WO2000023463A9 - Fluorescent dye binding peptides - Google Patents

Fluorescent dye binding peptides

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Publication number
WO2000023463A9
WO2000023463A9 PCT/US1999/024266 US9924266W WO0023463A9 WO 2000023463 A9 WO2000023463 A9 WO 2000023463A9 US 9924266 W US9924266 W US 9924266W WO 0023463 A9 WO0023463 A9 WO 0023463A9
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WO
WIPO (PCT)
Prior art keywords
seq
peptide
fluorette
dye
group
Prior art date
Application number
PCT/US1999/024266
Other languages
French (fr)
Other versions
WO2000023463A3 (en
WO2000023463A2 (en
Inventor
Garry P Nolan
Michael N Rozinov
Original Assignee
Univ Leland Stanford Junior
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Univ Leland Stanford Junior filed Critical Univ Leland Stanford Junior
Priority to AU11202/00A priority Critical patent/AU1120200A/en
Publication of WO2000023463A2 publication Critical patent/WO2000023463A2/en
Publication of WO2000023463A3 publication Critical patent/WO2000023463A3/en
Publication of WO2000023463A9 publication Critical patent/WO2000023463A9/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6827—Total protein determination, e.g. albumin in urine
    • G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green

Definitions

  • the invention relates to peptides that bind to fluorescent dyes, termed "fluorettes", and to methods of making and using the fluorettes
  • the fluorettes can be used in detection and assay systems in vitro and in vivo
  • Fluorophore dyes due to their extraordinar sensitivity and ease of use, are widely used in numerous approaches in fluorescent microscopy, flow cytometry and other detection systems (Haugland Handbook of fluorescent probes and research chemicals (sixth edition) Molecular Probes, Inc , Eugene OR (1996) and ref therein)
  • the invention provides peptides that bind to fluorophore dyes
  • the peptides are made of naturally occunng ammo acids, non-naturally occurring ammo acids, or combinations thereof
  • methods are provided for isolating and identifying peptides that bind to fluorophore dyes.
  • the method comprises creating and screening peptide libraries that bind to fluorophores
  • complexes of fluorettes bound to fluorophore dyes The binding of the fluorophore by the fluorette may alter the excitation and/or the emission spectrum of the fluorophore
  • the present invention provides methods for detecting a fluorette by binding a fluorette to a fluorophore dye and detecting the fluorette/fluorophore dye complex
  • Fluorophore dye carriers Fluorescein, Oregon Green 514, Rhodamine Red and Texas Red activated derivatives were covalently linked to the polymer carrier Ultrahnk Immobilized DADPA via a 12-atom diaminodipropylamine spacer A spacer is shown as a horizontal thick bar Digits with arrows m the chemical structures of all fluorophore dyes, except Oregon Green 514, show that "mixed isomers" of these fluorophore dye activated derivatives were used for a coupling Figure 2 Biopanning with a peptide phage display library against Fluorescein, Oregon Green 514, Rhodamine Red and Texas Red carriers A Ph D -12 phage display peptide library (New England Biolabs) is a combinatorial library of 12-mer peptides fused to the ammo-terminus of a minor coat pill protein of the bacte ⁇ ophage M13mp19 Bound phage
  • FIG. 5A and 5B Peptide - Texas Red binding Excitation and emission spectra Peptides at the noted concentrations were incubated with Texas Red (50nM), or without, in 0 6ml of TBS for 1 hr at room temperature (RT) Samples were scanned on a spectrofluo ⁇ meter (SPEX Fluoromax (Jobin Yvon-SPEX Instruments Co )) Peak excitation or emission positions are shown in the figure using vertical bars Excitation and excitation/emission peak shifts for PepTR401(10 ⁇ M)/Texas Red and PepTRS311 (10 ⁇ M)/Texas Red complexes, respectively (A), were reproducible in three independent experiments The results of a single experiment are presented
  • FIG. 7A-D Peptide - Texas Red binding Texas Red-specific peptides TR401 (linear) ( Figure 6B), TRP501 (SKVILFE-flanked) ( Figure 6C), TRP512 (SKVILFE-flanked) ( Figure 6D), and nonspecific peptide (SKVILFE-flanked) ( Figure 6A) as a negative control were bound via polyhistidme (H ⁇ s 6 ) tag to cobalt ion-containing Sepharose beads
  • the peptide-coated beads were washed and incubated with 0 5 ⁇ M Texas Red in TBS buffer for 1 hour at room temperature followed by several washings of beads in order to remove unbound dye Fluorescent and nonfluorescent control beads were photographed on fluorescent microscope Axiophot (Zeiss) using Rhodamine Red/Texas Red filter with the same time exposure for every sample All binding experiments were accomplished in duplicate and results of a single experiment are shown
  • the present invention provides novel peptides or "fluorettes” that specifically bind fluorophore dyes
  • the present invention also provides novel compositions comprising multimenc fluorettes and fluorettes fused or linked to other compounds or molecules Also provided are methods to create and modify fluorettes
  • the present invention further provides methods for the use of fluorettes in detecting biological materials, molecules, or target analytes, intracellular events, and intermolecular and intramolecular interactions
  • the fluorettes also find use in in vitro assays and high throughput screens
  • the methods provided are based on the formation and detection of a fluorette/fluorophore dye complex
  • compositions and methods of the present invention provide a significant improvement over conventional light-emitting techniques Fluorettes, due to their small size, are not intrusive to the systems being studied and, therefore, permit detection and analysis of a target moiety or molecule while minimizing target modification
  • Current light-emitting detection methods involve the use of enzyme-generated fluorochromes, luciferase-generated light, or reporter systems using engineered, inherently fluorescent proteins such as Aequona victoria green fluorescent protein (GFP)
  • GFP Aequona victoria green fluorescent protein
  • the present invention provides a peptide, sometimes termed a "fluorette” herein, that will bind a fluorescent dye
  • peptide or “fluorette” herein is meant at least about 8 covalently attached ammo acids
  • the peptide may be made up of naturally occurring am o acids and peptide bonds, or synthetic peptidomimetic structures
  • “ammo acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic ammo acids
  • homo-phenylalanme, citrul ne and noreleucme are considered ammo acids for the purposes of the invention
  • Ammo acid also includes ir ⁇ ino acid residues such as proline and hydroxyproline
  • the side chains may be in either the (R) or the (S) configuration In the preferred embodiment, the am o acids are in the (S) or L-configuration If non-naturally occurring side chains are used, non-ammo acid substituents may be used, for example to prevent or retard in vivo degradations
  • the peptides can be linear or branched Ammo acids includes naturally occurring ammo acids or non-naturally occurring amino acids
  • Naturally-occurring ammo acids herein is meant ammo acids that are produced by living organisms Naturally-occurring am o acids are preferred in in vivo embodiments
  • Non-naturally-occurnng ammo acids am o acids not produced by a living organism but that can be chemically synthesized
  • Non-naturally-occurnng ammo acids include, for example, stereoisomers or enantiomers of the naturally-occurring ammo acids in which the ammo side chains, are in either the (D)((R)) or (S)((L)) configuration, ammo acids in which an amine group is bonded to any other but the alpha-carbon or may have more than one amine group bonded to the alpha-carbon, and ammo acids having functional groups or "R" groups not known to occur in nature
  • the length of the peptide or fluorette, i e the number of ammo acids, will vary In general, the number of amino acids varies from about 8 to about 50, with from about 8 to about 40 being preferred, from about 8 to about 25 being particularly preferred, and from about 8 to about 12 being especially preferred
  • "peptide" includes peptide
  • the fluorette can be monomenc or multimenc That is, a monome ⁇ c fluorette binds a single fluorophore
  • a multimenc fluorette is two or more associated monomenc fluorettes
  • the association of a multimenc fluorette can be either covalent or non-covalent
  • the monomers can be joined directly together, for example as a linear fusion (i e , the carboxy terminus of one is joined to the ammo terminus of the second), or as a branched fusion, wherein the attachment of the second monomer is other than to the backbone of the first
  • a chemical cross-linker or inclusion of specific reactive group on a fluorette be used to join fluorette monomers
  • the monomers may be non-covalently associated, for example, as is more fully outlined below, dime ⁇ zation sequences can be used to associate two monomers
  • a multimenc fluorette can be homomultime ⁇ c, i e , all the monomers are the same, and bind the same fluorophore dye, or heteromultimeric, having at least two different fluorette monomers and can bind different fluorophores
  • the fluorette peptides bind to at least one fluorophore dye
  • binding herein generally is meant a non-covalent association or interaction between a fluorette and a fluorescent dye
  • the non-covalent interactions between the fluorette and dye may involve various types of electrostatic, hydrophilic, and hydrophobic interactions
  • Binding may also involve forming one or more covalent bonds between the fluorette and the dye
  • Covalent bonds can be formed directly between the fluorette and the dye or can be formed by a cross linker or by inclusion of a specific reactive group on either the fluorette or dye or both molecules
  • Binding may also involve a combination of covalent and non-covalent interactions
  • the binding is specific
  • specific binding herein is meant that the fluorophores will preferentially bind to a fluorophore dye with a binding constant in the range of at least about 10 "6 M " to about 10 10 M 1 , with a preferred range being from at least about 10 "6 M 1 to about 10 "7 M “1 , with an especially preferred range of from at least about 10 "7 M “1 to about 10 10 M “1
  • the fluorettes do not specifically bind to other fluorophore dyes or compounds or moieties, that is, a fluorette is specific to one fluorophore
  • a fluorette may bind two or more fluorescent dyes (bind together 2 or more fluorophores or more or capable of binding independently two or more fluorophores), that is, a fluorette may specifically bind another compound or moiety, including another fluorophore, if these molecules have a common structural feature(s) that specifically interact with the fluorette
  • a fluorette may bind two or more fluorescent dyes
  • a dye is non-fluorescent but becomes fluorescent when bound by a fluorette
  • fluorette binding to a non-fluorescent dye causes the release of a fluorescent moiety When the fluorescent moiety is released, the fluorette may bind to the released fluorescent moiety, may bind to the non-fluorescent dye, or may be released and therefore free to bind a second molecule of the non-fluorescent dye and repeat this process
  • fluorescent dye or “fluorophore” or “fluorophore dye” herein is meant a compound that absorbs an incident light of a characteristic range of wavelengths or excitation spectrum and dissipates the absorbed energy by emitting light of a characteristic range of wavelengths or emission spectrum
  • excitation spectrum herein is meant the wavelength of incident light absorbed by the fluorophore dye that causes the fluorophore dye to fluoresce
  • emission spectrum herein is meant the characteristic wavelengths of the emitted or fluorescent light produced as the energy of the absorbed incident light is released
  • the excitation and emission spectra for a fluorophore dye may or may not overlap In a preferred embodiment the incident light is in the ultraviolet spectrum and the emitted light is in the visible spectrum
  • Preferred fluorophores include, but are not limited to, Texas Red, Rhodamine Red, Oregon Green 514, and Fluorescein Examples of fluorescent dyes are found in the Molecular Probes Catalog, 6th Ed , Richard Haugland, Ed , which is expressly incorporated by reference in its entirety
  • the fluorette can further comprise additional components, such as a fusion partner or functional group
  • fusion partner or “functional group” herein is meant a sequence that is associated with the fluorettes, that confers upon the fluorette a function or ability Fusion partners can be heterologous (i e not native to a host cell), or synthetic (not native to any cell)
  • Suitable fusion partners include, but are not limited to a) presentation structures, as defined below, which provide the fluorettes in a conformationally restricted or stable form, b) targeting sequences, defined below, which allow the localization of the flourette into a subcellular or extracellular compartment, c) rescue sequences as defined below, which allows the purification or isolation of the fluorette, d) stability sequences, which confer stability or protection from degradation to the fluorette, for example resistance to proteolytic degradation, e) dimenzation sequences, to allow for fluorette dimenzation or multime ⁇ zation, or f) any combination of a), b), c), d
  • the fusion partner is a presentation structure
  • presentation structure or grammatical equivalents herein is meant a sequence, which, when fused to the fluorette, causes the fluorette to assume a conformationally restricted form Proteins interact with other proteins and molecules largely through conformationally constrained domains
  • small peptides with freely rotating ammo- and carboxy-termmi can have potent functions as is known in the art, the conversion of such peptide structures into active agents can be difficult due to the inability to predict side-chain positions for peptidomimetic synthesis Therefore the presentation of fluorettes in conformationally constrained structures will likely lead to higher affinity interactions of the fluorette with its target fluorophore dye This fact has been recognized in the combinatorial library generation systems using biologically generated short peptides in bacterial phage systems A number of workers have constructed small domain molecules in which one might present biased-combinatorial libraries of peptide structures
  • presentation structures are preferably peptides or proteins
  • synthetic presentation structures i e artificial polypeptides
  • synthetic presentation structures are capable of presenting fluorettes as a conformationally-rest ⁇ cted domain
  • presentation structures comprise a first portion joined to the N-termmal end of the flourette, and a second portion joined to the C-termmal end of the flourette , that is, the flourette is inserted into the presentation structure, although variations may be made, as outlined below
  • the presentation structures are selected or designed to have minimal biologically activity when expressed in the target cell
  • presentation structures maximize accessibility to the flourette by presenting it on an exterior loop
  • suitable presentation structures include, but are not limited to, minibody structures, loops on beta-sheet turns and coiled-coil stem structures in which residues not critical to structure are randomized, zinc-finger domains, cysteine-lmked (disulfide) structures, transglutaminase linked structures, cyclic peptides, B-loop structures, helical barrels or bundles, leucme zipper motifs, etc
  • presentation structures include dimenzation sequences, as defined below
  • the presentation structure is a coiled-coil structure, allowing the presentation of the flourette on an exterior loop (See, for example, Myszka et al , Biochem
  • coiled- coil structures allow for between 6 to 20 ammo acids
  • a preferred coiled-coil presentation structure is as follows
  • the underlined regions represent a coiled-coil leucme zipper region defined previously (see Martin et al , EMBO J 13(22) 5303-5309 (1994), incorporated by reference)
  • the bolded X (n) region represents the loop structure and when appropriately replaced with peptides (i e fluorettes , generally depicted herein as (X) n , where X is an ammo acid residue and n is an integer of at least 5 or 6) can be of variable length
  • the replacement of the bolded region is facilitated by encoding restriction endonuclease sites in the underlined regions, which allows the direct incorporation of oligonucleotides at these positions
  • a preferred embodiment generates an Xhol site at the double underlined LE site and a Hindlll site at the double-underlined KL
  • the presentation structure is a minibody structure
  • a "minibody” is essentially composed of a minimal antibody complementarity region
  • a preferred minibody presentation structure is as follows MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTSQSILYLQK KKGPP (SEQ ID NO 50)
  • the bold, underline regions are the regions which may be contain fluorette sequences
  • the italicized phenylalanine must be invariant in the first randomizing region
  • the entire peptide is cloned in a three-oligonucleotide variation of the coiled-coil embodiment, thus allowing two different randomizing regions to be incorporated simultaneously
  • This embodiment utilizes non-pa ndromic BstXI sites on the termini
  • the presentation structure is a sequence that contains generally two cysteine residues, such that a disulfide bond may be formed, resulting in a conformationally constrained sequence
  • fluorettes are to be secreted from a cell
  • any number of flourettes, with or without spacer or linking sequences may be flanked with cysteine residues
  • effective presentation structures may be generated by the flourettes themselves
  • the flourettes may be "doped" with cysteine residues which, under the appropriate redox conditions, may result in highly crosslmked structured conformations, similar to a presentation structure
  • the flourettes may be controlled to contain a certain number of residues to confer ⁇ -sheet or ⁇ -helical structures.
  • a presentation structure is neuropeptide head activator.
  • Bodenmuller et al. EMBO J. 5(8), 1825-1829 (1983) showed that neuropeptide head activator (HAv) dimerized to yield a biologically inactive form of the peptide at concentration as low as 10- 13 M, thus indicating extremely high self-binding affinity.
  • Aldwin et al. U.S. Patent No. 5,491 ,074 observed that a fragment containing the last six amino acids of this peptide's carboxyl terminus (SKVILF) (SEQ ID NO:51) resulted in dimers that were even more stable than the HA itself.
  • SKVILF carboxyl terminus
  • the USPN 5,491 ,074 shows that the last amino acid of SKVILF, the F (phenylalanine), must be on the carboxyl terminus for a proper peptide dimenzation activity. However, if F is not on the carboxyl terminus, it must be followed by one of two amino acids with free carboxyl group, E or D, to maintain the peptide dimenzation. Therefore, if the dimerizing peptide positioned inside a carrier protein, 6-mer SKVILF sequence must be converted to 7-mer SKVILFE (SEQ ID NO:51) or SKVILFD (SEQ ID NO:53) sequences.
  • F phenylalanine
  • Linkers or spacers as known in the art are optionally placed between the HAv peptides and the variable ((X) n ) region and optionally placed at the amino and carboxy terminus of the structure.
  • peptides in constrained conformations usually have higher affinity for a ligand than the same peptide in linear conformation.
  • the constrained peptide has a higher protease resistance than the linear peptide as a result of the formation of a "rigid surface" structure.
  • transfer of a constrained peptide as a cassette to a protein carrier will not dramatically change its original conformation (and consequently its binding activity) while a linear peptide is more likely to show a decrease in binding activity.
  • the fusion partner is a targeting sequence.
  • the localization of proteins within a cell is a simple method for increasing effective concentration.
  • the concentration of a protein or polypeptide can also be simply increased by nature of the localization. For example, shuttling the fluorettes into the nucleus confines them to a smaller space thereby increasing the concentration.
  • the ligand or target may simply be localized to a specific compartment.
  • the targeting sequence can be fused to the protein partner as well
  • suitable targeting sequences include, but are not limited to, binding sequences capable of causing binding of the fluorette to a predetermined molecule or class of molecules while retaining activity of the fluorette, (for example by using enzyme inhibitor or substrate sequences to target a class of relevant enzymes), sequences signalling selective degradation, of itself or co-bound proteins, and signal sequences capable of constitutively localizing the fluorette to a predetermined cellular locale, including a) subcellular locations such as the Golgi, endoplasmic reticulum, nucleus, nucleoli, nuclear membrane, mitochondria, chloroplast, secretory vesicles, lysosome, and cellular membrane, and b) extracellular locations via a secretory signal Particularly preferred is localization to either subcellular locations or to the outside of the cell via secretion
  • the targeting sequence is a nuclear localization signal (NLS)
  • NLSs are generally short, positively charged (basic) domains that serve to direct the entire protein in which they occur to the cell's nucleus
  • NLS ammo acid sequences have been reported including single basic NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro Lys Lys Lys Arg Lys Val) (SEQ ID NO 54), Kalderon (1984), et al , Cell, 39 499-509, the human retinoic acid receptor- ⁇ nuclear localization signal (ARRRRP) (SEQ ID NO 55), NFKB p50 (EEVQRKRQKL SEQ ID NO 56), Ghosh et al , Cell 62 1019 (1990), NFKB p65 (EEKRKRTYE (SEQ ID NO 57, Nolan et al , Cell 64 961 (1991), and others (see for example Boulikas, J Cell Biochem 55(1) 32-58 (19
  • the targeting sequence is a membrane anchoring signal sequence
  • a membrane anchoring region is provided at the carboxyl terminus of the peptide presentation structure
  • a membrane anchoring region is provided at the ammo terminus of the peptide presentation structure
  • the ammo terminal anchoring region functions as the anchoring region of a Type 2 glycoprotem
  • the fluorette is expressed on the cell surface and presented to the extracellular space, such that it can bind its target fluorophore
  • the binding of a fluorette, espescially when fused to a polypeptide or protein could confer a function on the cells expressing the fluor
  • Membrane-anchoring sequences are well known in the art and are based on the geometry of mammalian transmembrane molecules Peptides are inserted into the membrane based on a signal sequence (designated herein as ssTM) and require a hydrophobic transmembrane domain (herein TM)
  • ssTM signal sequence
  • TM hydrophobic transmembrane domain
  • the transmembrane proteins are inserted into the membrane such that the regions encoded 5' of the transmembrane domain are extracellular and the sequences 3' become intracellular
  • these transmembrane domains are placed 5' of the variable region, they will serve to anchor it as an intracellular domain, which may be desirable in some embodiments sTMs and TMs are known for a wide variety of membrane bound proteins, and these sequences may be used accordingly, either as pairs from a particular protein or with each component being taken from a different protein, or alternatively, the sequences may be synthetic, and derived entirely from consensus as artificial delivery domains
  • membrane-anchoring sequences including both ssTM and TM, are known for a wide variety of proteins and any of these may be used Particularly preferred membrane-anchoring sequences include, but are not limited to, those derived from CD8,
  • Useful sequences include sequences from 1) class I integral membrane proteins such as IL-2 receptor beta-chain (residues 1-26 are the signal sequence, 241-265 are the transmembrane residues, see Hatakeyama et al , Science 244 551 (1989) and von Heijne et al, Eur J Biochem 174 671 (1988)) and insulin receptor beta chain (residues 1-27 are the signal, 957-959 are the transmembrane domain and 960-1382 are the cytoplasmic domain, see Hatakeyama, supra, and Ebina et al , Cell 40 747 (1985)), 2) class II integral membrane proteins such as neutral endopeptidase (residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmic domain, see Malfroy et al , Biochem Biophys Res Commun 144 59 (1987)), 3) type III proteins such as human cytochrome P450 NF25 (Hatakeyama, supra);
  • CD8 and ICAM-2 are particularly preferred.
  • the signal sequences from CD8 and ICAM-2 lie at the extreme 5' end of the transcript. These consist of the amino acids 1-32 in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; (SEQ ID NO:59) Nakauchi et al., PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2
  • membrane anchoring sequences include the GPI anchor, which results in a covalent bond between the molecule and the lipid bilayer via a glycosyl-phosphatidylinositol bond for example in DAF (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO:63), with the bolded serine the site of the anchor; see Homans et al., Nature 333(6170):269-72 (1988), and Moran et al., J. Biol. Chem. 266:1250 (1991)).
  • the GPI sequence from Thy-1 can be cassetted 3' of the fluorette in place of a transmembrane sequence.
  • myristylation sequences can serve as membrane anchoring sequences. It is known that the myristylation of c-src recruits it to the plasma membrane. This is a simple and effective method of membrane localization, given that the first 14 amino acids of the protein are solely responsible for this function: MGSSKSKPKDPSQR (SEQ ID NO:64) (see Cross et al., Mol. Cell. Biol. 4(9): 1834 (1984); Spencer et al., Science 262:1019-1024 (1993), both of which are hereby incorporated by reference). This motif has already been shown to be effective in the localization of reporter genes and can be used to anchor the zeta chain of the TCR.
  • This motif is placed 5' of the fluorette in order to localize it to the plasma membrane.
  • Other modifications such as palmitoylation can be used to anchor fluorettes in the plasma membrane; for example, palmitoylation sequences from the G protein-coupled receptor kinase GRK6 sequence (LLQRLFSRQDCCGNCSDSEEELPTRL (SEQ ID NO:65), with the bold cysteines being palmitolyated; Stoffel et al., J. Biol. Chem 269:27791 (1994)); from rhodopsin
  • KQFRNCMLTSLCCGKNPLGD (SEQ ID NO:66); Bamstable et al., J. Mol. Neurosci. 5(3):207 (1994)); and the p21 H-ras 1 protein (LNPPDESGPGCMSCKCVLS (SEQ ID NO:67); Capon et al., Nature 302:33 (1983)).
  • the targeting sequence is a lysozomal targeting sequence, including, for example, a lysosomal degradation sequence such as Lamp-2 (KFERQ (SEQ ID NO:68); Dice, Ann. N.Y. Acad. Sci. 674:58 (1992); or lysosomal membrane sequences from Lamp-1 (MLIPIAGFFALAGL W /AY /GRKRSHAGYQTI (SEQ ID NO:69), Uthayakumar et al., Cell. Mol. Biol. Res.
  • a lysosomal degradation sequence such as Lamp-2 (KFERQ (SEQ ID NO:68); Dice, Ann. N.Y. Acad. Sci. 674:58 (1992); or lysosomal membrane sequences from Lamp-1 (MLIPIAGFFALAGL W /AY /GRKRSHAGYQTI (SEQ ID NO:69), Uthayakumar et al., Cell. Mol. Biol. Res.
  • Lamp-2 (LVPIA VGAALA G VLIL VLLA YF/GLKH H H AG YEQ F (SEQ ID NO:70), Konecki et la., Biochem. Biophys. Res. Comm. 205:1-5 (1994), both of which show the transmembrane domains in italics and the cytoplasmic targeting signal underlined).
  • the targeting sequence may be a mitrochondrial localization sequence, including mitochondrial matrix sequences (e.g. yeast alcohol dehydrogenase III;
  • MLRTSSLFTRRVQPSLFSRNILRLQST SEQ ID NO:71 ); Schatz, Eur. J. Biochem. 165:1-6 (1987)); mitochondrial inner membrane sequences (yeast cytochrome c oxidase subunit IV;
  • MLSLRQSIRFFKPATRTLCSSRYLL SEQ ID NO:72; Schatz, supra; mitochondrial intermembrane space sequences (yeast cytochrome d ;
  • mitochondrial outer membrane sequences yeast 70 kD outer membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK (SEQ ID NO:74);
  • the target sequences may also be endoplasmic reticulum sequences, including the sequences from calreticulin (KDEL; Pelham, Royal Society London Transactions B; 1-10 (1992)) or adenovirus E3/19K protein (LYLSRRSFIDEKKMP (SEQ ID NO:75); Jackson et al., EMBO J. 9:3153 (1990)).
  • KDEL calreticulin
  • LYLSRRSFIDEKKMP SEQ ID NO:75
  • targeting sequences also include peroxisome sequences (for example, the peroxisome matrix sequence from Luciferase; SKL; Keller et al., PNAS USA 4:3264 (1987)); farnesylation sequences (for example, P21 H-ras 1 ; LNPPDESGPGCMSCKCVLS (SEQ ID NO:76), with the bold cysteine farnesylated; Capon, supra); geranylgeranylation sequences (for example, protein rab-5A; LTEPTQPTRNQCCSN (SEQ ID NO:77), with the bold cysteines geranylgeranylated; Farnsworth, PNAS USA 91 :11963 (1994)); or destruction sequences (cyclin B1; RTALGDIGN (SEQ ID NO:78); Klotzbucher et al., EMBO J. 1 :3053 (1996)).
  • peroxisome sequences for example, the peroxisome matrix sequence from Luciferase; SKL; Keller et al.,
  • the targeting sequence is a secretory signal sequence capable of effecting the secretion of the fluorette.
  • secretory signal sequences There are a large number of known secretory signal sequences which are placed 5' to the fluorette sequence and are cleaved from the peptide region to effect secretion into the extracellular space. Secretory signal sequences and their transferability to unrelated proteins are well known, e.g., Silhavy, et al. (1985) Microbiol. Rev. 49, 398-418.
  • a fusion product is configured to contain, in series, secretion signal peptide-presentation structure-flourette-presentation structure
  • target cells grown in the vicinity of cells caused to express the fluorettes are bathed in secreted fluorette
  • Target cells exhibiting a phenotypic change in response to the presence of a fluorette, or internalization of the fluorette and binding to intracellular targets are localized by any of a variety of methods, such as, FRET analysis (Selvm et al Methods Enzymol 246 300-334 (1995)) as described below
  • Suitable secretory sequences are known, including signals from IL-2
  • a particularly preferred secretory signal sequence is the signal leader sequence from the secreted cytokme IL-4, which comprises the first 24 ammo acids of IL-4 as follows MGLTSQLLPPLFFLLACAGNFVHG (SEQ ID NO 83)
  • the fusion partner is a rescue sequence
  • a rescue sequence is a sequence which may be used to purify or isolate the fluorette (or, in some cases, the nucleic acid encoding it)
  • peptide rescue sequences include purification sequences such as the H ⁇ s 6 tag for use with Ni affinity columns and epitope tags for detection, immunoprecipitation or FACS (fluorescence-activated cell sorting)
  • Suitable epitope tags include myc (for use with the commercially available 9E10 antibody), the BSP biotmylation target sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST
  • a fluorette sequence functions as a rescue sequence
  • the fluorette because it binds a fluorophore dye can be used to purify or isolate a molecule to which it is fused or attached by affinity chromatography using fluorophore dye columns If the fluorette alters the excitation or emission spectrum of the bound fluorophore dye, as described below, this difference can be used to monitor attachment and/or elution of the fluorette from the affinity column
  • the fluorette/dye complex can be used in FACS
  • a linker joining the fluorette to its fusion partner also contains a convenient site or sites to seperate the fluorette from the fusion partner, such as, a unique protease recognition sequences
  • the affinity column can contain antibody reactive with the fluorette
  • the fusion partner is a stability sequence to confer stability to the fluorette
  • fluorettes may be stabilized by the incorporation of glycmes after an amino-terminal methionine (MG or MGGO), for protection of the fluorette to ubiquitination as per Varshavsky's N-End Rule, thus conferring long half-life in the cytoplasm.
  • MG or MGGO amino-terminal methionine
  • two prolines at the C-terminus impart upon peptides resistance to carboxypeptidase action.
  • the presence of two glycines prior to the prolines impart both flexibility and prevent structure initiating events in the di- proline to be propagated into the fluorette structure.
  • preferred stability sequences are as follows: MG(X) n GGPP (SEQ ID NO:84), where (X) n is a fluorette of any amino acid and n is an integer of at least about 8, as outlined above for fluorette length.
  • the fusion partner is a dimerization sequence.
  • a dimerization sequence allows the non-covalent association of one fluorette to another fluorette, with sufficient affinity to remain associated under normal physiological conditions.
  • the dimers may be homo- or heterodimers.
  • Dimerization sequences may be a single sequence that self-aggregates, or two sequences. That is, a first fluorette with dimerization sequence 1 , and a second fluorette with dimerization sequence 2, such that upon introduction into a cell and expression of the nucleic acids, dimerization sequence 1 associates with dimerization sequence 2 to form new random peptide structures.
  • dimerization sequences can be used as presentation structures. That is, by putting a first dimerization sequence at one terminus of the fluorette, and a second dimerization sequence at the other terminus, similar to some other presentation structures, a "cyclized" fluorette can be made.
  • dimerization sequences will encompass a wide variety of sequences. Any number of protein-protein interaction sites are known. In addition, dimerization sequences may also be elucidated using standard methods such as the yeast two hybrid system, and traditional biochemical affinity binding studies, or even using the present methods.
  • the fusion partners may be placed anywhere (i.e. N-terminal, C-terminal, internal) in the structure as the biology and activity permits, although in general, N- or C-terminal fusions are preferred to fusions internal to the fluorette sequence.
  • a fluorette occupies an internal position within the fusion partner as the biology and activity permits. This will allow a means to measure access of the fluorette for its fluorophore dye as a means of determining structure and function of the region in which the fluorette resides.
  • the fusion partner includes a linker or tethering sequence.
  • Linker sequences between various targeting sequences may be desirable to allow the candidate agents to interact with potential targets unhindered
  • useful linkers include glycme- serme polymers (including, for example, (GS) n (SEQ ID NO 85), (GSGGS) n (SEQ ID NO 86) and (GGGS) n (SEQ ID NO 87), where n is an integer of at least one), glycme-alanine polymers, alanine-se ⁇ ne polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art Glycine-serme polymers are preferred since both of these ammo acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components Secondly, s
  • linkers can also be sequences that are derived from other proteins and have no structure or an assumed structure
  • linkers distance the fluorette from a molecule to which they are attached, for example, a fusion protein
  • the fusion partners including presentation structures, may be modified, randomized, and/or matured to alter the presentation orientation of the fluorette
  • determinants at the base of the loop may be modified to slightly modify the internal loop tertiary structure, which maintains the fluorette ammo acid sequence
  • combinations of fusion partners are used
  • any number of combinations of presentation structures, targeting sequences, rescue sequences, and stability sequences may be used, with or without linker sequences
  • the fluorette is fused to a target analyte, for example, to monitor or follow the target analyte, as is more fully described below
  • target analyte or “analyte” or grammatical equivalents herein is meant any molecule, compound or particle to be detected
  • a large number of analytes may be detected using the present methods, basically, any target analyte to which a fluorette may be attached may be detected using the methods of the invention
  • Suitable analytes include organic and inorganic molecules, including biomolecules
  • the analyte may be an environmental pollutant (including pesticides, insecticides, toxins, etc ), a chemical (including solvents, polymers, organic materials, etc ), therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc ), biomolecules (including hormones, cytokmes, proteins, lipids, carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, etc), whole cells (including procaryotic (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells), viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc ), and spores, etc
  • Particularly preferred analytes are proteins
  • the target analyte is a protein and the fluorette is made as a fusion protein, using techniques well known in the art
  • the nucleic acid encoding the fluorette is linked in-frame with a nucleic acid encoding a target protein to produce a fusion nucleic acid
  • fusion proteins are produced by culturing a host cell transformed with an expression vector containing the fusion nucleic acid, under the appropriate conditions to induce or cause expression of the fusion protein
  • the conditions appropriate for fusion protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation
  • the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an mducible promoter requires the appropriate growth conditions for induction
  • the timing of expression is important
  • fluorettes can be produced by in vitro transcription of the encoding nucleic acid and translation of the RNA transcript, as known in the art
  • the target analyte is other than a protein, and the fusion of the fluorette to the target analyte is generally done chemically
  • the fluorette and the target analyte are attached through the use of functional groups on each that can then be used for attachment
  • Preferred functional groups for attachment are ammo groups, carboxy groups, oxo groups and thiol groups
  • linker Linkers are well known in the art, for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference)
  • carboxyl groups (either from the surface or from the candidate agent) may be denvatized using well known linkers (see the Pierce catalog)
  • carbodiimides activate carboxyl groups for attack by good nucleophiles such as amines (see Torchilin et al , Critical Rev
  • fluorettes and target analytes may be attached in a variety of ways, including those listed above. What is important is that manner of attachment does not significantly alter the functionality of the fluorette (and preferably the target analyte as well); that is, the attachment should be done in such a flexible manner as to allow the interaction of the fluorette peptide with a dye, and the target analyte to its binding partners.
  • the fluorette will bind to Texas Red.
  • Suitable fluorettes in this embodiment include, but are not limited to, KHVQYWTQMFYS (SEQ ID NO:1 ); DFLQWKLARQKP
  • KNVQYWTQMFYT (SEQ ID NO:17); KHVQYWTHMFYT (SEQ ID NO:18); KHVQYWTQMFYT
  • RTIWEPKEASNHT SEQ ID NO:105
  • WSKMGHTVT SEQ ID NO:106
  • RWTWEPISE SEQ ID NO:107
  • GNQKCLQHNRCST SEQ ID NO:108
  • SQTWSFPEH SEQ ID NO:109
  • EPMARPWERKQDR (SEQ ID NO:110); and GTLSATRPYGRQW (SEQ ID NO:111).
  • the fluorette will bind to Rhodamine.
  • Suitable fluorettes in this embodiment include, but are not limited to, IPHPPMYWTRVF (SEQ ID NO:3); IPHRPMYWTPVF (SEQ ID NO:22); and LPHPPMYWTRVF (SEQ ID NO:23).
  • the fluorette will bind to Oregon Green 514.
  • Suitable fluorettes in this embodiment include, but are not limited to, HGWDYYWDWTAW (SEQ ID NO:4); ASDYWDWEWYYS (SEQ ID NO:5); YPNDFEWWEYYF (SEQ ID NO:6); HTSHISWPPWYF (SEQ ID NO:7); LEPRWGFGWWLK (SEQ ID NO:8); QYYGWYYDHNFW (SEQ ID NO:9); YMYDEYQYWNFW (SEQ ID NO:10); HEWEYYWDWTAW (SEQ ID NO:24); HEWDYYWDWTAW (SEQ ID NO:25); HGWDYYWDWTDW (SEQ ID NO:26); HGWDYYWDWTPW (SEQ ID NO:27); HGWDYYWDWTTW (SEQ ID NO:28); HGWDYNWDWTAW (SEQ ID NO:29); and QGWDYYWDW
  • the fluorette will bind to Fluorescein.
  • Suitable fluorettes in this embodiment include, but are not limited to, YPNDFEWWEYYF (SEQ ID NO:6); ASDYWDWEWYYS (SEQ ID NO:5); WYDDWNDWHAWP (SEQ ID NO:11); WHMSPSWGWGYW
  • a peptide is a fluorette if the overall homology of the peptide sequence to the amino acid sequences shown above (SED ID NOS 1-40 and 104-110) is preferably greater than about 70%, more preferably greater than about 75% even more preferably greater than about 80% and most preferably greater than 90%, with homologies of greater than 95 to 98% being especially preferred.
  • Homology in this context means sequence similarity or identity, with identity being preferred.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989).
  • Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
  • Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol.
  • WU-BLAST-2 uses several search parameters, most of which are set to the default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
  • a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "shorter" sequence in the aligned region.
  • the "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+/c; X u set to 16, and X g set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to -22 bits.
  • a percent amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region.
  • the "longer” sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
  • percent sequence identity is determined by inspection in which only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0". Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.
  • amino acid sequences of the fluorettes may be modified as needed. Sequence modifications include substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the fluorette are desired, substitutions are generally made in accordance with the following chart: Chart 1
  • substitutions that are less conservative than those shown in Chart I, although these generally are not preferred.
  • substitutions may be made which more significantly affect the structure of the fluorette backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule; or the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the fluorette's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • leucyl isoleucyl, phenylalanyl, valyl or alanyl
  • a cysteine or proline is substituted for (or by) any other residue
  • a residue having an electropositive side chain e.g. lysyl, arginyl, or histidyl
  • an electronegative residue e.g. glutamyl or aspartyl
  • a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
  • not more than about three substitutions or deletions will be made, and that the change will not be more than about 20 number %, usually not more than about 10 number %, of the number of amino acids in the fluorette, although in some instances higher numbers of alterations may be made.
  • conservative substitutions as known in the art, including substitutions within the large hydrophobic group isoleucme, leucme, valme and phenylalanme between senne and threonme, glycine and alanme, asparagme and glutamine, aspartic acid and glutamic acid, or lysme, argmine and histidme.
  • non-conservative alterations are done
  • such libraries are based on, for example, either bacte ⁇ ophage (Matthews and Wells Science 260 1113-1117 (1993), Koivunen et al Biotechnology 13 265-270 (1995), Yu and Smith Methods Enzymol 267 3- 27 (1996), Harrison ef al Methods Enzymol 267 83-109 (1996), Rebar and Pabo Science 2
  • fluorettes are created, isolated, or selected from aptamer library phage display approaches Such libraries are based on, for example, M13 filamentous bactenophage (Smith Science 228 1315-1317 (1985), Cwirla ef al Proc Natl Acad Sci U S A 87 6378-6382 (1990), Devlin et al Science 249 404-406 (1990), Scott and Smith Science 249 386-390 (1990))
  • fluorettes are identified by screening a phage display peptide library that contains a combinatorial library of peptides interposed betwen the leader sequence and the ammo terminus of mature form of bactenophage M13amp9 minor coat protein, pill
  • the recombinato ⁇ al library can be produced by a number of methods as known m the art
  • the library is produced by annealing and cloning an oligonucleotide library of sequences into the M13amp9 pill gene
  • Annealing of the oligonucleotide library to produce double-stranded DNA preferably produces "sticky ends" to facilitate directional cloning into the appropriate reading frame of the M13amp9
  • the annealed oligonucleotide library contains unique restriction enzyme sites, which are digested to produce "sticky ends" to facilitate directional cloning
  • the oligonucleotide library preferably also encodes an in-frame linker, tethering sequence, or spacer as described herein The function of the linker is to decrease or minimize ste ⁇ c hindrance and promote accessibility of the fluorette for its fluorophore dye The linker also promotes structural and functional independence of the fluorette Phage particles bearing the peptide library are produced by
  • oligonucleotide library produces a library of peptides either directly fused to the pill protein or, preferably, fused via a linker or spacer
  • the linker or spacer can be, for example, sequence, as described herein, interposed between the fluorette and the pill protein sequences
  • the nucleic acids which give rise to the fluorettes are chemically synthesized, and thus may incorporate any nucleotide at any position Thus, when the nucleic acids are expressed to form peptides, any ammo acid residue may be incorporated at any position
  • the synthetic process can be designed to generate randomized nucleic acids, to allow the formation of all or most of the possible combinations over the length of the nucleic acid, thus forming a library of randomized nucleic acids and peptides
  • random DNA fragments may be made from fragmentation of genomic DNA, followed by sizing, and ligation into an appropriate vector for expression and selection
  • the library should provide a sufficiently structurally diverse population of randomized expression products to effect a probabilistically sufficient range to allow isolation of a fluorette Accordingly, a library must be large enough so that at least one of its members will have a structure that gives it affinity for a fluorophore dye Although it is difficult to gauge the required absolute size of a library, nature provides a hint with the immune response a diversity of 10 7 -10 8 different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organism Published in vitro selection techniques have also shown that a library size of 10 7 to 10 8 is sufficient to find structures with affinity for the target A library of all combinations of a peptide from about 7 to 20 ammo acids in length has the potential to code for 20 7 (10 9 ) to 20 20 Thus, with libraries of 10 7 to 10 8 per ml of phage particles the present methods allow a "working" subset of a theoretically complete interaction library for 7 ammo acids, and a subset of shapes for the 20 20 library Thus, in a
  • bactenophage expressing fluorettes are selected by biopanning, which consists of four sequential steps i) phage binding with a fluorophore dye carrier, n) removal of unbound or weakly bound phage, in) elution of bound phage and (iv) amplification of bound phage Certain steps can be omitted, for example, such as washing and amplification if the peptide library is expressed in eukaryotic or mammalian cells and fluorescence is detected and cells are shorted according to fluorescence or spectral shifts If necessary or desirable, the amplified phage are used for the subsequent round(s) of biopanning against the corresponding fluorophore dye carrier until apparent enrichment for binding is observed over background Preferably, after about four rounds of biopanning, phage bearing sequences that bind specifically to a fluorophore dye can be identified Following biopann
  • the fluorophore dyes can be bound to solid-phase carriers
  • the dyes are covalently attached to a target bead This facilitates washing and removal of non-specifically bound or weakly bound phage
  • a linker or spacer molecule is interposed between the fluorophore dye and the target bead, for example, succinimidy esters and derivatives of fluorophores
  • a spacer molecule increases the accessibility of the fluorophore dye and minimizes potential ste ⁇ c hindrance that may interfere with the interactions between the fluorophore dye and the bactenophage particles that bear the peptide library
  • the number of fluorophore dye molecules bound to the carrier will vary In general, there are up to about 1 micromole Higher or lower amounts of dye can be attached per ml of carrier beads, as needed, for example to increase or decrease the stringency of the biopanning conditions
  • a fluorette Once a fluorette is identified, it can be mutagenized or denvatized to isolate fluorettes with altered properties
  • the de ⁇ vatize fluorettes have a higher affinity or specificity, thereby, providing, for example, increased sensitivity for the assay or system in which the fluorette is employed This also allows the use of lower amounts of fluorescent dye, thereby, limiting toxicity of the fluorescent dye to cells, if needed
  • affinity maturation (Yu and Smith supra) can be used to produce mutant or derivative fluorettes with improved affinities over their corresponding parental fluorettes
  • a second oligonucleotide library is produced that is, preferabbly, based That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities
  • the nucleotides or ammo acid residues are randomized within a defined class, for example, of hydrophobic ammo acids, hydrophilic residues, ste ⁇ cally biased (either small or large) residues, towards the creation of cystemes, for cross-linking, prolines for SH-3 domains, se ⁇ nes, threonmes, tyrosines or histidmes for phosphorylation sites, etc , or to purmes, etc
  • the ammo acid sequences of the fluorettes are subjected to biased mutagenesis at a desired mutagenesis rate using a number of methods as known in the art
  • the sequences can be mutagenized by systemmatic alteration of the codons encoding the fluorettes
  • Oligonucleotide mutagenesis is preferablly, performed during the synthesis of the oligonucleotide library
  • specific nucleotides such as A and C, can be omitted from the third position of each codon
  • the oligonucleotide library is preferably annealed and cloned into an expression vector of choice
  • the biopanning conditions are preferably altered to be more stringent by, for example, changing one or more of the following conditions This also provides a method to maximize selection against the parental fluorette
  • the concentration of the fluorophore dye bound to the carrier beads can be decreased
  • the phage concentration represented in the binding step and/or the binding time also can be reduced
  • the stringency of the washing conditions is increased Washing stringency can be increased by, for example, increasing the volume of washing buffer per ml of carrier beads, increasing the wash temperature, altering the ionic strength or pH of the wash buffer, increasing the detergent concentration or by using a stronger, ionizing detergent (Sambrook ef al , (1989) Molecular Cloning A Laboratory Manual (2nd Edition) Cold Spring Harabor Laboratory, Cold Spring Harbor, NY)
  • Other parameters that can be altered to increase stringency are, for example, increasing the temperature and competition for binding to the fluorophore dye by related
  • Fluorette binding to a fluorophore dye can be assessed in several ways
  • binding of the bacte ⁇ ophage-bea ⁇ ng peptide library to the fluorophore dye-carrier can be determined by comparing the infectious particle titers of input phage to eluted phage
  • a known concentration of free fluorophore dye is mixed with a known number of purified phage particles The number of particles is preferably calculated from the optical densities of the pu ⁇ fed phage at 260 nm and 280 nm but other methods, such as, infectious titer can be used
  • Phage-fluorophore dye complexes are separated by, for example, precipitation to remove unbound dye, spotted onto a solid-support filter, such as, nitrocellulose and scanned for fluorescence
  • Other methods to remove unbound dye include, for example, competition with related molecules, washing, and increased stringency
  • Fluorette/fluorophore dye dissociation constants can be determined in several ways
  • phage-fluorophore dye dissociation constants can be determined by, for example, incubating for several hours subsaturatmg amounts of phage with a fluorophore dye bound to a carrier, for example, a bead as described above The bead suspensions are removed, for example, by centnfugation, and the unbound phage in the supernatants can be titrated
  • visualization Dissociation constants can be measured via a standard linear Scatchard plot Nonspecific background binding is determined using phage that does not express fluorette sequences
  • fluorettes are encoded by nucleic acids and produced by recombmant molecular biology techniques (Sambrook et al (1989) Molecular Cloning A Laboratory Manual (2nd Edition) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Ausubel et al (1994) Current Protocols in Molecular Biology Massachusettes General Hospital and Harvard Medical School Boston, MA)
  • nucleic acids By "nucleic acids” herein is meant a nucleic acid, DNA or RNA depending on the delivery or expression vehicle or vector used, which can be manipulated to express fluorettes, that is, the nucleic acids encoding the fluorettes and the fusion partners and linkers if present
  • the nucleic acids will also generally contain enough extra sequence to effect translation or transcription, as necessary
  • the nucleic acid generally contains cloning sites which are placed to allow in frame expression of the fluorettes, and any fusion partners, if present, such as presentation structures
  • any fusion partners if present, such as presentation structures
  • nucleic acids are introduced and expressed within the cells to produce fluorettes
  • introduction into or grammatical equivalents herein is meant that the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid
  • the method of introduction is largely dictated by the targeted cell type, discussed below.
  • Exemplary methods include CaP0 4 precipitation, liposome fusion, lipofectm®, electroporation, viral infection, etc
  • the candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction), or may exist either transiently or stably in the cytoplasm (i e through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc ) As many pharmaceutically important screens require human or model mammalian cell targets, retroviral vectors capable of transfectmg such targets are preferred
  • the fluorettes functions within the cell in which they are expressed
  • fluorettes may be expressed within a cell or secreted from a cell, and purified If desired, the fluorette can be introduced into another cell by electroporation, calcium phosphate precipitation, microinjectiom, liposome fusion, lipofectm, viral infection, etc
  • Fluorettes also may be produced in vitro by transcription/translation reactions Alternatively, fluorettes are chemically synthesized using any of a number of methods
  • the present invention provides a complex or composition of a fluorette bound to a fluorophore dye
  • the binding of the fluorette to the fluorophore dye leads to four possible outcomes in regards to the excitation and emission spectra of the fluorophore dye i) little to no change in the fluorophore emission or excitation spectra, n) change in fluorophore emission, in) change in fluorophore excitation and (iv) change in both fluorophore emission and excitations
  • binding of the fluorette to the fluorophore dye may prevent fluorophore excitation or emission In either scenario, the fluorophore dye, no longer fluoresces when bound to the fluorette In another embodiment, the excitation spectrum and/or the emission spectrum of the fluorophore dye is shifted, to a shorter or longer wavelength Alternatively, the excitation spectrum and/or emission spectrum can be broadened
  • the excitation spectrum is altered up to about 5 nm, about 10 nm, being preferred, 20 nm particularly preferred, and about 100 nm being especially preferred
  • Fluorettes in their various forms find use in detector systems and various types of assays and techniques using a large set of potential fluorophore dyes, for example as, in detection of viral delivery systems, diagnostics, high-throughput assays
  • compositions find use in a number of applications These applications include binding a fluorophore dye to the fluorette, removing unbound dye and detecting fluorescence
  • fluorophore dye binding a fluorophore dye to the fluorette, removing unbound dye and detecting fluorescence
  • the emission and/or excitation spectrum of the fluorophore dye is altered when bound to a fluorette Therefore, it is not necessary to remove unbound dye
  • FRET fluorescence resonance energy transfer
  • FRET applications based on fluorette/fluorophore dye interactions provide methods to demonstrate the proximity or interactions of two or more molecules These molecules include, for example, biological molecules, such as proteins, lipids, nucleic acids, carbohydrates In another embodiment, FRET can be applied to detect or induce the localized activation of a produrg
  • the interactions of two or more proteins, each containing a unique fluorette sequence can be demonstrated as follows First, the fluorophore dyes are bound by their corresponding fluorettes Next, one of the fluorette/fluorophore dye complexes or donor complex is excited and fluoresces When the two proteins containing the fluorette sequences are in close proximity or in some way have interacted, the emitted light of the first donor complex causes the second fluorette/fluorophore dye complex (acceptor) to fluoresce
  • FRET fluorette/fluorophore dye complexes that are covalently or associately linked to target biomate ⁇ als via small ligands
  • the demonstration of FRET as described above indicates an interaction or the proximity of the target biomate ⁇ als when bound to their respective ligands
  • Other applications include, "trigger dependent FRET", in which the use and design of trigger molecules, such as, heavy metal ions, drugs, etc , that specifically bind to the ligand or target biomatenals, causing dissociation of one or more of the fluorette/dye complexes from the target biomatenals and a decrease or loss of FRET
  • FRET may also be employed to detect intramolecular interactions or events, such as, changes in molecular structure that occurs in a molecule with the binding and/or dissociation of a ligand
  • double or higher order fluorettes may be linked, for example, via a rigid connector, such as polyalanme or polysenne, to provide the correct angle of orientation of corresponding donor and acceptor fluorette/fluorophore dye complexes This may be of importance for efficient and quantitative FRET output
  • the spacer may be desirable for the spacer to contain a peptide sequence or other material which, upon binding a specific fluorophore dye, brings the donor and acceptor comlexes into proximity such that FRET can ensue
  • FRET applications also include dual fluorophore dye quenching due to fluorette binding
  • fluorettes specific for donor and acceptor fluorophores are joined by a protease cleavable spacer
  • This fluorette dimer is bound to its two fluorophore dyes targets and FRET ensues as described above
  • FRET decreases after protease cleavage of the fluorette dimer
  • novel assays are based on fluorophore dye quenching due to its binding of a specific fluorette
  • a target protein is labeled in vitro by a fluorophore dye and is assembled into a complex particle or structure with other materials (e g , other proteins, DNA or RNA) whose conformation or structure is to be investigated
  • the addition of a specific fluorette after the complex is assembled and measuring fluoresence indicates if the fluorette had access (binding) or no access (no binding) to its target dye
  • Novel reporter gene assays based on fluorette-induced Stokes shift changes also can be created
  • a fluorette fused with a given carrier protein can be used as a gene activity reporter for the presence of the protein Cell extracts, or living cells expressing the protein, can be monitored for fluorette activity when the specific fluorophore dye is bound by the peptide Binding of the fluorophore dye by the fluorette/peptide modifies the excitation and/or emitted wavelength
  • Fluorophore Dye Carriers Four fluorophore dyes, Texas Red, Fluorescein, Rhodamine Red and Oregon Green 514, were chosen, for selection of fluorettes from a phage display library (Example 2) The chemical structures of the dye conjugates are shown in Figure 1
  • the first three dyes, Texas Red, Fluorescein and Rhodamine Red fall into one group (i) they have a relatively high molar extinction coefficient for absorption and excellent quantum fluorescence yields (Haugland supra), (n) they have non-overlapping spectral characteristics, (in) they have the potential for cross-quenching and/or FRET analysis
  • the fourth dye, Oregon Green 514, related structurally to Fluorescein was chosen to determine if structure-function relationships could be discerned between fluorette peptides that bound related dyes
  • fluorophore dyes 6-(fluoresce ⁇ n-5-(and-6)-carboxam ⁇ do)hexano ⁇ c acid succinimidyl ester (5(6)-SFX), "mixed isomers", Oregon Green 514 carboxylic acid, succinimidyl ester, Rhodamine Red-X, succinimidyl ester, “mixed isomers” and Texas Red-X, succinimidyl ester, “mixed isomers” were purchased from Molecular Probes
  • a phage display peptide library was screened that contained a combinatorial library of 12-mer peptides fused via a short glycine linker spacer (GGG) to the ammo-terminus of a minor coat pill protein (5 copies per particle, Li et al (1980) J Biol Chem 255 10331-10337) of the filamentous bactenophage M13mp19 (Ph D phage display, New England Biolabs) During phage maturation the leader secretory sequence is removed This results in the 12-mer peptide positioned immediately at the ammo-terminus of the mature protein
  • E coli ER2537 strain [F' lacl q ⁇ (lacZ)M15 proA + B + /fhuA2 supE thi ⁇ (lac-proAB) ⁇ (hsdSM-mcrB)5 (rk mk mcrBC )] and E coli TG-1 strain [supE th ⁇ -1 ⁇ (lac-proAB) ⁇ (mcrB-hsdSM)5 (rk mk ) (F' traD36 proAB lacl q Z ⁇ M15)] were purchased from New England Biolabs and Stratagene, respectively, and used as bactenophage M13mp19 hosts All bacteriological techniques were performed as described (Sambrook ef al , supra, Ausubel ef al , supra)
  • each biopanning round consisted of four sequential steps (i) phage binding with a fluorophore dye carrier, (n) washing unbound phage from the beads, (in) nonspecific elution of bound phage and (iv) amplification of bound phage
  • the Ph D -12 phage display peptide library (based on modified M13mp19 bactenophage, 1 9x10 9 independent transformants) as a part of the Ph D -12 phage display peptide library kit was purchased from New England Biolabs All procedures were carried out at room temperature unless noted First, 0 15ml of cent ⁇ fuged fluorophore dye carrier beads were blocked with 3ml of TBS + 2mg/ml BSA (blocking buffer) for 1 5hr with gentle rotating, beads were washed with 15ml TBS + 0 1% Tween-20 + 0 5mg/ml BSA (binding buffer), cent ⁇ fuged, mixed with 1 8x10 11 plaque-forming units (p
  • the amplified phage from round 1 were further selected against the corresponding fluorophore dye carrier
  • the next biopanning rounds were performed in the same manner as round 1 , except that (i) input phage in the binding reaction were increased to 4 5x10 11 pfu, ( ) time of binding was reduced to 2hr in round 4, (in) Tween-20 concentration in a washing buffer was increased to 0 2% in round 3 and 0 4% in round 4, (iv) eluted phage from last round 4 were not amplified, but rather served as a source of independent phage clones used for sequencing
  • TR-4, RhR-4, OG-4 and Flu-4 phage sets were used as a source of independent phage clones that were sequenced for further analysis
  • Phage single-stranded DNAs (ss DNAs) and double-stranded DNAs (ds DNAs) were purified as previously described (Sambrook ef al , supra) Fluorette-cod g portions of the DNA and adjacent DNA regions were sequenced with -96 gill sequencing primer CCCTCATAGTTAGCGTAACG (New England Biolabs) (SEQ ID NO 115) using an Applied Biosystems 391 automated DNA sequencer
  • TR401 and RhR401 phage were selected against the polymer linker moiety rather than a fluorophore dye moiety of the dye carriers
  • the TR-4 and RhR-4 phage were cross-bound to Rhodamine Red and Texas Red carriers, respectively Cross-binding of each phage to the inappropriate dye carrier did not exceed the nonspecific phage background binding (data not shown)
  • the TR401 and RhR401 fluorettes are specific by this comparison for their respective conjugated dyes, despite the similarity of the compound core ring structures Second, they had affinity for the corresponding conjugated fluorophore dyes rather than only a polymer linker moiety of Texas Red and Rhodamine Red carriers or other chemical features of the carriers themselves
  • phage clones were isolated after four biopanning rounds with random combinatorial Ph.D. -12 phage display peptide library (see text), b) partial consensus (shown underlined) for Oregon Green 514- and Fluorescein-specific peptide fluorettes, (YWDW (SEQ ID NO:113); W(D/E)YY
  • Oregon Green 514 is structurally related to Fluorescein, and is considered a Fluorescein pentafluoride (see Figure 1), independent selection of the same peptide fluorettes against Oregon Green 514 and Fluorescein carriers was predicted. The same fluorettes selected independently against these fluorophores possibly bind to similar domains of the Oregon Green 514 and Fluorescein dye molecules.
  • the selected phage had been originally selected as being capable of binding fluorophore dyes that had been covalently linked to a polymer carrier.
  • the nature of carrier-crosslinked fluorophores, while useful for initial selection of phage, is inappropriate to study affinity of the phage except in relative terms.
  • Each bactenophage particle contains 5 copies of plll-fluorette fusion protein. Therefore phage binding is governed by avidity considerations.
  • RhR401 , OG402, OG403 (1.3-1.8x10 phage particles; calculated from optical densities of the purified phage at 260 and 280nm) in TBS + 0.05% sodium azide buffer were separately mixed with an equal volume of 20 ⁇ M solution of the corresponding free dyes in the same buffer; mixtures were adjusted by Tween-20 to 0.1% and incubated for 3hr.
  • Phage-fluorophore dye complexes were precipitated three times by PEG-8000 in order to remove unbound dye and dissolved in 6.6 ⁇ l of TBS + 0.05% sodium azide buffer.
  • phage-fluorophore dye complexes (1 ⁇ l of each) were spotted to nitrocellulose filter and filter was scanned on the Storm 840 scanner (Molecular
  • GAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO 90), OG403-91CL CTCCCCTTCGGCCGAACCTCCACCCCAAGCAGTCCAATCCCAATAATAATCCCACCCATGA
  • nucleotides in a regular case shows no degeneracy
  • boldfaced nucleotide designates 91% of shown nucleotide and 3x3% of each of the other three nucleotides
  • boldfaced and underlined nucleotide designates 91 % of A or C and 9% of C or A, respectively
  • Ph D 121 was a randomly picked phage clone from the Ph D -12 phage display peptide library) in a total volume 0 1 ml Ligations were electroporated into 0 5ml of TG-1 electrocompetent cells (Stratagene) according to manufacturer's protocol and phage library complexities were determined by immediate mixing of several 10-fold dilutions of transfected TG-1 cells with ER2537 cells and plating Phage libraries were further amplified in 400ml of liquid LB media for 4hr at 37°C with vigorous shaking, concentrated by PEG (see above) and, finally, suspended in 0 5ml TBS + 0 05% sodium azide buffer
  • a key difference of the secondary biopanning versus the primary biopanning with the original phage library is the strict need to maximize selection against the originating, parental fluorette
  • phage clones were isolated after three biopanning rounds with TR401-91CL, RhR401- 91 CL, OG403-91CL and OG402-91CL nonrandom combinatorial phage display peptide libraries (see text), b) ammo acid substitutions in mutant peptide fluorette vs the corresponding parent are shown, mutant peptide fluorette sequence is shown as many times as it was found (see phage clone frequency), dots designate the same ammo acid as that in the corresponding parent, dash in a second position of the FluS304 fluorette designates m- frame deletion, c) original parental clones and the parental clones carrying silent mutat ⁇ on(s) in peptide fluorette moiety
  • PEG-purified phage (input 5x10 8 -2 5x10 10 pfu with 2 to 5-fold increments) were bound to the respective fluorophore dye carriers essentially in the same manner as described above for Texas Red-specific phage RhR401 and RhRS308, OG403 and OGS316, OG402 and FluS303 phage, were bound to the Rhodamine Red, Oregon Green 514 or Fluorescein carriers, respectively Following binding the beads were quickly washed twice by 0 25ml of TBS + 0 1% Tween-20 and suspended in 10ml TBS 10 ⁇ l of suspension was mixed with " 2x10 8 log-phase ER2537 cells and incubated 1 hr at 4°C with slight shaking to allow phage adsorption Several ten-fold dilutions of infected cells were mixed with non fected ER2537 cells and plated in standard plaque assay (Sambrook ef al , supra) All binding experiments and titrations were accomplished in
  • the TR401 peptide is the progenitor of the TRS311 peptide
  • the two substitutions H2P and S12T result not only in an increased binding, but also in an apparent differences in the excitation spectra elicited
  • PepTRS311 had a peak emission spectra shift
  • one or both of the two substitutions, H2P or S12T are critical for shifting the spectra of the emission profile
  • peptide names reflect the names of phage clones carrying respective fluorettes except Pep Control (see Tables 1 and 2)
  • first twenty ammo acids of the peptides are the same as in the pill fusion protein of the corresponding phage clones except Pep Control (see Tables 1 and 2 and also New England Biolabs "Ph D -12 Phage Display Peptide Library Kit” manual), thus, amino acids 1-12 are the fluorette potion (double underlined) and ammo acids 13-20 are the distal pill fusion part (underlined)
  • Ammo acids 21-29 contain a small GGG spacer followed by H ⁇ s 6 tag (shown in regular case) (SEQ ID NOS 41-48) c) specific for both Oregon Green 514 and Fluorescein (see Table 1) d) nonspecific control peptide synthesized for specificity check
  • Constrained Texas Red-Binding Peptides were identified via five rounds of biopanning of a mixture of two constrained phage display libraries against a polymer carrier with covalently bound Texas Red Two libraries contained SKVILFE-flanked nine or thirteen ammo acid variable region in the N-termmal part of M13 bactenophage pill protein The structure of the libraries were as follows
  • GGGSKVILFEGPAG (X) 9or 13 GAPGSKVILFEGGPG (SEQ ID NO 93)- (pill protein)
  • SKVILFE-dimenzers are underlined Flexible linkers or spacers GGG, GPAG ( SEQ ID NO 94), GAPG (SEQ ID NO 95) and GGPG (SEQ ID NO 96) are double underlined X represents any
  • the flexible linker or spacer GPAG is encoded by a nucleotide sequence containing Fsel restriction endonuclease site
  • the flexible linker or spacer GAPG is encoded by a nucleotide sequence containing Ascl restriction endonuclease site
  • Fsel and Ascl restriction enzymes are rare eight-cutters
  • Consensus motifs can be observed within these peptides (e g , RXXWEP (SEQ ID NO 104), WEP and TW, see Table 5) that suggests structural features common to the peptides that allow for efficient binding Interestingly, no said constrained peptide has a significant homology with linear Texas Red-binding peptides
  • Binding affinity of phage clones revealed the most avid binders, TRP512 phage and TRP501 phage, with K ⁇ equal to 25 pM and 80 pM, respectively Other five phage clones were much less avid with K ⁇ , ⁇ 5 nM (see Table 5)
  • Binding affinity of synthetic TRP512 peptide is measured as I 10 nM ( « 160-fold more avid than linear TR401 and TRS311 peptides)
  • phage clones were isolated after five biopanning rounds with a mixture of combinatorial P71 R4-CL-9 & P71 R4-CI-13 phage display peptide libraries b) -1 (D,E) or +1 (K,R) c) number of hydrophobic amino acids (A,V,L,I,W,Y,F) per total number of fluorette amino acids *) phage clone is represented in the corresponding group at least twice

Abstract

The present invention is directed to novel polypeptides, termed fluorettes, that bind with high avidity to fluorophore dyes. The peptides find use in a variety of methods and approaches involving fluorophore dyes.

Description

FLUORESCENT DYE BINDING PEPTIDES
This application is a contmuation-in-part of application Serial No 60/104,465 filed October 16 1998
FIELD OF THE INVENTION
The invention relates to peptides that bind to fluorescent dyes, termed "fluorettes", and to methods of making and using the fluorettes In particular, the fluorettes can be used in detection and assay systems in vitro and in vivo
BACKGROUND OF THE INVENTION
Fluorophore dyes, due to their exquisite sensitivity and ease of use, are widely used in numerous approaches in fluorescent microscopy, flow cytometry and other detection systems (Haugland Handbook of fluorescent probes and research chemicals (sixth edition) Molecular Probes, Inc , Eugene OR (1996) and ref therein)
Detection of proteins in living cells using fluorescence approaches has been accomplished in a variety of settings For instance, it is possible to use ligands (or naturally-derived antibodies) conjugated directly or indirectly to fluorophores as probes of the expression levels of nearly any given surface-expressed protein on living cells In some particular cases for proteins within cells, it is possible to use permeable ligands for individual target proteins In these cases the ligand is either self-fluorescent, becomes fluorescent upon binding or is conjugated to fluorescent adducts In other cases, it has been possible for many years to genetically fuse reporter enzymes such as β-galactosidase, β-glucuronidase, and β-glucosidase to proteins and use a fluorogenic dye, acted upon by the reporter enzyme(s), to assay enzymatic activity on a cell by cell basis (Nolan ef al Proc Natl Acad Sci U S A 85 2603-2607 (1988), Lonncz et al Cytometry 24 321-329 (1996), Krasnow et al Science 251 81-85 (1991) Other systems, including β-lactamase (Zlokarnik ef al Science 279 84-88(1998)) build upon those findings by applying dyes with increased cell permeability or having radiometπc fluorescent qualities that might have advantages in some applications In recent years proteins with inherent fluorescence, such as Green Fluorescent Protein (Welsh and Kay Curr Opin Biotechnol 8 617-622 (1997), Misteli and Spector Nat Biotechnol 15 961-964 (1997)) have become widespread in their application owing to ease of use, the availability of mutant proteins with differing spectral qualities in either excitation or emission, and the relative non-toxicity of the approach
However, in the aforementioned cases the approaches are limited by a need to genetically fuse a relatively bulky reporter protein to the molecule under study This can have detrimental consequences to the functionality of the protein in question or interfere mechanistically with cellular constituents with which the protein interacts While it would be best to directly measure a given target protein using a specific fluorescent dye that recognized any given target moiety, no technology exists as yet to create such reagents
There is a need, therefore, to develop approaches that provide the building blocks for specific biomateπal detection
SUMMARY OF THE INVENTION
The invention provides peptides that bind to fluorophore dyes In one aspect of the invention the peptides are made of naturally occunng ammo acids, non-naturally occurring ammo acids, or combinations thereof
In another aspect of the invention, methods are provided for isolating and identifying peptides that bind to fluorophore dyes The method comprises creating and screening peptide libraries that bind to fluorophores
In another aspect of the invention, methods are provided for increasing the binding affinity of the fluorette for a fluorophore
In a further aspect of the invention are provided complexes of fluorettes bound to fluorophore dyes The binding of the fluorophore by the fluorette may alter the excitation and/or the emission spectrum of the fluorophore
In an additional aspect, the present invention provides methods for detecting a fluorette by binding a fluorette to a fluorophore dye and detecting the fluorette/fluorophore dye complex
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Fluorophore dye carriers Fluorescein, Oregon Green 514, Rhodamine Red and Texas Red activated derivatives were covalently linked to the polymer carrier Ultrahnk Immobilized DADPA via a 12-atom diaminodipropylamine spacer A spacer is shown as a horizontal thick bar Digits with arrows m the chemical structures of all fluorophore dyes, except Oregon Green 514, show that "mixed isomers" of these fluorophore dye activated derivatives were used for a coupling Figure 2 Biopanning with a peptide phage display library against Fluorescein, Oregon Green 514, Rhodamine Red and Texas Red carriers A Ph D -12 phage display peptide library (New England Biolabs) is a combinatorial library of 12-mer peptides fused to the ammo-terminus of a minor coat pill protein of the bacteπophage M13mp19 Bound phage in four biopanning rounds were calculated as a percentage of eluted phage plaque forming unit (plaque forming unit (pfu)) yield vs input phage plaque forming unit (pfu) All experiments were performed in duplicate Standard deviations did not exceed 19%
Figure 3 Phage-fluorophore dye binding Fluorophore dye-specific OG403, OG402, RhR401 and TR401 phage were bound to Oregon Green 514, Fluorescein, Rhodamine Red and Texas Red "free" dyes, respectively (see text) The normalized particle amounts of amplified Ph D -12 phage display peptide library (nonspecific phage) were bound in solution with the same dyes as controls Phage-fluorophore dye complexes were precipitated three times by PEG to remove unbound dye and spotted to a nitrocellulose filter The filter was scanned on the Storm 840 scanner (Molecular Dynamics) The presented data is from a single scan and image enhancements were done simultaneously on the complete set of dye binding results using NIH Image 1 59 software
Specific/nonspecific signal ratios were quantified by densitometry of spot images using NIH Image 1 59 software (see Materials and Methods for details) All binding experiments shown were completed in duplicate Densitometry was carried out prior to contrast enhancement Brightness and contrast values were modified to 50 and 65, respectively in Adobe Photoshop Individual elements of the figure were arranged as a composite from the same original scan from which the binding ratios were determined
Figure 4 Biopanning with four biased combinatorial phage display peptide libraries based on degenerated fluorettes against Fluorescein, Oregon Green 514, Rhodamine Red and Texas Red carriers OG402-91CL, OG403-91CL, RhR401-91CL and TR401-91CL biased combinatorial phage display peptide libraries (see text) were subjected to a biopanning against Fluorescein,
Oregon Green 514, Rhodamine Red and Texas Red carriers, respectively Bound phage in three biopanning rounds were calculated as described in a legend to Figure 2 All experiments were performed in duplicate Standard deviations did not exceed 21 %
Figure 5A and 5B Peptide - Texas Red binding Excitation and emission spectra Peptides at the noted concentrations were incubated with Texas Red (50nM), or without, in 0 6ml of TBS for 1 hr at room temperature (RT) Samples were scanned on a spectrofluoπmeter (SPEX Fluoromax (Jobin Yvon-SPEX Instruments Co )) Peak excitation or emission positions are shown in the figure using vertical bars Excitation and excitation/emission peak shifts for PepTR401(10μM)/Texas Red and PepTRS311 (10μM)/Texas Red complexes, respectively (A), were reproducible in three independent experiments The results of a single experiment are presented
Figure 6 Texas Red-specific peptide PepTR401 (Panel A), PepTRS311 (Panel B), PepTR406 (Panel C) and nonspecific PepControl (Panel D) (see peptide sequences in Table 4) were bound via Hιs6 tag to cobalt ion-containing Sepharose beads The peptide coated beats were washed and incubated with 0 5 micromolar Texas Red in TBS for 1 hour at room temperature followed by several washings of beads in order to remove unbound dye Fluorescent and nonflourescent control beads were photographed on fluorescent microscope Axiophot (Zeiss) using Rhodamine Red/Texas Red filter with the same time exposure for every sample All binding experiments were accomplished in three parallels and results of a single experiments are shown
Figure 7A-D Peptide - Texas Red binding Texas Red-specific peptides TR401 (linear) (Figure 6B), TRP501 (SKVILFE-flanked) (Figure 6C), TRP512 (SKVILFE-flanked) (Figure 6D), and nonspecific peptide (SKVILFE-flanked) (Figure 6A) as a negative control were bound via polyhistidme (Hιs6) tag to cobalt ion-containing Sepharose beads The peptide-coated beads were washed and incubated with 0 5 μM Texas Red in TBS buffer for 1 hour at room temperature followed by several washings of beads in order to remove unbound dye Fluorescent and nonfluorescent control beads were photographed on fluorescent microscope Axiophot (Zeiss) using Rhodamine Red/Texas Red filter with the same time exposure for every sample All binding experiments were accomplished in duplicate and results of a single experiment are shown
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel peptides or "fluorettes" that specifically bind fluorophore dyes The present invention also provides novel compositions comprising multimenc fluorettes and fluorettes fused or linked to other compounds or molecules Also provided are methods to create and modify fluorettes
The present invention further provides methods for the use of fluorettes in detecting biological materials, molecules, or target analytes, intracellular events, and intermolecular and intramolecular interactions The fluorettes also find use in in vitro assays and high throughput screens The methods provided are based on the formation and detection of a fluorette/fluorophore dye complex
The compositions and methods of the present invention provide a significant improvement over conventional light-emitting techniques Fluorettes, due to their small size, are not intrusive to the systems being studied and, therefore, permit detection and analysis of a target moiety or molecule while minimizing target modification Current light-emitting detection methods involve the use of enzyme-generated fluorochromes, luciferase-generated light, or reporter systems using engineered, inherently fluorescent proteins such as Aequona victoria green fluorescent protein (GFP) These systems are unwieldy and bulky and are intrusive to the system being studied, often necessitating the synthesis of large fusion proteins or out of context genomic constructions Thus, conventional techniques can involve substantial genetic manipulations of the targets one is trying to study, which may disrupt, interfere, or alter the process being measured Fluorettes minimize or avoid these limitations
Accordingly, the present invention provides a peptide, sometimes termed a "fluorette" herein, that will bind a fluorescent dye
By "peptide" or "fluorette" herein is meant at least about 8 covalently attached ammo acids The peptide may be made up of naturally occurring am o acids and peptide bonds, or synthetic peptidomimetic structures Thus "ammo acid", or "peptide residue", as used herein means both naturally occurring and synthetic ammo acids For example, homo-phenylalanme, citrul ne and noreleucme are considered ammo acids for the purposes of the invention "Ammo acid" also includes irπino acid residues such as proline and hydroxyproline The side chains may be in either the (R) or the (S) configuration In the preferred embodiment, the am o acids are in the (S) or L-configuration If non-naturally occurring side chains are used, non-ammo acid substituents may be used, for example to prevent or retard in vivo degradations The peptides can be linear or branched Ammo acids includes naturally occurring ammo acids or non-naturally occurring amino acids
By "naturally-occurring ammo acids" herein is meant ammo acids that are produced by living organisms Naturally-occurring am o acids are preferred in in vivo embodiments
By "non-naturally-occurnng ammo acids" herein is meant am o acids not produced by a living organism but that can be chemically synthesized Non-naturally-occurnng ammo acids include, for example, stereoisomers or enantiomers of the naturally-occurring ammo acids in which the ammo side chains, are in either the (D)((R)) or (S)((L)) configuration, ammo acids in which an amine group is bonded to any other but the alpha-carbon or may have more than one amine group bonded to the alpha-carbon, and ammo acids having functional groups or "R" groups not known to occur in nature The length of the peptide or fluorette, i e , the number of ammo acids, will vary In general, the number of amino acids varies from about 8 to about 50, with from about 8 to about 40 being preferred, from about 8 to about 25 being particularly preferred, and from about 8 to about 12 being especially preferred Thus, "peptide" includes peptides, oligopeptides, and in some cases, proteins
The fluorette can be monomenc or multimenc That is, a monomeπc fluorette binds a single fluorophore A multimenc fluorette is two or more associated monomenc fluorettes The association of a multimenc fluorette can be either covalent or non-covalent For example, the monomers can be joined directly together, for example as a linear fusion (i e , the carboxy terminus of one is joined to the ammo terminus of the second), or as a branched fusion, wherein the attachment of the second monomer is other than to the backbone of the first In addition, a chemical cross-linker or inclusion of specific reactive group on a fluorette be used to join fluorette monomers Alternatively the monomers may be non-covalently associated, for example, as is more fully outlined below, dimeπzation sequences can be used to associate two monomers
A multimenc fluorette can be homomultimeπc, i e , all the monomers are the same, and bind the same fluorophore dye, or heteromultimeric, having at least two different fluorette monomers and can bind different fluorophores
The fluorette peptides bind to at least one fluorophore dye
By "binding" herein generally is meant a non-covalent association or interaction between a fluorette and a fluorescent dye The non-covalent interactions between the fluorette and dye may involve various types of electrostatic, hydrophilic, and hydrophobic interactions Binding may also involve forming one or more covalent bonds between the fluorette and the dye Covalent bonds can be formed directly between the fluorette and the dye or can be formed by a cross linker or by inclusion of a specific reactive group on either the fluorette or dye or both molecules Binding may also involve a combination of covalent and non-covalent interactions
In a preferred embodiment, the binding is specific By "specific binding" herein is meant that the fluorophores will preferentially bind to a fluorophore dye with a binding constant in the range of at least about 10"6 M" to about 1010 M 1, with a preferred range being from at least about 10"6 M 1 to about 10"7 M"1, with an especially preferred range of from at least about 10"7 M"1 to about 10 10 M"1 In a preferred embodiment, the fluorettes do not specifically bind to other fluorophore dyes or compounds or moieties, that is, a fluorette is specific to one fluorophore Alternatively, a fluorette may bind two or more fluorescent dyes (bind together 2 or more fluorophores or more or capable of binding independently two or more fluorophores), that is, a fluorette may specifically bind another compound or moiety, including another fluorophore, if these molecules have a common structural feature(s) that specifically interact with the fluorette Preferably, however, a fluorette will not appreciably bind compounds other than fluorescent dyes
In other embodiments, a dye is non-fluorescent but becomes fluorescent when bound by a fluorette In further embodiments, fluorette binding to a non-fluorescent dye causes the release of a fluorescent moiety When the fluorescent moiety is released, the fluorette may bind to the released fluorescent moiety, may bind to the non-fluorescent dye, or may be released and therefore free to bind a second molecule of the non-fluorescent dye and repeat this process
By "fluorescent dye" or "fluorophore" or "fluorophore dye" herein is meant a compound that absorbs an incident light of a characteristic range of wavelengths or excitation spectrum and dissipates the absorbed energy by emitting light of a characteristic range of wavelengths or emission spectrum By "excitation spectrum" herein is meant the wavelength of incident light absorbed by the fluorophore dye that causes the fluorophore dye to fluoresce By "emission spectrum" herein is meant the characteristic wavelengths of the emitted or fluorescent light produced as the energy of the absorbed incident light is released The excitation and emission spectra for a fluorophore dye may or may not overlap In a preferred embodiment the incident light is in the ultraviolet spectrum and the emitted light is in the visible spectrum
Preferred fluorophores include, but are not limited to, Texas Red, Rhodamine Red, Oregon Green 514, and Fluorescein Examples of fluorescent dyes are found in the Molecular Probes Catalog, 6th Ed , Richard Haugland, Ed , which is expressly incorporated by reference in its entirety
In addition, the fluorette can further comprise additional components, such as a fusion partner or functional group By "fusion partner" or "functional group" herein is meant a sequence that is associated with the fluorettes, that confers upon the fluorette a function or ability Fusion partners can be heterologous (i e not native to a host cell), or synthetic (not native to any cell) Suitable fusion partners include, but are not limited to a) presentation structures, as defined below, which provide the fluorettes in a conformationally restricted or stable form, b) targeting sequences, defined below, which allow the localization of the flourette into a subcellular or extracellular compartment, c) rescue sequences as defined below, which allows the purification or isolation of the fluorette, d) stability sequences, which confer stability or protection from degradation to the fluorette, for example resistance to proteolytic degradation, e) dimenzation sequences, to allow for fluorette dimenzation or multimeπzation, or f) any combination of a), b), c), d), and e), as well as linker sequences as needed
In a preferred embodiment, the fusion partner is a presentation structure By "presentation structure" or grammatical equivalents herein is meant a sequence, which, when fused to the fluorette, causes the fluorette to assume a conformationally restricted form Proteins interact with other proteins and molecules largely through conformationally constrained domains Although small peptides with freely rotating ammo- and carboxy-termmi can have potent functions as is known in the art, the conversion of such peptide structures into active agents can be difficult due to the inability to predict side-chain positions for peptidomimetic synthesis Therefore the presentation of fluorettes in conformationally constrained structures will likely lead to higher affinity interactions of the fluorette with its target fluorophore dye This fact has been recognized in the combinatorial library generation systems using biologically generated short peptides in bacterial phage systems A number of workers have constructed small domain molecules in which one might present biased-combinatorial libraries of peptide structures
While the fluorettes are peptides, presentation structures are preferably peptides or proteins Thus, synthetic presentation structures, i e artificial polypeptides, are capable of presenting fluorettes as a conformationally-restπcted domain Generally such presentation structures comprise a first portion joined to the N-termmal end of the flourette, and a second portion joined to the C-termmal end of the flourette , that is, the flourette is inserted into the presentation structure, although variations may be made, as outlined below To increase the affinity or specificity of the fluorette, the presentation structures are selected or designed to have minimal biologically activity when expressed in the target cell
Preferred presentation structures maximize accessibility to the flourette by presenting it on an exterior loop Accordingly, suitable presentation structures include, but are not limited to, minibody structures, loops on beta-sheet turns and coiled-coil stem structures in which residues not critical to structure are randomized, zinc-finger domains, cysteine-lmked (disulfide) structures, transglutaminase linked structures, cyclic peptides, B-loop structures, helical barrels or bundles, leucme zipper motifs, etc In addition, presentation structures include dimenzation sequences, as defined below
In a preferred embodiment, the presentation structure is a coiled-coil structure, allowing the presentation of the flourette on an exterior loop (See, for example, Myszka et al , Biochem
33 2362-2373 (1994), hereby incorporated by reference) Using this system investigators have isolated peptides capable of high affinity interaction with the appropriate target In general, coiled- coil structures allow for between 6 to 20 ammo acids A preferred coiled-coil presentation structure is as follows
MGCAALESEVSALESEVASLESEVAALX|P,LAAVKSKLSAVKSKLASVKSKLAACGPP (SEQ ID NO 49) The underlined regions represent a coiled-coil leucme zipper region defined previously (see Martin et al , EMBO J 13(22) 5303-5309 (1994), incorporated by reference) The bolded X(n) region represents the loop structure and when appropriately replaced with peptides (i e fluorettes , generally depicted herein as (X)n, where X is an ammo acid residue and n is an integer of at least 5 or 6) can be of variable length The replacement of the bolded region is facilitated by encoding restriction endonuclease sites in the underlined regions, which allows the direct incorporation of oligonucleotides at these positions For example, a preferred embodiment generates an Xhol site at the double underlined LE site and a Hindlll site at the double-underlined KL site
In a preferred embodiment, the presentation structure is a minibody structure A "minibody" is essentially composed of a minimal antibody complementarity region The minibody presentation structure generally provides two randomizing regions that in the folded protein are presented along a single face of the tertiary structure (See for example Bianchi et al , J Mol Biol 236(2) 649-59 (1994), and references cited therein, all of which are incorporated by reference) Investigators have shown this minimal domain is stable in solution and have used phage selection systems in combinatorial libraries to select mmibodies with peptide regions exhibiting high affinity, Kd = 107, for the pro-inflammatory cytokme IL-6
A preferred minibody presentation structure is as follows MGRNSQATSGFTFSHFYMEWVRGGEYIAASRHKHNKYTTEYSASVKGRYIVSRDTSQSILYLQK KKGPP (SEQ ID NO 50) The bold, underline regions are the regions which may be contain fluorette sequences The italicized phenylalanine must be invariant in the first randomizing region The entire peptide is cloned in a three-oligonucleotide variation of the coiled-coil embodiment, thus allowing two different randomizing regions to be incorporated simultaneously This embodiment utilizes non-pa ndromic BstXI sites on the termini
In a preferred embodiment, the presentation structure is a sequence that contains generally two cysteine residues, such that a disulfide bond may be formed, resulting in a conformationally constrained sequence This embodiment is particularly preferred when fluorettes are to be secreted from a cell As will be appreciated by those in the art, any number of flourettes, with or without spacer or linking sequences, may be flanked with cysteine residues In other embodiments, effective presentation structures may be generated by the flourettes themselves For example, the flourettes may be "doped" with cysteine residues which, under the appropriate redox conditions, may result in highly crosslmked structured conformations, similar to a presentation structure Similarly, the flourettes may be controlled to contain a certain number of residues to confer β-sheet or α-helical structures.
In a preferred embodiment, a presentation structure is neuropeptide head activator. Bodenmuller et al. EMBO J. 5(8), 1825-1829 (1983) showed that neuropeptide head activator (HAv) dimerized to yield a biologically inactive form of the peptide at concentration as low as 10-13M, thus indicating extremely high self-binding affinity. Aldwin et al. U.S. Patent No. 5,491 ,074 observed that a fragment containing the last six amino acids of this peptide's carboxyl terminus (SKVILF) (SEQ ID NO:51) resulted in dimers that were even more stable than the HA itself. The USPN 5,491 ,074 shows that the last amino acid of SKVILF, the F (phenylalanine), must be on the carboxyl terminus for a proper peptide dimenzation activity. However, if F is not on the carboxyl terminus, it must be followed by one of two amino acids with free carboxyl group, E or D, to maintain the peptide dimenzation. Therefore, if the dimerizing peptide positioned inside a carrier protein, 6-mer SKVILF sequence must be converted to 7-mer SKVILFE (SEQ ID NO:51) or SKVILFD (SEQ ID NO:53) sequences. For example, a presentation structure comprising neuropeptide head activator peptides comprises: SKVILFE/D(X)nSKVILFE/D (SEQ ID NO:112); wherein X represents any amino acid, and n is at least about 8 amino acids. Linkers or spacers as known in the art are optionally placed between the HAv peptides and the variable ((X)n) region and optionally placed at the amino and carboxy terminus of the structure.
Positioning of peptides of between two SKVILFE(D) sequences makes the peptides constrained. Upon SKVILFE(D) -SKVILFE(D) intramolecular interaction, the peptide structure is converted from linear to a loop or mini-domain such that the peptide becomes structurally constrained. This provides three important advantages. First, peptides in constrained conformations usually have higher affinity for a ligand than the same peptide in linear conformation. Second, the constrained peptide has a higher protease resistance than the linear peptide as a result of the formation of a "rigid surface" structure. Third, and more importantly, transfer of a constrained peptide as a cassette to a protein carrier will not dramatically change its original conformation (and consequently its binding activity) while a linear peptide is more likely to show a decrease in binding activity.
In a preferred embodiment, the fusion partner is a targeting sequence. As will be appreciated by those in the art, the localization of proteins within a cell is a simple method for increasing effective concentration. The localization of a peptide to a specific cellular location, such as a membrane, limits its search for its ligand to that limited dimensional space. Alternatively, the concentration of a protein or polypeptide can also be simply increased by nature of the localization. For example, shuttling the fluorettes into the nucleus confines them to a smaller space thereby increasing the concentration. Finally, the ligand or target may simply be localized to a specific compartment. As will be appreciated by those in the art when a fluorette is fused to another protein, as outlined herein, the targeting sequence can be fused to the protein partner as well
Thus, suitable targeting sequences include, but are not limited to, binding sequences capable of causing binding of the fluorette to a predetermined molecule or class of molecules while retaining activity of the fluorette, (for example by using enzyme inhibitor or substrate sequences to target a class of relevant enzymes), sequences signalling selective degradation, of itself or co-bound proteins, and signal sequences capable of constitutively localizing the fluorette to a predetermined cellular locale, including a) subcellular locations such as the Golgi, endoplasmic reticulum, nucleus, nucleoli, nuclear membrane, mitochondria, chloroplast, secretory vesicles, lysosome, and cellular membrane, and b) extracellular locations via a secretory signal Particularly preferred is localization to either subcellular locations or to the outside of the cell via secretion
In a preferred embodiment, the targeting sequence is a nuclear localization signal (NLS) NLSs are generally short, positively charged (basic) domains that serve to direct the entire protein in which they occur to the cell's nucleus Numerous NLS ammo acid sequences have been reported including single basic NLS's such as that of the SV40 (monkey virus) large T Antigen (Pro Lys Lys Lys Arg Lys Val) (SEQ ID NO 54), Kalderon (1984), et al , Cell, 39 499-509, the human retinoic acid receptor-β nuclear localization signal (ARRRRP) (SEQ ID NO 55), NFKB p50 (EEVQRKRQKL SEQ ID NO 56), Ghosh et al , Cell 62 1019 (1990), NFKB p65 (EEKRKRTYE (SEQ ID NO 57, Nolan et al , Cell 64 961 (1991), and others (see for example Boulikas, J Cell Biochem 55(1) 32-58 (1994), hereby incorporated by reference) and double basic NLS's exemplified by that of the Xenopus (African clawed toad) protein, nucleoplasmin (Ala Val Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gin Ala Lys Lys Lys Lys Leu Asp) (SEQ ID NO 58), Dmgwall, et al , Cell, 30 449-458, 1982 and Dmgwall, et al , J Cell Biol , 107 641-849, 1988) Numerous localization studies have demonstrated that NLSs incorporated into peptides or grafted onto proteins not normally targeted to the cell nucleus cause these peptides and proteins to be concentrated in the nucleus See, for example, Dmgwall, and Laskey, Ann, Rev Cell Biol , 2 367- 390, 1986, Bonnerot, et al , Proc Natl Acad Sci USA, 84 6795-6799, 1987, Galileo, et al , Proc Natl Acad Sci USA, 87 458-462, 1990
In a preferred embodiment, the targeting sequence is a membrane anchoring signal sequence This is particularly useful since many parasites and pathogens bind to the membrane, in addition to the fact that many intracellular events originate at the plasma membrane Thus, membrane- bound fluorettes are useful for both the identification of important elements in these processes as well as for the discovery of effective inhibitors The invention provides methods for presenting the fluorettes extracellularly or in the cytoplasmic space For extracellular presentation, a membrane anchoring region is provided at the carboxyl terminus of the peptide presentation structure In another embodiment, for extracellular presentation, a membrane anchoring region is provided at the ammo terminus of the peptide presentation structure Preferably, the ammo terminal anchoring region functions as the anchoring region of a Type 2 glycoprotem The fluorette is expressed on the cell surface and presented to the extracellular space, such that it can bind its target fluorophore The binding of a fluorette, espescially when fused to a polypeptide or protein could confer a function on the cells expressing the fluorette The cytoplasmic region could be neutral or could contain a domain that, when the fluorette is bound, confers a function on the cells (activation of a kinase, phosphatase, binding of other cellular components to effect function) Similarly, the fluorette could be contained within a cytoplasmic region, and the transmembrane region and extracellular region remain constant or have a defined function
Membrane-anchoring sequences are well known in the art and are based on the geometry of mammalian transmembrane molecules Peptides are inserted into the membrane based on a signal sequence (designated herein as ssTM) and require a hydrophobic transmembrane domain (herein TM) The transmembrane proteins are inserted into the membrane such that the regions encoded 5' of the transmembrane domain are extracellular and the sequences 3' become intracellular Of course, if these transmembrane domains are placed 5' of the variable region, they will serve to anchor it as an intracellular domain, which may be desirable in some embodiments ssTMs and TMs are known for a wide variety of membrane bound proteins, and these sequences may be used accordingly, either as pairs from a particular protein or with each component being taken from a different protein, or alternatively, the sequences may be synthetic, and derived entirely from consensus as artificial delivery domains
As will be appreciated by those in the art, membrane-anchoring sequences, including both ssTM and TM, are known for a wide variety of proteins and any of these may be used Particularly preferred membrane-anchoring sequences include, but are not limited to, those derived from CD8,
ICAM-2, IL-8R, CD4 and LFA-1
Useful sequences include sequences from 1) class I integral membrane proteins such as IL-2 receptor beta-chain (residues 1-26 are the signal sequence, 241-265 are the transmembrane residues, see Hatakeyama et al , Science 244 551 (1989) and von Heijne et al, Eur J Biochem 174 671 (1988)) and insulin receptor beta chain (residues 1-27 are the signal, 957-959 are the transmembrane domain and 960-1382 are the cytoplasmic domain, see Hatakeyama, supra, and Ebina et al , Cell 40 747 (1985)), 2) class II integral membrane proteins such as neutral endopeptidase (residues 29-51 are the transmembrane domain, 2-28 are the cytoplasmic domain, see Malfroy et al , Biochem Biophys Res Commun 144 59 (1987)), 3) type III proteins such as human cytochrome P450 NF25 (Hatakeyama, supra); and 4) type IV proteins such as human P- glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 and ICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at the extreme 5' end of the transcript. These consist of the amino acids 1-32 in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP; (SEQ ID NO:59) Nakauchi et al., PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2
(MSSFGYRTLTVALFTLICCPG (SEQ ID NO:60); Staunton et al., Nature (London) 339:61 (1989)). These leader sequences deliver the fluorette to the membrane while the hydrophobic transmembrane domains, placed 3' of the fluorette region, serve to anchor the construct in the membrane. These transmembrane domains are encompassed by amino acids 145-195 from CD8 (PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR (SEQ ID N0.61);
Nakauchi, supra) and 224-256 from ICAM-2 (MVIIVTWSVLLSLFVTSVLLCFIFGQHLRQQR (SEQ ID NO:62); Staunton, supra).
Alternatively, membrane anchoring sequences include the GPI anchor, which results in a covalent bond between the molecule and the lipid bilayer via a glycosyl-phosphatidylinositol bond for example in DAF (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO:63), with the bolded serine the site of the anchor; see Homans et al., Nature 333(6170):269-72 (1988), and Moran et al., J. Biol. Chem. 266:1250 (1991)). In order to do this, the GPI sequence from Thy-1 can be cassetted 3' of the fluorette in place of a transmembrane sequence.
Similarly, myristylation sequences can serve as membrane anchoring sequences. It is known that the myristylation of c-src recruits it to the plasma membrane. This is a simple and effective method of membrane localization, given that the first 14 amino acids of the protein are solely responsible for this function: MGSSKSKPKDPSQR (SEQ ID NO:64) (see Cross et al., Mol. Cell. Biol. 4(9): 1834 (1984); Spencer et al., Science 262:1019-1024 (1993), both of which are hereby incorporated by reference). This motif has already been shown to be effective in the localization of reporter genes and can be used to anchor the zeta chain of the TCR. This motif is placed 5' of the fluorette in order to localize it to the plasma membrane. Other modifications such as palmitoylation can be used to anchor fluorettes in the plasma membrane; for example, palmitoylation sequences from the G protein-coupled receptor kinase GRK6 sequence (LLQRLFSRQDCCGNCSDSEEELPTRL (SEQ ID NO:65), with the bold cysteines being palmitolyated; Stoffel et al., J. Biol. Chem 269:27791 (1994)); from rhodopsin
(KQFRNCMLTSLCCGKNPLGD (SEQ ID NO:66); Bamstable et al., J. Mol. Neurosci. 5(3):207 (1994)); and the p21 H-ras 1 protein (LNPPDESGPGCMSCKCVLS (SEQ ID NO:67); Capon et al., Nature 302:33 (1983)).
In a preferred embodiment, the targeting sequence is a lysozomal targeting sequence, including, for example, a lysosomal degradation sequence such as Lamp-2 (KFERQ (SEQ ID NO:68); Dice, Ann. N.Y. Acad. Sci. 674:58 (1992); or lysosomal membrane sequences from Lamp-1 (MLIPIAGFFALAGL W /AY /GRKRSHAGYQTI (SEQ ID NO:69), Uthayakumar et al., Cell. Mol. Biol. Res. 41 :405 (1995)) or Lamp-2 (LVPIA VGAALA G VLIL VLLA YF/GLKH H H AG YEQ F (SEQ ID NO:70), Konecki et la., Biochem. Biophys. Res. Comm. 205:1-5 (1994), both of which show the transmembrane domains in italics and the cytoplasmic targeting signal underlined).
Alternatively, the targeting sequence may be a mitrochondrial localization sequence, including mitochondrial matrix sequences (e.g. yeast alcohol dehydrogenase III;
MLRTSSLFTRRVQPSLFSRNILRLQST (SEQ ID NO:71 ); Schatz, Eur. J. Biochem. 165:1-6 (1987)); mitochondrial inner membrane sequences (yeast cytochrome c oxidase subunit IV;
MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO:72); Schatz, supra); mitochondrial intermembrane space sequences (yeast cytochrome d ;
MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSLTAEAMTA
(SEQ ID NO:73); Schatz, supra) or mitochondrial outer membrane sequences (yeast 70 kD outer membrane protein; MKSFITRNKTAILATVAATGTAIGAYYYYNQLQQQQQRGKK (SEQ ID NO:74);
Schatz, supra).
The target sequences may also be endoplasmic reticulum sequences, including the sequences from calreticulin (KDEL; Pelham, Royal Society London Transactions B; 1-10 (1992)) or adenovirus E3/19K protein (LYLSRRSFIDEKKMP (SEQ ID NO:75); Jackson et al., EMBO J. 9:3153 (1990)).
Furthermore, targeting sequences also include peroxisome sequences (for example, the peroxisome matrix sequence from Luciferase; SKL; Keller et al., PNAS USA 4:3264 (1987)); farnesylation sequences (for example, P21 H-ras 1 ; LNPPDESGPGCMSCKCVLS (SEQ ID NO:76), with the bold cysteine farnesylated; Capon, supra); geranylgeranylation sequences (for example, protein rab-5A; LTEPTQPTRNQCCSN (SEQ ID NO:77), with the bold cysteines geranylgeranylated; Farnsworth, PNAS USA 91 :11963 (1994)); or destruction sequences (cyclin B1; RTALGDIGN (SEQ ID NO:78); Klotzbucher et al., EMBO J. 1 :3053 (1996)).
In a preferred embodiment, the targeting sequence is a secretory signal sequence capable of effecting the secretion of the fluorette. There are a large number of known secretory signal sequences which are placed 5' to the fluorette sequence and are cleaved from the peptide region to effect secretion into the extracellular space. Secretory signal sequences and their transferability to unrelated proteins are well known, e.g., Silhavy, et al. (1985) Microbiol. Rev. 49, 398-418. This is particularly useful to generate a fluorette that is expressed at the surface of a cell In a preferred approach, a fusion product is configured to contain, in series, secretion signal peptide-presentation structure-flourette-presentation structure In this manner, target cells grown in the vicinity of cells caused to express the fluorettes, are bathed in secreted fluorette Target cells exhibiting a phenotypic change in response to the presence of a fluorette, or internalization of the fluorette and binding to intracellular targets, are localized by any of a variety of methods, such as, FRET analysis (Selvm et al Methods Enzymol 246 300-334 (1995)) as described below
Suitable secretory sequences are known, including signals from IL-2
(MYRMQLLSCIALSLALVTNS (SEQ ID NO 79), Villmger et al , J Immunol 155 3946 (1995)), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT (SEQ ID NO 80), Roskam et al , Nucleic Acids Res 7 30 (1979)), prepromsulm (MALWMRLLPLLALLALWGPDPAAAFVN (SEQ ID NO 81), Bell et al , Nature 284 26 (1980)), and influenza HA protein (MKAKLLVLLYAFVAGDQI (SEQ ID NO 82), Sekiwawa et al , PNAS 80 3563)), with cleavage between the non-underlmed- underlmed junction A particularly preferred secretory signal sequence is the signal leader sequence from the secreted cytokme IL-4, which comprises the first 24 ammo acids of IL-4 as follows MGLTSQLLPPLFFLLACAGNFVHG (SEQ ID NO 83)
In a preferred embodiment, the fusion partner is a rescue sequence A rescue sequence is a sequence which may be used to purify or isolate the fluorette (or, in some cases, the nucleic acid encoding it) Thus, for example, peptide rescue sequences include purification sequences such as the Hιs6 tag for use with Ni affinity columns and epitope tags for detection, immunoprecipitation or FACS (fluorescence-activated cell sorting) Suitable epitope tags include myc (for use with the commercially available 9E10 antibody), the BSP biotmylation target sequence of the bacterial enzyme BirA, flu tags, lacZ, and GST
In a preferred embodiment, a fluorette sequence functions as a rescue sequence The fluorette because it binds a fluorophore dye can be used to purify or isolate a molecule to which it is fused or attached by affinity chromatography using fluorophore dye columns If the fluorette alters the excitation or emission spectrum of the bound fluorophore dye, as described below, this difference can be used to monitor attachment and/or elution of the fluorette from the affinity column Alternatively, the fluorette/dye complex can be used in FACS If desired, a linker joining the fluorette to its fusion partner also contains a convenient site or sites to seperate the fluorette from the fusion partner, such as, a unique protease recognition sequences Alternatively, the affinity column can contain antibody reactive with the fluorette
In a preferred embodiment, the fusion partner is a stability sequence to confer stability to the fluorette Thus, for example, fluorettes may be stabilized by the incorporation of glycmes after an amino-terminal methionine (MG or MGGO), for protection of the fluorette to ubiquitination as per Varshavsky's N-End Rule, thus conferring long half-life in the cytoplasm. Similarly, two prolines at the C-terminus impart upon peptides resistance to carboxypeptidase action. The presence of two glycines prior to the prolines impart both flexibility and prevent structure initiating events in the di- proline to be propagated into the fluorette structure. Thus, preferred stability sequences are as follows: MG(X)nGGPP (SEQ ID NO:84), where (X)n is a fluorette of any amino acid and n is an integer of at least about 8, as outlined above for fluorette length.
In a preferred embodiment, the fusion partner is a dimerization sequence. A dimerization sequence allows the non-covalent association of one fluorette to another fluorette, with sufficient affinity to remain associated under normal physiological conditions. The dimers may be homo- or heterodimers.
Dimerization sequences may be a single sequence that self-aggregates, or two sequences. That is, a first fluorette with dimerization sequence 1 , and a second fluorette with dimerization sequence 2, such that upon introduction into a cell and expression of the nucleic acids, dimerization sequence 1 associates with dimerization sequence 2 to form new random peptide structures. In addition, dimerization sequences can be used as presentation structures. That is, by putting a first dimerization sequence at one terminus of the fluorette, and a second dimerization sequence at the other terminus, similar to some other presentation structures, a "cyclized" fluorette can be made.
Suitable dimerization sequences will encompass a wide variety of sequences. Any number of protein-protein interaction sites are known. In addition, dimerization sequences may also be elucidated using standard methods such as the yeast two hybrid system, and traditional biochemical affinity binding studies, or even using the present methods.
The fusion partners may be placed anywhere (i.e. N-terminal, C-terminal, internal) in the structure as the biology and activity permits, although in general, N- or C-terminal fusions are preferred to fusions internal to the fluorette sequence.
In another embodiment, a fluorette occupies an internal position within the fusion partner as the biology and activity permits. This will allow a means to measure access of the fluorette for its fluorophore dye as a means of determining structure and function of the region in which the fluorette resides.
In a preferred embodiment, the fusion partner includes a linker or tethering sequence. Linker sequences between various targeting sequences (for example, membrane targeting sequences) and the other components of the constructs and fluorettes may be desirable to allow the candidate agents to interact with potential targets unhindered For example, useful linkers include glycme- serme polymers (including, for example, (GS)n (SEQ ID NO 85), (GSGGS)n (SEQ ID NO 86) and (GGGS)n (SEQ ID NO 87), where n is an integer of at least one), glycme-alanine polymers, alanine-seπne polymers, and other flexible linkers such as the tether for the shaker potassium channel, and a large variety of other flexible linkers, as will be appreciated by those in the art Glycine-serme polymers are preferred since both of these ammo acids are relatively unstructured, and therefore may be able to serve as a neutral tether between components Secondly, senne is hydrophilic and therefore able to solubilize what could be a globular glycine chain Third, similar chains have been shown to be effective in joining subunits of recombmant proteins such as single chain antibodies As is more fully outlined below, linkers such as these may also be used in some embodiments between fluorettes and the moiety to which they are fused
In alternative embodiments linkers can also be sequences that are derived from other proteins and have no structure or an assumed structure In another embodiment the linkers distance the fluorette from a molecule to which they are attached, for example, a fusion protein
In addition, the fusion partners, including presentation structures, may be modified, randomized, and/or matured to alter the presentation orientation of the fluorette For example, determinants at the base of the loop may be modified to slightly modify the internal loop tertiary structure, which maintains the fluorette ammo acid sequence
In a preferred embodiment, combinations of fusion partners are used Thus, for example, any number of combinations of presentation structures, targeting sequences, rescue sequences, and stability sequences may be used, with or without linker sequences
In a preferred embodiment, the fluorette is fused to a target analyte, for example, to monitor or follow the target analyte, as is more fully described below By "target analyte" or "analyte" or grammatical equivalents herein is meant any molecule, compound or particle to be detected As will be appreciated by those in the art, a large number of analytes may be detected using the present methods, basically, any target analyte to which a fluorette may be attached may be detected using the methods of the invention
Suitable analytes include organic and inorganic molecules, including biomolecules In a preferred embodiment, the analyte may be an environmental pollutant (including pesticides, insecticides, toxins, etc ), a chemical (including solvents, polymers, organic materials, etc ), therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc ), biomolecules (including hormones, cytokmes, proteins, lipids, carbohydrates, cellular membrane antigens and receptors (neural, hormonal, nutrient, and cell surface receptors) or their ligands, etc), whole cells (including procaryotic (such as pathogenic bacteria) and eukaryotic cells, including mammalian tumor cells), viruses (including retroviruses, herpesviruses, adenoviruses, lentiviruses, etc ), and spores, etc Particularly preferred analytes are proteins
In a preferred embodiment, the target analyte is a protein and the fluorette is made as a fusion protein, using techniques well known in the art For example, the nucleic acid encoding the fluorette is linked in-frame with a nucleic acid encoding a target protein to produce a fusion nucleic acid Preferably, fusion proteins are produced by culturing a host cell transformed with an expression vector containing the fusion nucleic acid, under the appropriate conditions to induce or cause expression of the fusion protein The conditions appropriate for fusion protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation For example, the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an mducible promoter requires the appropriate growth conditions for induction In addition, in some embodiments, the timing of expression is important
Alternatively, fluorettes can be produced by in vitro transcription of the encoding nucleic acid and translation of the RNA transcript, as known in the art
In a preferred embodiment, the target analyte is other than a protein, and the fusion of the fluorette to the target analyte is generally done chemically In general, the fluorette and the target analyte are attached through the use of functional groups on each that can then be used for attachment Preferred functional groups for attachment are ammo groups, carboxy groups, oxo groups and thiol groups These functional groups can then be attached, either directly or indirectly through the use of a linker Linkers are well known in the art, for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference) In an additional embodiment, carboxyl groups (either from the surface or from the candidate agent) may be denvatized using well known linkers (see the Pierce catalog) For example, carbodiimides activate carboxyl groups for attack by good nucleophiles such as amines (see Torchilin et al , Critical Rev Therapeutic Drug Carrier Systems, 7(4) 275-308 (1991 ), expressly incorporated herein) Protemaceous species may also be attached using other techniques known in the art, for example for the attachment of antibodies to polymers, see Slinkm et al , Bioconj Chem 2 342-348 (1991 ), Torchilin et al., supra; Trubetskoy et al., Bioconj. Chem. 3:323-327 (1992); King et al., Cancer Res. 54:6176-6185 (1994); and Wilbur et al., Bioconjugate Chem. 5:220-235 (1994), all of which are hereby expressly incorporated by reference). It should be understood that the fluorettes and target analytes may be attached in a variety of ways, including those listed above. What is important is that manner of attachment does not significantly alter the functionality of the fluorette (and preferably the target analyte as well); that is, the attachment should be done in such a flexible manner as to allow the interaction of the fluorette peptide with a dye, and the target analyte to its binding partners.
In a preferred embodiment the fluorette will bind to Texas Red. Suitable fluorettes in this embodiment include, but are not limited to, KHVQYWTQMFYS (SEQ ID NO:1 ); DFLQWKLARQKP
(SEQ ID NO:2); KPVQYWTQMFYT (SEQ ID NO:15); KPAQYWTQMFYS (SEQ ID NO:16);
KNVQYWTQMFYT (SEQ ID NO:17); KHVQYWTHMFYT (SEQ ID NO:18); KHVQYWTQMFYT
(SEQ ID NO:19); NHVHYWTQMFYS (SEQ ID NO:20); THVQYWTQMFYS (SEQ ID NO:21);
RTIWEPKEASNHT (SEQ ID NO:105): WSKMGHTVT (SEQ ID NO:106); RWTWEPISE (SEQ ID NO:107); GNQKCLQHNRCST (SEQ ID NO:108); SQTWSFPEH (SEQ ID NO:109);
EPMARPWERKQDR (SEQ ID NO:110); and GTLSATRPYGRQW (SEQ ID NO:111).
In a preferred embodiment the fluorette will bind to Rhodamine. Suitable fluorettes in this embodiment include, but are not limited to, IPHPPMYWTRVF (SEQ ID NO:3); IPHRPMYWTPVF (SEQ ID NO:22); and LPHPPMYWTRVF (SEQ ID NO:23).
In a preferred embodiment the fluorette will bind to Oregon Green 514. Suitable fluorettes in this embodiment include, but are not limited to, HGWDYYWDWTAW (SEQ ID NO:4); ASDYWDWEWYYS (SEQ ID NO:5); YPNDFEWWEYYF (SEQ ID NO:6); HTSHISWPPWYF (SEQ ID NO:7); LEPRWGFGWWLK (SEQ ID NO:8); QYYGWYYDHNFW (SEQ ID NO:9); YMYDEYQYWNFW (SEQ ID NO:10); HEWEYYWDWTAW (SEQ ID NO:24); HEWDYYWDWTAW (SEQ ID NO:25); HGWDYYWDWTDW (SEQ ID NO:26); HGWDYYWDWTPW (SEQ ID NO:27); HGWDYYWDWTTW (SEQ ID NO:28); HGWDYNWDWTAW (SEQ ID NO:29); and QGWDYYWDWTAW (SEQ ID NO:30).
In a preferred embodiment the fluorette will bind to Fluorescein. Suitable fluorettes in this embodiment include, but are not limited to, YPNDFEWWEYYF (SEQ ID NO:6); ASDYWDWEWYYS (SEQ ID NO:5); WYDDWNDWHAWP (SEQ ID NO:11); WHMSPSWGWGYW
(SEQ ID NO:12); HMSWWEFYLVPP (SEQ ID NO:13); YWDYSWHYYAPY (SEQ ID NO:14); YPNEFDWWDYYY (SEQ ID NO:31); YPNDFEWWDYYY (SEQ ID NO:32); YHNDYEWWEYYY (SEQ ID NO:33); YPNDFEWWEYYY (SEQ ID NO:34); YPNDFDWWEYYL (SEQ ID NO:35); YTHDYEWWEYYF (SEQ ID NO:36); YPNDYEWWEYYF (SEQ ID NO:37); YPSDFEWWEYYF (SEQ ID NO:38); YHDFEWWEYYF (SEQ ID NO:39); and YPYDFEWWEYYM (SEQ ID NO:40).
Included within the definition of fluorettes are derivative or variant fluorettes. Accordingly, as used herein, a peptide is a fluorette if the overall homology of the peptide sequence to the amino acid sequences shown above (SED ID NOS 1-40 and 104-110) is preferably greater than about 70%, more preferably greater than about 75% even more preferably greater than about 80% and most preferably greater than 90%, with homologies of greater than 95 to 98% being especially preferred. Homology in this context means sequence similarity or identity, with identity being preferred.
This homology will be determined using standard techniques known in the art, including, but not limited to, the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biool. 48:443 (1970), by the search for similarity method of Pearson & Lipman, PNAS USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wl), the Best Fit sequence program described by Devereux ef al., Nucl. Acid Res. 72:387-395 (1984), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1 ; gap penalty of 1 ; gap size penalty of 0.33; and joining penalty of 30, "Current Methods in Sequence Comparison and
Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.
One example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol.
Biol. 215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzvmoloαv. 266: 460-480 (1996); http://blast.wustl/edu/blast/ README.html]. WU-BLAST-2 uses several search parameters, most of which are set to the default values. The adjustable parameters are set with the following values: overlap span =1 , overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "shorter" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
An additional useful algorithm is gapped BLAST as reported by Altschul ef al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62 substitution scores; threshold T parameter set to 9; the two-hit method to trigger ungapped extensions; charges gap lengths of k a cost of 10+/c; Xu set to 16, and Xg set to 40 for database search stage and to 67 for the output stage of the algorithms. Gapped alignments are triggered by a score corresponding to -22 bits.
A percent amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).
Alternatively, percent sequence identity is determined by inspection in which only identities are scored positively (+1) and all forms of sequence variation including gaps are assigned a value of "0". Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one having the most actual residues in the aligned region.
Once identified, the amino acid sequences of the fluorettes may be modified as needed. Sequence modifications include substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the fluorette are desired, substitutions are generally made in accordance with the following chart: Chart 1
Original Residue Exemolarv Substitutions
Ala Ser Arg Lys Asn Gin, His Asp Glu Cys Ser Gin Asn Glu Asp Gly Pro His Asn, Gin lie Leu, Val Leu lie, Val Lys Arg, Gin, Glu Met Leu, He Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val lie, Leu
Substantial changes in function are made by selecting substitutions that are less conservative than those shown in Chart I, although these generally are not preferred. For example, substitutions may be made which more significantly affect the structure of the fluorette backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the fluorette's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue having an electropositive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
In a preferred embodiment, not more than about three substitutions or deletions will be made, and that the change will not be more than about 20 number %, usually not more than about 10 number %, of the number of amino acids in the fluorette, although in some instances higher numbers of alterations may be made.
Similarly, if the function or affinity of the fluorette is to be decreased, the amount of changes may also be greater Preferred are conservative substitutions, as known in the art, including substitutions within the large hydrophobic group isoleucme, leucme, valme and phenylalanme between senne and threonme, glycine and alanme, asparagme and glutamine, aspartic acid and glutamic acid, or lysme, argmine and histidme In some embodiments, non-conservative alterations are done
As will be appreciated by those skilled in the art, a number of methods exist for the creation, isolation, or selection of fluorettes from a library of sequences (Oldenburg ef al Proc Natl Acad Sci U S A 89 5393-5397 (1992), Scott ef al , Proc Natl Acad Sci U S A 89 5398-5402 (1992), Cull ef al , Proc Natl Acad Sci U S A 89 1865-1869 (1992), Devlin ef al Science 249 404-406 (1990), Schatz Biotechnology 11 1138-1143 (1993)) In preferred embodiments, such libraries are based on, for example, either bacteπophage (Matthews and Wells Science 260 1113-1117 (1993), Koivunen et al Biotechnology 13 265-270 (1995), Yu and Smith Methods Enzymol 267 3- 27 (1996), Harrison ef al Methods Enzymol 267 83-109 (1996), Rebar and Pabo Science 263 671-673 (1994), Rebar et al , Methods Enzymol 267 129-149 (1996), Saggio and Laufer Biochem J 293 613-616 (1993), Katz Biochemistry 34 15421-15429 (1995), Caparon ef al Mol Divers 1 241-246 (1996), Parmley and Smith Gene 73 305-318 (1988)), for example M13 or plasmids (Cull ef al Proc Natl Acad Sci U S A 89 1865-1869 (1992), Schatz ef al Methods Enzymol 267 171-191 (1996), ) or polysomes (Mattheakis ef al Proc Natl Acad Sci U S A 91 9022-9026, Hanes and Pluckthun Proc Natl Acad Sci U S A 94 4937-4942, Roberts and Szostak Proc Natl Acad Sci U S A 94 12297-12301)) Alternatively, peptide libraries can be expressed on the surface of a eukaryotic or prokaryotic cell and directly bound to a fluorophore dye and sorted, for example, by FACS Alternatively, the cell expressing a fluorette is bound to a fluorophore dye that is bound to bead or a magnetic bead and purified and amplified as known in the art
In a preferred embodiment, fluorettes are created, isolated, or selected from aptamer library phage display approaches Such libraries are based on, for example, M13 filamentous bactenophage (Smith Science 228 1315-1317 (1985), Cwirla ef al Proc Natl Acad Sci U S A 87 6378-6382 (1990), Devlin et al Science 249 404-406 (1990), Scott and Smith Science 249 386-390 (1990)) In a preferred embodiment, fluorettes are identified by screening a phage display peptide library that contains a combinatorial library of peptides interposed betwen the leader sequence and the ammo terminus of mature form of bactenophage M13amp9 minor coat protein, pill
The recombinatoπal library can be produced by a number of methods as known m the art In a preferred embodiment, the library is produced by annealing and cloning an oligonucleotide library of sequences into the M13amp9 pill gene Annealing of the oligonucleotide library to produce double-stranded DNA preferably produces "sticky ends" to facilitate directional cloning into the appropriate reading frame of the M13amp9 Alternatively, the annealed oligonucleotide library contains unique restriction enzyme sites, which are digested to produce "sticky ends" to facilitate directional cloning The oligonucleotide library preferably also encodes an in-frame linker, tethering sequence, or spacer as described herein The function of the linker is to decrease or minimize steπc hindrance and promote accessibility of the fluorette for its fluorophore dye The linker also promotes structural and functional independence of the fluorette Phage particles bearing the peptide library are produced by introducing the M13amp9 vector into a permissive bacterial host, such as, Eschenchia coli and other gram negative bacteria According to the above strategy for cloning the peptide library, during phage maturation, the leader secretory sequence of pill is removed, resulting in the peptide library sequence positioned immediately at the amino- termmus of the mature protein
Cloning and expression of the oligonucleotide library produces a library of peptides either directly fused to the pill protein or, preferably, fused via a linker or spacer The linker or spacer can be, for example, sequence, as described herein, interposed between the fluorette and the pill protein sequences
The nucleic acids which give rise to the fluorettes are chemically synthesized, and thus may incorporate any nucleotide at any position Thus, when the nucleic acids are expressed to form peptides, any ammo acid residue may be incorporated at any position The synthetic process can be designed to generate randomized nucleic acids, to allow the formation of all or most of the possible combinations over the length of the nucleic acid, thus forming a library of randomized nucleic acids and peptides In an alternative embodiment, random DNA fragments may be made from fragmentation of genomic DNA, followed by sizing, and ligation into an appropriate vector for expression and selection
The library should provide a sufficiently structurally diverse population of randomized expression products to effect a probabilistically sufficient range to allow isolation of a fluorette Accordingly, a library must be large enough so that at least one of its members will have a structure that gives it affinity for a fluorophore dye Although it is difficult to gauge the required absolute size of a library, nature provides a hint with the immune response a diversity of 107-108 different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organism Published in vitro selection techniques have also shown that a library size of 107 to 108 is sufficient to find structures with affinity for the target A library of all combinations of a peptide from about 7 to 20 ammo acids in length has the potential to code for 207 (109) to 2020 Thus, with libraries of 107 to 108 per ml of phage particles the present methods allow a "working" subset of a theoretically complete interaction library for 7 ammo acids, and a subset of shapes for the 2020 library Thus, in a preferred embodiment, at least 106, preferably at least 108, more preferably at least 1010 and most preferably at least 1012 different expression products are simultaneously analyzed in the subject methods Preferred methods maximize library size and diversity
It is important to understand that in any library system encoded by oligonucleotide synthesis one cannot have complete control over the codons that will eventually be incorporated into the peptide structure This is especially true in the case of codons encoding stop signals (TAA, TGA, TAG) In a synthesis with NNN as the random region, there is a 3/64, or 4 69%, chance that the codon will be a stop codon Thus, in a peptide of 10 residues, there is an unacceptable high likelihood that 46 7% of the peptides will prematurely terminate For free peptide structures this is perhaps not a problem But for larger structures, such as those envisioned here, such termination will lead to sterile peptide expression To alleviate this, random residues are encoded as NNK, where K= T or G This allows for encoding of all potential ammo acids (changing their relative representation slightly), but importantly preventing the encoding of two stop residues TAA and TGA Thus, libraries encoding a 10 ammo acid peptide will have a 15 6% chance to terminate prematurely For candidate nucleic acids which are not designed to result in peptide expression products, this is not necessary In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position
Once generated a peptide library is screened for binding to a fluorescent dye In a preferred embodiment, bactenophage expressing fluorettes are selected by biopanning, which consists of four sequential steps i) phage binding with a fluorophore dye carrier, n) removal of unbound or weakly bound phage, in) elution of bound phage and (iv) amplification of bound phage Certain steps can be omitted, for example, such as washing and amplification if the peptide library is expressed in eukaryotic or mammalian cells and fluorescence is detected and cells are shorted according to fluorescence or spectral shifts If necessary or desirable, the amplified phage are used for the subsequent round(s) of biopanning against the corresponding fluorophore dye carrier until apparent enrichment for binding is observed over background Preferably, after about four rounds of biopanning, phage bearing sequences that bind specifically to a fluorophore dye can be identified Following biopanning, the individual phage are isolated, amplified and the sequence of the fluorette can be determined by various methods as known in the art In another embodiment, eukaryotic or prokaryotic cells expressing a peptide library on their surface can be sorted by FACS and/or magnetic beads to isolate cells expressing fluorettes
For biopanning, the fluorophore dyes can be bound to solid-phase carriers In a preferred embodiment, the dyes are covalently attached to a target bead This facilitates washing and removal of non-specifically bound or weakly bound phage Preferably, a linker or spacer molecule is interposed between the fluorophore dye and the target bead, for example, succinimidy esters and derivatives of fluorophores A spacer molecule increases the accessibility of the fluorophore dye and minimizes potential steπc hindrance that may interfere with the interactions between the fluorophore dye and the bactenophage particles that bear the peptide library The number of fluorophore dye molecules bound to the carrier will vary In general, there are up to about 1 micromole Higher or lower amounts of dye can be attached per ml of carrier beads, as needed, for example to increase or decrease the stringency of the biopanning conditions
Once a fluorette is identified, it can be mutagenized or denvatized to isolate fluorettes with altered properties In a preferred embodiment, the deπvatize fluorettes have a higher affinity or specificity, thereby, providing, for example, increased sensitivity for the assay or system in which the fluorette is employed This also allows the use of lower amounts of fluorescent dye, thereby, limiting toxicity of the fluorescent dye to cells, if needed
In a preferred embodiment, affinity maturation (Yu and Smith supra) can be used to produce mutant or derivative fluorettes with improved affinities over their corresponding parental fluorettes To create, select or isolate fluorettes with a higher affinity, a second oligonucleotide library is produced that is, preferabbly, based That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities For example, in a preferred embodiment, the nucleotides or ammo acid residues are randomized within a defined class, for example, of hydrophobic ammo acids, hydrophilic residues, steπcally biased (either small or large) residues, towards the creation of cystemes, for cross-linking, prolines for SH-3 domains, seπnes, threonmes, tyrosines or histidmes for phosphorylation sites, etc , or to purmes, etc
In a preferred embodiment, the ammo acid sequences of the fluorettes are subjected to biased mutagenesis at a desired mutagenesis rate using a number of methods as known in the art The sequences can be mutagenized by systemmatic alteration of the codons encoding the fluorettes Oligonucleotide mutagenesis, is preferablly, performed during the synthesis of the oligonucleotide library To increase the probability that all amino acids will be represented at each position and to limit the generation of stop codons within the fluorette encoding sequences, specific nucleotides, such as A and C, can be omitted from the third position of each codon As described above, the oligonucleotide library is preferably annealed and cloned into an expression vector of choice
To select fluorettes with higher binding affinity than the parental fluorette, the biopanning conditions are preferably altered to be more stringent by, for example, changing one or more of the following conditions This also provides a method to maximize selection against the parental fluorette To increase the stringency of the biopanning, the concentration of the fluorophore dye bound to the carrier beads can be decreased In addition, the phage concentration represented in the binding step and/or the binding time also can be reduced Alternatively, the stringency of the washing conditions is increased Washing stringency can be increased by, for example, increasing the volume of washing buffer per ml of carrier beads, increasing the wash temperature, altering the ionic strength or pH of the wash buffer, increasing the detergent concentration or by using a stronger, ionizing detergent (Sambrook ef al , (1989) Molecular Cloning A Laboratory Manual (2nd Edition) Cold Spring Harabor Laboratory, Cold Spring Harbor, NY) Other parameters that can be altered to increase stringency are, for example, increasing the temperature and competition for binding to the fluorophore dye by related molecules
Fluorette binding to a fluorophore dye can be assessed in several ways For example, when a phage display system is used, binding of the bacteπophage-beaπng peptide library to the fluorophore dye-carrier can be determined by comparing the infectious particle titers of input phage to eluted phage To measure phage-fluorophore dye binding in solution, a known concentration of free fluorophore dye is mixed with a known number of purified phage particles The number of particles is preferably calculated from the optical densities of the puπfed phage at 260 nm and 280 nm but other methods, such as, infectious titer can be used Phage-fluorophore dye complexes are separated by, for example, precipitation to remove unbound dye, spotted onto a solid-support filter, such as, nitrocellulose and scanned for fluorescence Other methods to remove unbound dye include, for example, competition with related molecules, washing, and increased stringency To determine nonspecific binding, controls using phage particles that do not display recombinatonal library sequences are run in parallel In addition, specific to nonspecific binding ratios also can be quantitated
Fluorette/fluorophore dye dissociation constants can be determined in several ways For example, when a phage display system is used, phage-fluorophore dye dissociation constants can be determined by, for example, incubating for several hours subsaturatmg amounts of phage with a fluorophore dye bound to a carrier, for example, a bead as described above The bead suspensions are removed, for example, by centnfugation, and the unbound phage in the supernatants can be titrated Other methods for measuring unbound phage are known in the art, for example, visualization Dissociation constants can be measured via a standard linear Scatchard plot Nonspecific background binding is determined using phage that does not express fluorette sequences
In a preferred embodiment, fluorettes are encoded by nucleic acids and produced by recombmant molecular biology techniques (Sambrook et al (1989) Molecular Cloning A Laboratory Manual (2nd Edition) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, Ausubel et al (1994) Current Protocols in Molecular Biology Massachusettes General Hospital and Harvard Medical School Boston, MA) Thus, the present invention provides nucleic acids encoding fluorettes By "nucleic acids" herein is meant a nucleic acid, DNA or RNA depending on the delivery or expression vehicle or vector used, which can be manipulated to express fluorettes, that is, the nucleic acids encoding the fluorettes and the fusion partners and linkers if present In addition, the nucleic acids will also generally contain enough extra sequence to effect translation or transcription, as necessary For a fluorette, the nucleic acid generally contains cloning sites which are placed to allow in frame expression of the fluorettes, and any fusion partners, if present, such as presentation structures For example, when presentation structures are used, the presentation structure will generally contain the initatmg ATG, as a part of the parent vector For example, for retrovirus expression, the nucleic acids are generally constructed with an internal CMV promoter, tRNA promoter or cell specific promoter designed for immediate and appropriate expression of the fluorette at the initiation site of RNA synthesis The RNA is expressed anti-sense to the direction of retroviral synthesis and is terminated as known, for example with an orientation specific terminator sequence Interference from upstream transcription is alleviated in the target cell with the self-mactivation deletion, a common feature of certain retroviral expression systems In another embodiment, other expression vectors or systems also can be used, such as, adenovirus, adenoassociated virus, or transposons
Generally, the nucleic acids are introduced and expressed within the cells to produce fluorettes By "introduced into " or grammatical equivalents herein is meant that the nucleic acids enter the cells in a manner suitable for subsequent expression of the nucleic acid The method of introduction is largely dictated by the targeted cell type, discussed below. Exemplary methods include CaP04 precipitation, liposome fusion, lipofectm®, electroporation, viral infection, etc The candidate nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction), or may exist either transiently or stably in the cytoplasm (i e through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc ) As many pharmaceutically important screens require human or model mammalian cell targets, retroviral vectors capable of transfectmg such targets are preferred
In a preferred embodiment the fluorettes functions within the cell in which they are expressed Alternatively, fluorettes may be expressed within a cell or secreted from a cell, and purified If desired, the fluorette can be introduced into another cell by electroporation, calcium phosphate precipitation, microinjectiom, liposome fusion, lipofectm, viral infection, etc Fluorettes also may be produced in vitro by transcription/translation reactions Alternatively, fluorettes are chemically synthesized using any of a number of methods
In a preferred embodiment, the present invention provides a complex or composition of a fluorette bound to a fluorophore dye The binding of the fluorette to the fluorophore dye leads to four possible outcomes in regards to the excitation and emission spectra of the fluorophore dye i) little to no change in the fluorophore emission or excitation spectra, n) change in fluorophore emission, in) change in fluorophore excitation and (iv) change in both fluorophore emission and excitations
In one embodiment, binding of the fluorette to the fluorophore dye may prevent fluorophore excitation or emission In either scenario, the fluorophore dye, no longer fluoresces when bound to the fluorette In another embodiment, the excitation spectrum and/or the emission spectrum of the fluorophore dye is shifted, to a shorter or longer wavelength Alternatively, the excitation spectrum and/or emission spectrum can be broadened
In a preferred embodiment the excitation spectrum is altered up to about 5 nm, about 10 nm, being preferred, 20 nm particularly preferred, and about 100 nm being especially preferred
Fluorettes in their various forms find use in detector systems and various types of assays and techniques using a large set of potential fluorophore dyes, for example as, in detection of viral delivery systems, diagnostics, high-throughput assays
Once made, the compositions find use in a number of applications These applications include binding a fluorophore dye to the fluorette, removing unbound dye and detecting fluorescence In a preferred embodiment, the emission and/or excitation spectrum of the fluorophore dye is altered when bound to a fluorette Therefore, it is not necessary to remove unbound dye In a preferred embodiment, are applications that in some manner tethers the excitation and emission of two or more flourette/fluorescent dye complexes in such a way that they can be detected by fluorescence resonance energy transfer (FRET)
FRET applications based on fluorette/fluorophore dye interactions provide methods to demonstrate the proximity or interactions of two or more molecules These molecules include, for example, biological molecules, such as proteins, lipids, nucleic acids, carbohydrates In another embodiment, FRET can be applied to detect or induce the localized activation of a produrg
In a preferred embodiment, the interactions of two or more proteins, each containing a unique fluorette sequence can be demonstrated as follows First, the fluorophore dyes are bound by their corresponding fluorettes Next, one of the fluorette/fluorophore dye complexes or donor complex is excited and fluoresces When the two proteins containing the fluorette sequences are in close proximity or in some way have interacted, the emitted light of the first donor complex causes the second fluorette/fluorophore dye complex (acceptor) to fluoresce
Another example of a FRET application employs fluorette/fluorophore dye complexes that are covalently or associately linked to target biomateπals via small ligands The demonstration of FRET as described above indicates an interaction or the proximity of the target biomateπals when bound to their respective ligands Other applications include, "trigger dependent FRET", in which the use and design of trigger molecules, such as, heavy metal ions, drugs, etc , that specifically bind to the ligand or target biomatenals, causing dissociation of one or more of the fluorette/dye complexes from the target biomatenals and a decrease or loss of FRET
FRET may also be employed to detect intramolecular interactions or events, such as, changes in molecular structure that occurs in a molecule with the binding and/or dissociation of a ligand
In FRET applications, double or higher order fluorettes may be linked, for example, via a rigid connector, such as polyalanme or polysenne, to provide the correct angle of orientation of corresponding donor and acceptor fluorette/fluorophore dye complexes This may be of importance for efficient and quantitative FRET output In some cases, it may be desirable for the spacer to contain a peptide sequence or other material which, upon binding a specific fluorophore dye, brings the donor and acceptor comlexes into proximity such that FRET can ensue
FRET applications also include dual fluorophore dye quenching due to fluorette binding For example, fluorettes specific for donor and acceptor fluorophores are joined by a protease cleavable spacer This fluorette dimer is bound to its two fluorophore dyes targets and FRET ensues as described above However, FRET decreases after protease cleavage of the fluorette dimer
Other novel assays are based on fluorophore dye quenching due to its binding of a specific fluorette For example, a target protein is labeled in vitro by a fluorophore dye and is assembled into a complex particle or structure with other materials (e g , other proteins, DNA or RNA) whose conformation or structure is to be investigated The addition of a specific fluorette after the complex is assembled and measuring fluoresence indicates if the fluorette had access (binding) or no access (no binding) to its target dye Novel reporter gene assays based on fluorette-induced Stokes shift changes also can be created For example, a fluorette fused with a given carrier protein can be used as a gene activity reporter for the presence of the protein Cell extracts, or living cells expressing the protein, can be monitored for fluorette activity when the specific fluorophore dye is bound by the peptide Binding of the fluorophore dye by the fluorette/peptide modifies the excitation and/or emitted wavelength permitting specific detection of the expressed protein Consequently, the more Stokes shift changes introduced by binding of peptide the more sensitive the assay can become The simplicity of such an approach surpasses the currently available enzymatic reporter gene assays (e g , luciferase and beta-galactosidase assays)
The following examples serve to more fully describe the manner of using the above-described invention, as well as to set forth the best modes contemplated for carrying out various aspects of the invention It is understood that these examples in no way serve to limit the true scope of this invention, but rather are presented for illustrative purposes All references, patents, patent applications, and publications cited herein are incorporated by reference in their entirety
EXAMPLE 1
Synthesis of Fluorophore Dye Carriers Four fluorophore dyes, Texas Red, Fluorescein, Rhodamine Red and Oregon Green 514, were chosen, for selection of fluorettes from a phage display library (Example 2) The chemical structures of the dye conjugates are shown in Figure 1 The first three dyes, Texas Red, Fluorescein and Rhodamine Red fall into one group (i) they have a relatively high molar extinction coefficient for absorption and excellent quantum fluorescence yields (Haugland supra), (n) they have non-overlapping spectral characteristics, (in) they have the potential for cross-quenching and/or FRET analysis The fourth dye, Oregon Green 514, related structurally to Fluorescein, was chosen to determine if structure-function relationships could be discerned between fluorette peptides that bound related dyes
The extinction coefficient and quantum yield is important for sensitivity of detection and was a major factor determining choice of fluorophores Fluorescence detection by the outlined approach, at its simplest conception, is stochiometπc in nature In addition, peptide binding of the target dye might lead to some fluorescence quenching Finally, if fluorettes can be created that each can specifically bind different dyes then it is possible to measure proximal interactions between molecules by FRET relative to the binding of specific dyes having accommodating overlapping fluorescence spectra Succinimidyl esters of the four chosen fluorophore dyes (with 3- or 7-atorπ spacer between the dye molecule and the reactive succinimidyl group) were separately and covalently linked to target beads Linkages were established through the formation of a stable peptide bond to UltraLmk Immobilized DADPA carrier beads (capacity - 45μMoles of free amino groups per ml of beads) containing a 12-atom diaminodipropylamine spacer and a terminal ammo-group (Pierce)
Activated derivatives of fluorophore dyes 6-(fluoresceιn-5-(and-6)-carboxamιdo)hexanoιc acid, succinimidyl ester (5(6)-SFX), "mixed isomers", Oregon Green 514 carboxylic acid, succinimidyl ester, Rhodamine Red-X, succinimidyl ester, "mixed isomers" and Texas Red-X, succinimidyl ester, "mixed isomers" were purchased from Molecular Probes
Covalent coupling of activated derivatives of fluorophore dyes to a polymer carrier was performed in accordance with both manufacturer's protocols, with some modifications Briefly, 1ml of each dye derivative (5mg/ml in anhydrous dimethylformamide) was separately mixed with 15ml of 50% slurry of UltraLmk Immobilized DADPA in 0 2M sodium bicarbonate buffer, pH 8 3 (the coupling yield was at least 95% and, consequently, four fluorophore dye carriers contained 0 7-1μMoles of covalently bound dyes per ml of beads), unreacted ammo groups of the carriers were extensively acetylated by an addition of 18-fold molar excess of acetic acid N-hydroxysuccinimide ester (Sigma) followed by hydroxylamine treatment of the fluorophore dye carriers in order to destroy any unstable intermolecular intermediates of fluorophore dyes via their hydroxyl groups (for Fluorescein- and Oregon Green 514 carriers only) Finally, fluorophore dye carriers were quenched in 0 75M Tns-HCI, pH 8 7, loaded to the columns and extensively washed in high salt buffer, 50mM Tns-HCI, pH 7 5 + 1 M NaCI followed by a storage buffer, 50mM Tns-HCI, pH 7 5 + 150mM NaCI (TBS) + 0 05% sodium azide Fluorophore dye carriers were stored as 50% slurries at 4°C
The chemical structures of the dye conjugates are shown in Figure 1 The resulting total spacer between the dye molecule and polymer carrier was 19-atoms long for Texas Red-, Fluorescein-, Rhodamine Red-conjugates and 15-atoms long for Oregon Green 514-conjugate This allows maximal reduction of stenc hindrance for potential interactions with the large bactenophage particles that bear the peptide libraries After preparation we determined that the fluorophore dye carriers contained from 0 7-1 μMoles of covalently bound dye per ml of carrier beads
EXAMPLE 2
Selection for peptide aptamers that bind small molecule fluorophores A phage display peptide library was screened that contained a combinatorial library of 12-mer peptides fused via a short glycine linker spacer (GGG) to the ammo-terminus of a minor coat pill protein (5 copies per particle, Li et al (1980) J Biol Chem 255 10331-10337) of the filamentous bactenophage M13mp19 (Ph D phage display, New England Biolabs) During phage maturation the leader secretory sequence is removed This results in the 12-mer peptide positioned immediately at the ammo-terminus of the mature protein
E coli ER2537 strain [F' laclq Δ(lacZ)M15 proA+B+ /fhuA2 supE thi Δ(lac-proAB) Δ(hsdSM-mcrB)5 (rk mk mcrBC )] and E coli TG-1 strain [supE thι-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5 (rk mk ) (F' traD36 proAB laclqZΔM15)] were purchased from New England Biolabs and Stratagene, respectively, and used as bactenophage M13mp19 hosts All bacteriological techniques were performed as described (Sambrook ef al , supra, Ausubel ef al , supra)
Each biopanning round consisted of four sequential steps (i) phage binding with a fluorophore dye carrier, (n) washing unbound phage from the beads, (in) nonspecific elution of bound phage and (iv) amplification of bound phage The Ph D -12 phage display peptide library (based on modified M13mp19 bactenophage, 1 9x109 independent transformants) as a part of the Ph D -12 phage display peptide library kit was purchased from New England Biolabs All procedures were carried out at room temperature unless noted First, 0 15ml of centπfuged fluorophore dye carrier beads were blocked with 3ml of TBS + 2mg/ml BSA (blocking buffer) for 1 5hr with gentle rotating, beads were washed with 15ml TBS + 0 1% Tween-20 + 0 5mg/ml BSA (binding buffer), centπfuged, mixed with 1 8x1011 plaque-forming units (pfu) of the Ph D -12 phage display peptide library ("95- fold library size) in 3ml of binding buffer and suspension was gently rotated for 4hr For washing, bead suspensions were centπfuged and beads were transferred to the microcolumns and slowly washed by 100-fold beads volume of TBS + 0 1 % Tween-20 The bound phage were nonspecifically eluted by 1ml of 0 2M glycme-HCI, pH 2 2 + 1mg/ml BSA (elution buffer) for 10' and the eluates were immediately adjusted to a neutral pH by 0 15ml of 1M Tns-HCI, pH 9 1 Bound phage yields were determined by a titration of the eluates on the ER2537 host strain (Sambrook ef al , supra) Amplification step all bound phage (1 1ml) were added to 27 ml of 1/100 diluted overnight ER2537 culture and amplified for 4 5hr at 37°C with vigorous shaking The amplified phage were precipitated from cell supernatants by 1/5 V of 20% PEG + 2 5M NaCI at 4°C, re-precipitated again and, finally, suspended in 0 5ml TBS + 0 05% sodium azide The resultant "50-fold concentrated amplified phage were stored at 4°C and their titers usually were in the range 0 5-1 5x1013 pfu/ml For long-term storage amplified phage were adjusted to 50% glycerol and stored at -20 °C
The amplified phage were used for the next biopanning rounds against the corresponding fluorophore dye carrier, if necessary, until apparent enrichment for binding was observed over background We observed a significant increase in the amounts of bound phage after 3-4 rounds of biopanning (for all of the fluorophore dye carriers see Figure 2) This was compared to the nonspecific phage background in the first and second biopanning rounds Eluted phage from round 4 selected against four different fluorophore dye carriers are termed herein as TR-4, RhR-4, OG-4 and Flu-4 (standing for Texas Red, Rhodamine Red, Oregon Green 514 and Fluorescein carriers, respectively)
The amplified phage from round 1 were further selected against the corresponding fluorophore dye carrier The next biopanning rounds were performed in the same manner as round 1 , except that (i) input phage in the binding reaction were increased to 4 5x1011 pfu, ( ) time of binding was reduced to 2hr in round 4, (in) Tween-20 concentration in a washing buffer was increased to 0 2% in round 3 and 0 4% in round 4, (iv) eluted phage from last round 4 were not amplified, but rather served as a source of independent phage clones used for sequencing
The TR-4, RhR-4, OG-4 and Flu-4 phage sets were used as a source of independent phage clones that were sequenced for further analysis Phage single-stranded DNAs (ss DNAs) and double-stranded DNAs (ds DNAs) were purified as previously described (Sambrook ef al , supra) Fluorette-cod g portions of the DNA and adjacent DNA regions were sequenced with -96 gill sequencing primer CCCTCATAGTTAGCGTAACG (New England Biolabs) (SEQ ID NO 115) using an Applied Biosystems 391 automated DNA sequencer
The sequences from the insert regions of the phage are grouped in Table 1 Two unique sequences were found that bound to the Texas Red conjugate beads, one for Rhodamine Red, seven for Oregon Green, and six for Fluorescein Note that the Oregon Green set and the Fluorescein set shared two sequences that were identical at the nucleotide level as well as the ammo acid sequence presented These and other issues relevant to such observations are explored later
Phage selected against the Texas Red carrier gave rise to the sequences KHVQYWTQMFYS and DFLQWKLARQKP at a 5 1 ratio (Table 1) Biopanning with the phage display library against the Rhodamine Red carrier gave rise to a single phage clone, RhR401 , carrying the ammo acid sequence IPHPPMYWTRVF Thus, TR-4 and RhR-4 phage sets may be considered to be nearly "pure" phage clone populations by the fourth round of selection
We tested the binding specificity and excluded the possibility that the TR401 and RhR401 phage were selected against the polymer linker moiety rather than a fluorophore dye moiety of the dye carriers The TR-4 and RhR-4 phage were cross-bound to Rhodamine Red and Texas Red carriers, respectively Cross-binding of each phage to the inappropriate dye carrier did not exceed the nonspecific phage background binding (data not shown) Thus the TR401 and RhR401 fluorettes are specific by this comparison for their respective conjugated dyes, despite the similarity of the compound core ring structures Second, they had affinity for the corresponding conjugated fluorophore dyes rather than only a polymer linker moiety of Texas Red and Rhodamine Red carriers or other chemical features of the carriers themselves
Table 1. Fluorophore dye-specific phage clones and peptide fluorette sequences.
Figure imgf000037_0001
Figure imgf000037_0002
a) phage clones were isolated after four biopanning rounds with random combinatorial Ph.D. -12 phage display peptide library (see text), b) partial consensus (shown underlined) for Oregon Green 514- and Fluorescein-specific peptide fluorettes, (YWDW (SEQ ID NO:113); W(D/E)YY
(SEQ ID NO:114) c) -1 (D, E) or +1 (K, R), d) number of hydrophobic amino acids (A, V, L, I, W, Y, F) per total number of peptide fluorette amino acids, phage clone is represented in the corresponding group at least twice.
Four rounds of biopanning with the phage display peptide library against Oregon Green 514 and Fluorescein carriers were undertaken. Three clones were predominant in the sequenced population selected against Oregon Green (OG401 , OG402, OG403) and four clones were represented once (Table 1). Similarly, when selected against Fluorescein three clones predominated and others were represented only once. Notably two of the three predominant Fluorescein-specific fluorettes had the same sequence as two of the predominant, independently selected fluorettes that had been found with the Oregon Green 514 carrier. These were OG402 fluorette (YPNDFEWWEYYF) and the OG401 fluorette (ASDYWDWEWYYS). Since Oregon Green 514 is structurally related to Fluorescein, and is considered a Fluorescein pentafluoride (see Figure 1), independent selection of the same peptide fluorettes against Oregon Green 514 and Fluorescein carriers was predicted. The same fluorettes selected independently against these fluorophores possibly bind to similar domains of the Oregon Green 514 and Fluorescein dye molecules.
The selected phage had been originally selected as being capable of binding fluorophore dyes that had been covalently linked to a polymer carrier. The nature of carrier-crosslinked fluorophores, while useful for initial selection of phage, is inappropriate to study affinity of the phage except in relative terms. We therefore tested whether the selected phage can bind to the corresponding free fluorophore dyes in solution. Each bactenophage particle contains 5 copies of plll-fluorette fusion protein. Therefore phage binding is governed by avidity considerations. We bound free fluorophore dyes to highly concentrated phage solutions and assayed for bound fluorophore dye after precipitation of the phage.
Four activated derivatives of fluorophore dyes (see above) were quenched in 0.5M Tris-HCI, pH
8.7 and used as "free" dyes. Ten μl of PEG-purified and 300-fold concentrated phage TR401 ,
12 RhR401 , OG402, OG403 (1.3-1.8x10 phage particles; calculated from optical densities of the purified phage at 260 and 280nm) in TBS + 0.05% sodium azide buffer were separately mixed with an equal volume of 20μM solution of the corresponding free dyes in the same buffer; mixtures were adjusted by Tween-20 to 0.1% and incubated for 3hr. Phage-fluorophore dye complexes were precipitated three times by PEG-8000 in order to remove unbound dye and dissolved in 6.6μl of TBS + 0.05% sodium azide buffer. Finally, phage-fluorophore dye complexes (1μl of each) were spotted to nitrocellulose filter and filter was scanned on the Storm 840 scanner (Molecular
Dynamics) in Blue Fluorescence/Chemifluorescence mode with 200μ pixel resolution. To determine nonspecific binding the normalized amounts of phage particles of amplified, unselected, and PEG-purified Ph.D. -12 phage display peptide library were separately incubated with the corresponding free dyes and treated in the same conditions. All binding experiments were accomplished in duplicate. Specific/nonspecific signal ratios were quantified by densitometry of spot images using "Measure" option of NIH Image 1 59 freeware package
We found that OG403, OG402, RhR401 and TR401 phage specifically bound their respective fluorophore dyes (see Figure 3) This result shows that phage selected against covalently bound fluorophore dye were still capable of interacting with their cognate free fluorophore dye in solution
EXAMPLE 3
Forced evolution of higher affinity fluorophore-bindinα peptides The total number of possible random 12-mer peptides is equal to 2012=4 1x1015 The complexity of the phage display peptide library used for biopanning against the fluorophore dye carriers was much smaller, containing onlyl 9x109 clones Thus, the library represents a fraction of all possible 12-mer peptides that could have been searched for binding We therefore sought to improve the present fluorettes We chose to introduce mutations into the fluorette peptide sequences at the DNA level in the phage and then select for phage that displayed peptides with higher affinity (compared to the parent) against the respective fluorophore dye carrier We chose the Texas Red- and Rhodamine Red-specific clones (TR401 and RhR401, respectively), as well as the predominant Oregon Green 514- and Fluorescein-specific fluorette clones (OG403 and OG402, respectively, see above), for this forced evolution based on initial indications of their affinities (data not shown) We set up oligonucleotide synthesis of the corresponding fluorette-coded DNA sequences in such a manner that the mutagenesis rate was 9% for every 36 nucleotide positions of the peptide fluorette Nucleotides A and C were omitted from the third position in each codon to improve the relative representation of all ammo acids and by limits stop codon generation
Four minus-strand oligonucleotides containing degenerated fluorette-coding sequences were synthesized (Protein & Nucleic Acids Facility, Stanford University Medical Center),
TR401-91CL CTCCCCTTCGGCCGAACCTCCACCAGAATAAAACATCTGCGTCCAATACTGCACATGCTTA GAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO 88),
RhR401-91CL
CTCCCCTTCGGCCGAACCTCCACCAAACACACGAGTCCAATACATAGGAGGATGCGGAAT AGAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO 89), OG402-91CL CTCCCCTTCGGCCGAACCTCCACCAAAATAATACTCCCACCACTCAAAATCATTCGGATAA
GAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO 90), OG403-91CL CTCCCCTTCGGCCGAACCTCCACCCCAAGCAGTCCAATCCCAATAATAATCCCACCCATGA
GAGTGAGAATAGAAAGGTACCACTCTCCC (SEQ ID NO 91)
Nucleotides in a regular case shows no degeneracy, boldfaced nucleotide designates 91% of shown nucleotide and 3x3% of each of the other three nucleotides, boldfaced and underlined nucleotide designates 91 % of A or C and 9% of C or A, respectively
Degenerated minus-strand oligonucleotides were separately annealed to the plus-strand oligonucleotide GGGAGAGTGGTACCTTTCTATTCTCAC (SEQ ID NO 92), partial duplexes were filled by T4 DNA Polymerase (Ausubel ef al , supra), cut by Kpnl and Eagl and, finally, 1 9 ng of double digested filled duplexes was ligated to 200ng of low-melting point agarose gel-purified large fragment of the rep cative form (RF) Ph D 121 DNA cut with the same pair of enzymes (the
Ph D 121 was a randomly picked phage clone from the Ph D -12 phage display peptide library) in a total volume 0 1 ml Ligations were electroporated into 0 5ml of TG-1 electrocompetent cells (Stratagene) according to manufacturer's protocol and phage library complexities were determined by immediate mixing of several 10-fold dilutions of transfected TG-1 cells with ER2537 cells and plating Phage libraries were further amplified in 400ml of liquid LB media for 4hr at 37°C with vigorous shaking, concentrated by PEG (see above) and, finally, suspended in 0 5ml TBS + 0 05% sodium azide buffer
Titers of four resultant nonrandom combinatorial phage libraries were 0 2-4 0x1013 pfu/ml For a long-term storage phage libraries were adjusted to 50% glycerol and stored at -20°C Twelve independent phage clones from each TR401-91CL and RhR401-91CL libraries were sequenced in order to determine the average level of ammo acid substitutions in the fluorette moiety
We determined the nucleotide sequences of twelve independent phage clones from each of the TR401-91CL and RhR401-91CL libraries in order to determine the average level of ammo acid substitutions in the displayed fluorettes These were determined to be 2 30 and 2 45, respectively These experimental values correlated well to the expected theoretical values (calculated from a degeneration frequency 0 09 on the nucleotide level, see above) The nucleotide mutation rate from the other two libraries, OG403-91 CL and OG402-91CL was not determined but likely had a comparable average level of ammo acid substitutions in the fluorette moiety as all four corresponding oligonucleotides were synthesized in parallel using the same batch of pre-mixed monomer nucleotides
A key difference of the secondary biopanning versus the primary biopanning with the original phage library is the strict need to maximize selection against the originating, parental fluorette For this purpose we significantly reduced concentrations of the fluorophore dye carrier We also reduced the phage concentrations present during the binding steps In addition, the binding time was reduced while the amounts of washing buffer per ml of carrier beads were increased Specifically, (i) input phage in the binding reaction were decreased to 1 0x1010 pfu, (n) time of binding was reduced to 10', (in) amount of fluorophore dye carrier beads in the binding reaction was reduced to 0 03ml, (iv) following binding to the phage beads were washed with 330-fold beads volume of washing buffer and (v) Tween-20 concentration in the washing buffer was increased to 04%
An increase in approximately 1 2-1 5 logs of magnitude in the total number of bound phage after 2-3 rounds of biopanning (compared to that of the first round) for all the fluorophore dye carriers was seen (see Figure 4) Eluted phage from round 3 were termed TRS-3, RhRS-3, OGS-3 and FluS-3 These phage represent a second generation of phage clones selected against the four fluorophore dyes, Texas Red, Rhodamine Red, Oregon Green 514 and Fluorescein, respectively
We isolated and sequenced a panel of independent phage clones from each of the TRS-3, RhRS- 3, OGS-3 and FluS-3 phage sets to determine the sequences of the mutants (Table 2) In the Texas Red selection half of those sequenced encoded the parental ammo acid structure This indicated successful, though partial, selection against the parental binding characteristics under these conditions Four of the sixteen TRS401 progeny cloned carried a conservative S12T substitution Each of these substitutions occurred independently as it can be observed they are associated with additional, independent mutations This suggests that the S12T substitution is probably an important change that enhances the affinity of the phage for the dye Three progeny Texas Red fluorettes showed substitution at the second amino acid of histidme to P (found twice) or N It is interesting to note that clone TRS311 carried both the H2P and the S12T mutations Fewer mutant clones, only two, were observed for Rhodamine Red The RhR401 phage clone might represent a local optima in affinity for Rhodamine Red We found numerous in-frame deletions in the RhR401 fluorette-coded DNA sequences (about 33% of independent clones carrying large and small in-frame deletions in the fluorette moiety) This is an indicator of potential toxicity of the substituted mutant RhR401 fluorette sequences for bactenophage growth and survival (data not shown) For Oregon Green 514 multiple different peptide sequences were selected, with only two independently containing the same, non-conservative, mutation (G2E) For Fluorescein the majority of peptide fluorettes selected were also not parental sequences Several independent mutations were independently selected at fluorette positions 3 (N to S found twice), 5 (conservative change of F to Y found three times), 6 (conservative change E to D found twice), 9 (conservative E to D found twice), and 12 (conservative F to Y found 4 times) Also, certain Table 2. Fluorophore dye-specific phage clones and peptide fluorette sequences. The second generation.
Figure imgf000042_0001
a) phage clones were isolated after three biopanning rounds with TR401-91CL, RhR401- 91 CL, OG403-91CL and OG402-91CL nonrandom combinatorial phage display peptide libraries (see text), b) ammo acid substitutions in mutant peptide fluorette vs the corresponding parent are shown, mutant peptide fluorette sequence is shown as many times as it was found (see phage clone frequency), dots designate the same ammo acid as that in the corresponding parent, dash in a second position of the FluS304 fluorette designates m- frame deletion, c) original parental clones and the parental clones carrying silent mutatιon(s) in peptide fluorette moiety
positions appeared favored for change, i e , 2, 3, and 12 with no strong bias for the substituted residue
These mutations may be important for increasing the overall affinity of the peptide fluorette- fluorophore interaction We tested one of each of the phage from the secondary screens for increases in avidity (Table 3) PEG-purified TR401 phage, TRS311 phage or the amplified Ph D - 12 phage display peptide library phage as a negative control (input 1 5x108-1 1x1010 pfu with 2 to 5-fold increments) were incubated with 23 5μl of BSA-blocked and washed Texas Red carrier beads in binding buffer (see above) in total volume 40μl for 3hr Beads suspensions were centπfuged, the supernatants (unbound phage) were titrated and dissociation constants were measured via a standard linear Scatchard plot For TR401 and TRS311 phage bound/unbound ratios were quite reliable (6 3-13 1 in the range of phage concentrations shown above) All binding experiments were performed in duplicate and titrations were performed in triplicate This direct binding assay can reliably measure K^ if it does not exceed "1 5-2nM For higher K^ bound/unbound ratios became unreliable (=1)
Other PEG-purified phage (input 5x108-2 5x1010 pfu with 2 to 5-fold increments) were bound to the respective fluorophore dye carriers essentially in the same manner as described above for Texas Red-specific phage RhR401 and RhRS308, OG403 and OGS316, OG402 and FluS303 phage, were bound to the Rhodamine Red, Oregon Green 514 or Fluorescein carriers, respectively Following binding the beads were quickly washed twice by 0 25ml of TBS + 0 1% Tween-20 and suspended in 10ml TBS 10μl of suspension was mixed with "2x108 log-phase ER2537 cells and incubated 1 hr at 4°C with slight shaking to allow phage adsorption Several ten-fold dilutions of infected cells were mixed with non fected ER2537 cells and plated in standard plaque assay (Sambrook ef al , supra) All binding experiments and titrations were accomplished in duplicate Nonspecific background of binding (determined with amplified Ph D -12 phage display peptide library phage) did not exceed more than 3% of specific binding in each case For all above phage pfu/particle ratio was in the range 0 4-0 5
The Texas Red progeny phage TRS311 , which contained the apparently important S12T substitution, had a threefold increased avidity as compared to the TR401 parent The affinity of the double mutant clone RhRS308 versus the parent was not even marginally improved (Table 3) For Oregon Green 514 the phage clone OGS316, which carried the G2E substitution, had a 2 7- fold higher avidity than its parent The multiply substituted clone FluS303, which contained two putatively important changes, had a 6 5 fold increase in relative avidity Thus, peptides can be matured to higher avidity This likely is due to corresponding affinity increases for the individual peptide fluorette against its respective dye EXAMPLE 4 Binding of peptides to fluorophores in solution To identify peptide fluorettes that bind fluorophore dye independent of the full context of the pill fusion bactenophage protein we used synthetic peptides corresponding to the sequence of the selected fluorette region We added to the peptide eight ammo acids derived from the adjacent sequence of the pill protein This would accommodate any partial contributions of context from the pill protein sequence These were followed by a short GGG spacer and Hιs6 tag (see Table 4) We synthesized peptides from the original library screening that had been capable in solution of specifically binding each of the three dyes (Texas Red, Oregon Green 514, Fluorescein) The Texas Red peptides chosen were TR401 and its higher avidity progeny TRS311 , a peptide corresponding to the unrelated Texas Red primary clone TR406 was also synthesized The Oregon Green 514 clones OG401 and OG403 were synthesized to test, in part, the observation that both contained a common motif YWDW which might represent a common binding motif for Oregon Green 514 Note that the OG401 clone also bound to Fluorescein in the original screening The other clone which bound to both Oregon Green and Fluorescein in the primary screen, OG402, was synthesized The peptide derived from a secondary screen against Fluorescein, FluS303, which demonstrated a 6 5-fold higher affinity was also synthesized No peptides for Rhodamine Red were synthesized
Peptides were bound to cobalt ion-coated Sepharose beads and the resultant peptide-coated beads were incubated with corresponding fluorophore dyes, washed, and visualized for dye binding The same amounts of Texas Red-specific peptides PepTR401 , PepTRS311 , PepTR406 as well as nonspecific peptide PepControl (see Table 4) were bound via Hιs6 tag to the TALON Metal Affinity Resin (Clontech) in TBS buffer essentially as described by manufacturer's protocol Beads (5μl) were washed twice with 0 5ml TBS to remove unbound peptide and incubated with 0 5μM Texas Red in 40μl of TBS buffer for 1 hr at RT Finally, the beads were washed three times with 0 3ml TBS to remove unbound dye Fluorescent and nonfluorescent control beads were photographed with 10-fold magnification on fluorescent microscope Axiophot (Zeiss) using Rhodamine Red/Texas Red filter with 5" exposure
Most peptides failed to bind dyes to any detectable degree However, Texas Red peptides PepTR401 and PepTRS311 showed significant binding to Texas Red while they bound to neither
Oregon Green 514 nor Fluorescein (data not shown) PepTR406 did not bind to Texas Red, however Lack of dye binding might be due to low peptide affinity for cognate dyes and/or because the binding requires contributions from the pill fusion protein not present in these peptides The above test only checks for bound dye that remains fluorescent at the wavelengths tested Binding of fluorophore dyes to specific peptides might result in changes in the fluorescence spectra of bound dye versus the spectra of free dye Or, one might observe an increase or quenching of fluorescence We mixed concentrated peptide with 50nM of the corresponding dye in solution and determined the excitation and emission spectra of the resulting mixture No detectable changes in peak excitation nor emission were observed for peptides selected against Oregon Green 514 or Fluorescein (data not shown), nor for PepTR406, nor for a control peptide (Figure 5B)
Table 3 Phage - fluorophore dye binding affinity Dissociation constants
Figure imgf000046_0001
a) amino acid substitutions in mutant peptide fluorette vs the corresponding parent are shown, dots designate the same ammo acid as that in the corresponding parent, b) mutant peptide fluorette vs the corresponding parental peptide fluorette However, the peak positions of the excitation and emission spectra of soluble Texas Red bound to 10μM PepTR401 was shifted relative to Texas Red alone or as compared to a mixture of Texas Red and a nonspecific control peptide (Figure 5A) A +1 9 nm peak excitation shift with PepTR401 was observed The peptide was checked against Fluorescein as a nonspecific control and showed no shift in the Fluorescein excitation or emission spectra An approximate peak excitation shift of +2 8 nm was effected upon dye binding with PepTRS311 Interestingly, although PepTR401 had a shift in its peak excitation, no emission shift change was elicited upon binding fluorophore However, PepTRS311 did manifest a significant +1 4nM peak emission shift in its spectra We checked all other excitation and emission wavelengths and the changes observed correspond to a largely global shift in the spectra No other significant changes in local optima were observed (data not shown)
Interestingly, the TR401 peptide is the progenitor of the TRS311 peptide Thus, the two substitutions H2P and S12T result not only in an increased binding, but also in an apparent differences in the excitation spectra elicited Moreover, though both show a shift in the excitation only the selected, higher affinity, PepTRS311 had a peak emission spectra shift Thus, one or both of the two substitutions, H2P or S12T, are critical for shifting the spectra of the emission profile
We attempted with these findings to obtain an affinity measurement for the higher affinity TRS311 peptide Peptides in different concentrations (1 3-20μM) were incubated with Texas Red (50nM) in 0 6ml of TBS buffer for 1 hr at RT Excitation and emission spectra of samples were obtained by using spectrofluoπmeter SPEX Fluoromax (Jobm Yvon-SPEX Instruments Co ) and DataMax software package Peak positions were automatically calculated by software List mode data was saved for other analyses
Reduction of the concentration of PepTRS311 from 10μM to 1 3μM reduced slightly, but not completely, the change in peak excitation position (Figure 5A) The narrow dynamic range of the shift and the broad emission spectra provided for a limited ability to obtain an approximate measure of the affinity Affinity measurements calculated roughly from these results place the affinity of the peptide for the fluorophore at 0 1-0 5μM Further examination of this issue by other approaches is required to obtain more accurate reflections of the affinity and off-rates However, together with the agarose bead-binding data we can conclude that we have successfully selected for peptides with sufficient affinity to bind small molecule fluorophore dyes in solution In addition, the binding is specific and the spectral qualities of the dyes can be modulated dependent upon modifications of the bound peptide Table 4 Fluorette-carrying peptides
Figure imgf000048_0001
a) peptide names reflect the names of phage clones carrying respective fluorettes except Pep Control (see Tables 1 and 2) b) first twenty ammo acids of the peptides are the same as in the pill fusion protein of the corresponding phage clones except Pep Control (see Tables 1 and 2 and also New England Biolabs "Ph D -12 Phage Display Peptide Library Kit" manual), thus, amino acids 1-12 are the fluorette potion (double underlined) and ammo acids 13-20 are the distal pill fusion part (underlined) Ammo acids 21-29 contain a small GGG spacer followed by Hιs6 tag (shown in regular case) (SEQ ID NOS 41-48) c) specific for both Oregon Green 514 and Fluorescein (see Table 1) d) nonspecific control peptide synthesized for specificity check
Example 5 Constrained Texas Red-Binding Peptides Constrained Texas Red-binding peptides were identified via five rounds of biopanning of a mixture of two constrained phage display libraries against a polymer carrier with covalently bound Texas Red Two libraries contained SKVILFE-flanked nine or thirteen ammo acid variable region in the N-termmal part of M13 bactenophage pill protein The structure of the libraries were as follows
GGGSKVILFEGPAG (X)9or 13 GAPGSKVILFEGGPG (SEQ ID NO 93)- (pill protein)
SKVILFE-dimenzers are underlined Flexible linkers or spacers GGG, GPAG ( SEQ ID NO 94), GAPG (SEQ ID NO 95) and GGPG (SEQ ID NO 96) are double underlined X represents any
The flexible linker or spacer GPAG is encoded by a nucleotide sequence containing Fsel restriction endonuclease site The flexible linker or spacer GAPG is encoded by a nucleotide sequence containing Ascl restriction endonuclease site Both Fsel and Ascl restriction enzymes are rare eight-cutters Thus, upon a double enzyme digestion, a universal cassette-library or cassettes containing specific sequences can be conveniently exchanged between different vector/host systems or different dimeπzers of a choice (e g , pair of cystemes, coiled coil structures, etc )
Biopanning against carrier with Texas Red ultimately revealed at least seven different Texas Red-bmdmg constrained peptides (both 9- and 13-mers in a variable part)
GGGSKVILFEGPAG RTIWEPKEASNHT GAPGSKVILFEGGPG (TRP501) SEQ ID NO 97
GGGSKVILFEGPAG WSKMGHTVT GAPGSKVILFEGGPG (TRP505) SEQ ID NO 98
GGGSKVILFEGPAG RWTWEPISE GAPGSKVILFEGGPG (TRP512) SEQ ID NO 99
GGGSKVILFEGPAG GNQKCLQHNRCST GAPGSKVILFEGGPG (TRP518) SEQ ID NO 100
GGGSKVILFEGPAG SQTWSFPEH GAPGSKVILFEGGPG (TRP526) SEQ ID NO 101 GGGSKVILFEGPAG EPMARPWERKQDR GAPGSKVILFEGGPG (TRP527)
SEQ ID NO 102
GGGSKVILFEGPAG GTLSATRPYGRQW GAPGSKVILFEGGPG (TRP541 )
SEQ ID NO 103
Consensus motifs can be observed within these peptides (e g , RXXWEP (SEQ ID NO 104), WEP and TW, see Table 5) that suggests structural features common to the peptides that allow for efficient binding Interestingly, no said constrained peptide has a significant homology with linear Texas Red-binding peptides
Binding affinity of phage clones revealed the most avid binders, TRP512 phage and TRP501 phage, with K^ equal to 25 pM and 80 pM, respectively Other five phage clones were much less avid with K^, ≤ 5 nM (see Table 5)
Binding of synthetic peptides (nonspecific peptide, TR401 , TRP501 and TRP512) to Texas Red is shown in Figure 6B-C
Binding affinity of synthetic TRP501 peptide is measured as K^ 200 nM (=8-fold more avid than linear TR401 and TRS311 peptides) Binding affinity of synthetic TRP512 peptide is measured as I 10 nM (« 160-fold more avid than linear TR401 and TRS311 peptides)
CONCLUDING REMARKS The foregoing description details specific methods which can be employed to practice the present invention Having detailed such specific methods, those skilled in the art will well enough know how to devise alternative reliable methods at arriving at the same information in using the fruits of the present invention Thus, however detailed the foregoing may appear in text, it should not be construed as limiting the overall scope thereof, rather, the ambit of the present invention is to be determined only by the lawful construction of the appended claims All documents cited herein are hereby expressly incorporated by reference Table 5. Texas Red - specific phage clones carrying SKVILFE - flanked fluorettes.
l_Λ o
Figure imgf000051_0001
a) phage clones were isolated after five biopanning rounds with a mixture of combinatorial P71 R4-CL-9 & P71 R4-CI-13 phage display peptide libraries b) -1 (D,E) or +1 (K,R) c) number of hydrophobic amino acids (A,V,L,I,W,Y,F) per total number of fluorette amino acids *) phage clone is represented in the corresponding group at least twice

Claims

CLAIMS We claim
1 A peptide comprising a sequence of at least about 8 ammo acids, wherein said peptide binds to a fluorophore dye
2 The peptide according to claim 1 , wherein said ammo acids are naturally-occurring ammo acids
3 The peptide according to claim 1 , wherein said fluorophore dye is selected from the group consisting of Texas Red, Rhodamine Red, Oregon Green 514, and Fluorescein
4 The peptide according to claim 1 , wherein said peptide is fused to a presentation structure
5 The peptide according to claim 4, wherein said presentation structure comprises the sequence shown in SEQ ID NO 112
6 A complex comprising a fluorophore dye and a peptide comprising a sequence of at least about 8 ammo acids, wherein said dye is bound by said peptide
7 The complex according to claim 6, wherein said composition has a dissociation constant less than or equal to about 0 5 micromolar
8 The complex according to claim 6, wherein said dye is Texas Red and said peptide has an ammo acid sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 97, SEQ ID NO 98, SEQ ID NO 99, SEQ ID NO 100, SEQ ID NO 101, SEQ ID NO 102, SEQ ID NO 103, SEQ ID NO 105, SEQ ID NO 106, SEQ ID NO 107, SEQ ID NO 108, SEQ ID NO 109, SEQ ID NO 110, SEQ ID NO 111
9 The complex according to claim 6, wherein said dye is Rhodamine Red and said peptide has an ammo acid sequence selected from the group consisting of SEQ ID NO 3, SEQ ID NO 22, and SEQ ID NO 23
10 The complex according to claim 6, wherein said dye is Oregon Green 514 and said peptide has an ammo acid sequence selected from the group consisting of SEQ ID NO 4, SEQ ID NO 5 SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 28, SEQ ID NO 29, and SEQ ID NO 30
11 The complex according to claim 6, wherein said dye is Fluorescein and said peptide has an ammo acid sequence selected from the group consisting of SEQ ID NO 5, SEQ ID NO 6, SEQ ID
NO 11 , SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 31 , SEQ ID NO 32, SEQ ID NO 33, SEQ ID NO 34, SEQ ID NO 35, SEQ ID NO 36, SEQ ID NO 37, SEQ ID NO 38, SEQ ID NO 39, and SEQ ID NO 40
12 The complex according to claim 6, wherein said composition has an excitation spectrum that differs from the excitation spectrum of said fluorophore dye in the absence of said peptide
13 The complex according to claim 6 or 8, wherein said composition has an emission spectrum that differs from the emission spectrum of said fluorophore dye in the absence of said peptide
14 A method of binding a peptide to a fluorophore dye, comprising, contacting said fluorophore dye with said peptide, wherein said peptide comprises a sequence of at least about 8 ammo acids that binds said fluorophore dye
15 The method according to claim 13, wherein said binding alters the emission spectrum of said fluorophore dye
16 The method according to claim 14 or 15, wherein said binding alters the excitation spectrum of said fluorophore dye
17 The method according to claim 14, wherein said peptide is fused to a protein
18 A method of detecting a fluorette comprising a) contacting a fluorophore dye and fluorette comprising at least 8 ammo acids under conditions wherein said dye and said fluorette bind to form a dye complex, and b) detecting the presence of said dye complex
19 The method according to claim 18, wherein said fluorette is fused to a target analyte
20 The method according to claim 18, wherein said fluorette is fused to a presentation structure
21 The method according to claim 20, wherein said presentation structure comprising the sequence shown in SEQ ID NO 112
22 A method according to claim 18 wherein said detecting comprises a) irradiating said dye bound to said peptide b) detecting an emission of said fluorophore dye bound to said peptide, whereby said peptide is detected
23 A method according to claim 18 wherein said peptide is fused to an exogenous protein to form a fusion protein
24 A peptide comprising an ammo acid sequence, X1-X2-X3-X4-Y-W-T-X5-M-F-Y-X6, wherein,
X, is selected from the group consisting of K, N and T,
X2 is selected from the group consisting of H, P, and N,
X3 is selected from the group consisting of A and V,
X4 is selected from the group consisting of H and Q, X5 is selected from the group consisting of H and Q,
Xe is selected from the group consisting of S and T
25 A peptide comprising an amino acid sequence, X.,-P-H-X2-P-M-Y-W-T-X3-V-F, wherein,
X, is selected from the group consisting of I and L, X2 is selected from the group consisting of P and R, X3 is selected from the group consisting of P and R
26 A peptide comprising an ammo acid sequence, X1-X2-W-X3-Y-X4-W-D-W-T-X5-F-W,
X, is selected from the group consisting of H and Q,
X2 is selected from the group consisting of E and G,
X3 is selected from the group consisting of D and E, X4 is selected from the group consisting of Y and N,
X5 is selected from the group consisting of A, D, P, and T,
27 A peptide comprising an amino acid sequence,
Figure imgf000054_0001
, wherein, X1 is selected from the group consisting of H, P, and T, or is optionally omitted;
X2 is selected from the group consisting of H, N, S, and Y;
X3 is selected from the group consisting of D and E;
X4 is selected from the group consisting of F and Y;
X5 is selected from the group consisting of D and E;
Xg is selected from the group consisting of D and E;
X7 is selected from the group consisting of F, L, M, and Y.
28. A peptide comprising an amino acid sequence that is 90% identical to the amino acid sequence of claims 19, 20, 21 , or 22.
PCT/US1999/024266 1998-10-16 1999-10-15 Fluorescent dye binding peptides WO2000023463A2 (en)

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WO2009005579A1 (en) * 2007-06-14 2009-01-08 The General Hospital Corporation High affinity fluorochrome binding peptides
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