binding domains may require supplements to the growth medium.
  • Zinc fingers for example, are stabilised by 50 ⁇ MZnCl in all media and ELISA binding and wash buffers. Incubate plates with orbital mixing at 250 rpm, for 16 hours at 30° C.
  • biotinylated nucleic acid target sites typically between 0 - 5 pmol
  • streptavidin-coated microtitre wells for the positive control, add an appropriate amount of the wild-type binding site to one well.
  • protocol recited above relates to phage clones expressing zinc fingers which have been selected
  • the protocol may readily be adapted to assay interactions between specific zinc finger polypeptides and DNA substrates.
  • the ELISA DNA binding assay described above may be used to determine the binding specificity of a particular zinc finger, or a series of zinc fingers. Similarly, either a single DNA sequence or a series of DNA sequences may be tested.
  • Figure 1 shows the results of such an ELISA assay. Seven zinc finger DNA-binding domains are designed to bind sequences in the HIV-1 promoter. The seven constructs and their respective binding sites are labelled A-G, and each clone is tested using all seven DNA sequences. Binding of zinc fingers to 0.4 pmol DNA per 50 ⁇ l well is plotted vertically from phage ELISA absorbance readings As can be seen from the figure, strong and specific binding of each zinc fmger is only observed to the DNA sequence against which it has been designed. See also Table 1.

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Abstract

L'invention concerne une bibliothèque de séquences d'ADN comportant 4N séquences avec N supérieur ou égal à trois, chaque séquence comportant une combinaison différente des 4N combinaisons possibles d'une séquence d'ADN de longueur N, et la bibliothèque de séquences d'ADN étant immobilisée sur un substrat solide. De préférence, chaque séquence occupe une position discrète, et la bibliothèque est disposée en deux ou trois sous-parties, de préférence 4N sous parties. Pour chaque sous-partie, une base dans la séquence d'ADN de longueur N est définie, et les autres N-1 bases sont rendues aléatoires. Ladite bibliothèque peut être utilisée dans des procédés de criblage pour l'identification et la caractérisation de doigts de zinc présentant une spécificité pour des séquences de nucléotides particulières.
EP00964464A 1999-10-01 2000-10-02 Banque d'adn et son utilisation dans des procedes de selection et conception des polypeptides Withdrawn EP1230355A2 (fr)

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GBGB9923327.2A GB9923327D0 (en) 1999-10-01 1999-10-01 DNA library
GB9923327 1999-10-01
GB0011068 2000-05-08
GB0011068A GB0011068D0 (en) 2000-05-08 2000-05-08 Molecules
GB0013106 2000-05-30
GB0013106A GB0013106D0 (en) 2000-05-30 2000-05-30 Molecules
PCT/GB2000/003765 WO2001025417A2 (fr) 1999-10-01 2000-10-02 Bibliotheque d'adn

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CA2561565C (fr) * 2004-04-08 2013-11-26 Sangamo Biosciences, Inc. Methodes de repression du gene phospholamban et de modulation de la contractilite cardiaque
WO2007136840A2 (fr) * 2006-05-20 2007-11-29 Codon Devices, Inc. Conception et création d'une banque d'acides nucléiques
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