(19) ~a)
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`(12)
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`Europaisches Patentamt
`
`European Patent Office
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`Office européen des brevets
`
`(11)
`
`EP1 522 582 A2
`
`EUROPEAN PATENT APPLICATION
`
`(43) Date of publication:
`13.04.2005 Bulletin 2005/15
`
`(51) Int CI.7: C12N 15/10, C12Q 1/68
`
`(21) Application number: 04078076.9
`
`(22) Date of filing: 07.01.1999
`
`
`(84) Designated Contracting States:
`AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU
`MC NL PT SE
`
`(30) Priority: 07.01.1999 GB 9900298
`
`(62) Document number(s) of the earlier application(s) in
`accordance with Art. 76 EPC:
`009000803 /1 141 272
`
`(71) Applicant: MEDICAL RESEARCH COUNCIL
`London W1N 4AL (GB)
`
`0 Tawfik, Dan, MRC Lab. of Molecular Biology
`Cambridge CBZ 20H (GB)
`- Sepp, Armin, Gendaq Limited
`London NW7 1AD (GB)
`
`(74) Representative: Crump, Julian Richard John et al
`Mintz Levin Cohn Ferris Glovsky and Popeo
`Intellectual Property LLP
`The Rectory
`9, Ironmonger Lane
`London EC2V 8EY (GB)
`
`Remarks:
`
`This application was filed on 08 - 11 - 2004 as a
`(72) Inventors:
`divisional application to the application mentioned
`- Griffiths, Andrew, MRC Lab. of Molecul. Biology
`under INID code 82.
`Cambridge C32 20H (GB)
`
`
`(54)
`
`Optical sorting method
`
`A method for increasing the concentration of a
`(57)
`nucleic acid molecule comprising the steps of:
`
`(a) forming aqueous microcapsules from a water-
`in-oil emulsion, wherein a plurality of microcapsules
`include a nucleic acid molecule, a bead capable of
`being linked to the nucleic acid molecule and an
`aqueous solution comprising components neces-
`sary to perform nucleic acid amplification;
`(b) amplifying the nucleic acid molecule in the mi-
`crocapsules to form amplified product copies of the
`
`nucleic acid molecule; and
`(c) capturing the amplified product copies to the
`bead in the microcapsules; thereby increasing the
`concentration of the nucleic acid molecule.
`
`EP1522582A2
`
`Printed by Jouve, 75001 PARlS (FR)
`
`

`

`Description
`
`EP 1 522 582 A2
`
`[0001] The present invention relates to methods for use in in vitro evolution of molecular libraries. In particular, the
`present invention relates to methods of selecting nucleic acids encoding gene products in which the nucleic acid and
`the activity of the encoded gene product are linked by compartmentation.
`[0002]
`Evolution requires the generation of genetic diversity (diversity in nucleic acid) followed by the selection of
`those nucleic acids which result in beneficial characteristics. Because the nucleic acid and the activity of the encoded
`gene product of an organism are physically linked (the nucleic acids being confined within the cells which they encode)
`multiple rounds of mutation and selection can result in the progressive survival of organisms with increasing fitness.
`Systems for rapid evolution of nucleic acids or proteins in vitro advantageously mimic this process at the molecular
`level in that the nucleic acid and the activity of the encoded gene product are linked and the activity of the gene product
`is selectable.
`
`[0003] Recent advances in molecular biology have allowed some molecules to be co-selected according to their
`properties along with the nucleic acids that encode them. The selected nucleic acids can subsequently be cloned for
`further analysis or use, or subjected to additional rounds of mutation and selection.
`[0004] Common to these methods is the establishment of large libraries of nucleic acids. Molecules having the desired
`characteristics (activity) can be isolated through selection regimes that select for the desired activity of the encoded
`gene product, such as a desired biochemical or biological activity, for example binding activity.
`[0005]
`Phage display technology has been highly successful as providing a vehicle that allows for the selection of
`a displayed protein by providing the essential link between nucleic acid and the activity of the encoded gene product
`(Smith, 1985; Bass et al.
`, 1990; McCafferty et al., 1990; for review see Clackson and Wells, 1994). Filamentous phage
`particles act as genetic display packages with proteins on the outside and the genetic elements which encode them
`on the inside. The tight linkage between nucleic acid and the activity of the encoded gene product is a result of the
`assembly of the phage within bacteria. As individual bacteria are rarely multiply infected, in most cases all the phage
`produced from an individual bacterium will carry the same genetic element and display the same protein.
`[0006] However, phage display relies upon the creation of nucleic acid libraries in vivo in bacteria. Thus, the practical
`limitation on library size allowed by phage display technology is of the order of 107 to 1011, even taking advantage of
`k phage vectors with excisable filamentous phage replicons. The technique has mainly been applied to selection of
`molecules with binding activity. A small number of proteins with catalytic activity have also been isolated using this
`technique, however, selection was not directly for the desired catalytic activity, but either for binding to a transition-
`state analogue (Widersten and Mannervik, 1995) or reaction with a suicide inhibitor (Soumillion et al., 1994; Janda et
`al., 1997). More recently there have been some examples of enzymes selected using phage-display by product for-
`mation (Atwell & Wells, 1999; Demartis et al., 1999; Jestin eta/., 1999; Pederson, et al., 1998), but in all these cases
`selection was not for multiple turnover.
`[0007]
`Specific peptide ligands have been selected for binding to receptors by affinity selection using large libraries
`of peptides linked to the C terminus of the lac repressor Lacl (Cull et al., 1992). When expressed in E. colithe repressor
`protein physically links the ligand to the encoding plasmid by binding to a lac operator sequence on the plasmid.
`[0008] An entirely in vitro polysome display system has also been reported (Mattheakis et al., 1994; Hanes and
`Pluckthun, 1997) in which nascent peptides are physically attached Via the ribosometo the RNA which encodes them.
`An alternative, entirely in Vitro system for linking genotype to phenotype by making RNA-peptide fusions (Roberts and
`Szostak, 1997; Nemoto et al., 1997) has also been described.
`[0009] However, the scope of the above systems is limited to the selection of proteins and furthermore does not
`allow direct selection for activities other than binding, for example catalytic or regulatory activity.
`[0010]
`In Vitro RNA selection and evolution (Ellington and Szostak, 1990), sometimes referred to as SELEX (sys-
`tematic evolution of ligands by exponential enrichment) (Tuerk and Gold, 1990) allows for selection for both binding
`and chemical activity, but only for nucleic acids. When selection is for binding, a pool of nucleic acids is incubated with
`immobilised substrate. Non-binders are washed away, then the binders are released, amplified and the whole process
`is repeated in iterative steps to enrich for better binding sequences. This method can also be adapted to allow isolation
`of catalytic RNA and DNA (Green and Szostak, 1992; for reviews see Chapman and Szostak, 1994; Joyce, 1994; Gold
`et al., 1995; Moore, 1995).
`[0011] However, selection for "catalytic" or binding activity using SELEX is only possible because the same molecule
`performs the dual role of carrying the genetic information and being the catalyst or binding molecule (aptamer). When
`selection is for "auto-catalysis" the same molecule must also perform the third role of being a substrate. Since the
`genetic element must play the role of both the substrate and the catalyst, selection is only possible for single turnover
`events. Because the "catalyst" is in this process itself modified, it is by definition not atrue catalyst. Additionally, proteins
`may not be selected using the SELEX procedure. The range of catalysts, substrates and reactions which can be
`selected is therefore severely limited.
`[0012] Those of the above methods that allow for iterative rounds of mutation and selection are mimicking in vitro
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`EP 1 522 582 A2
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`mechanisms usually ascribed to the process of evolution: iterative variation, progressive selection for a desired the
`activity and replication. However, none of the methods so far developed have provided molecules of comparable di-
`versity and functional efficacy to those that are found naturally. Additionally, there are no man-made "evolution" systems
`which can evolve both nucleic acids and proteins to effect the full range of biochemical and biological activities (for
`example, binding, catalytic and regulatory activities) and that can combine several processes leading to a desired
`product or activity.
`[0013] There is thus a great need for an in vitro system that overcomes the limitations discussed above.
`[0014]
`In Tawfik and Griffiths (1998), and in International patent application PCT/GB98/01889, we describe a system
`for in Vitro evolution that overcomes many of the limitations described above by using compartmentalisation in micro-
`capsules to link genotype and phenotype at the molecular level.
`International patent application PCT/
`[0015]
`In Tawfik and Griffiths (1998), and in several embodiments of
`GB98/O1889, the desired activity of a gene product results in a modification of the genetic element which encoded it
`(and is present in the same microcapsule). The modified genetic element can then be selected in a subsequent step.
`[0016] Here we describe a further invention in which the modification of the genetic element causes a change in the
`optical properties of the element itself, and which has many advantages over the methods described previously.
`
`BRIEF DESCRIPTION OF THE INVENTION
`
`[0017] According to a first aspect of the present invention, there is provided a method for isolating one or more
`genetic elements encoding a gene product having a desired activity the expression of which may result, directly or
`indirectly, in the modification of an optical property of a genetic element encoding the gene product, comprising the
`steps of:
`
`(a) compartmentalising genetic elements into microcapsules;
`(b) expressing the genetic elements to produce their respective gene products within the microcapsules;
`(c) sorting the genetic elements which produce the gene product(s) having the desired activity according to the
`changed optical properties of the genetic elements.
`
`[001 8] The microcapsules according to the present invention compartmentalise genetic elements and gene products
`such that they remain physically linked together.
`[001 9] As used herein, a genetic element is a molecule or molecular construct comprising a nucleic acid. The genetic
`elements of the present invention may comprise any nucleic acid (for example, DNA, RNA or any analogue, natural
`or artificial, thereof). The nucleic acid component of the genetic element may moreover be linked, covalently or non-
`covalently, to one or more molecules or structures, including proteins, chemical entities and groups, and solid—phase
`supports such as beads (including nonmagnetic, magnetic and paramagnetic beads), and the like.
`In the method of
`the invention, these structures or molecules can be designed to assist in the sorting and/or isolation of the genetic
`element encoding a gene product with the desired activity.
`[0020] Expression, as used herein, is used in its broadest meaning, to signify that a nucleic acid contained in the
`genetic element is converted into its gene product. Thus, where the nucleic acid is DNA, expression refers to the
`transcription of the DNA into RNA; where this RNA codes for protein, expression may also referto the translation of
`the RNA into protein. Where the nucleic acid is RNA, expression may refer to the replication of this RNA into further
`RNA copies, the reverse transcription of the RNA into DNA and optionally the transcription of this DNA into further
`RNA molecule(s), as well as optionally the translation of any of the RNA species produced into protein. Preferably,
`therefore, expression is performed by one or more processes selected from the group consisting of transcription, re-
`verse transcription, replication and translation.
`[0021] Expression of the genetic element may thus be directed into either DNA, RNA or protein, or a nucleic acid or
`protein containing unnatural bases or amino acids (the gene product) within the microcapsule of the invention, so that
`the gene product is confined within the same microcapsule as the genetic element.
`[0022] The genetic element and the gene product thereby encoded are linked by confining each genetic element
`and the respective gene product encoded by the genetic element within the same microcapsule. In this way the gene
`product in one microcapsule cannot cause a change in any other microcapsules. In addition, further linking means may
`be employed to link gene products to the genetic elements encoding them, as set forth below.
`[0023] The term "microcapsule" is used herein in accordance with the meaning normally assigned thereto in the art
`and further described hereinbelow. In essence, however, a microcapsule is an artificial compartment whose delimiting
`borders restrict the exchange of the components ofthe molecular mechanisms described herein which allowthe sorting
`of the genetic elements according to the function of the gene products which they encode.
`[0024]
`Preferably, the microcapsules used in the method of the present invention will be capable of being produced
`in very large numbers, and thereby to compartmentalise a library of genetic elements which encodes a repertoire of
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`EP 1 522 582 A2
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`gene products.
`[0025] As used herein, a change in optical properties of the genetic elements refers to any change in absorption or
`emission of electromagnetic radiation, including changes in absorbance, luminescence, phosphorescence or fluores-
`cence. All such properties are included in the term "optical". Genetic elements can be sorted, for example, by lumines-
`cence, fluorescence or phosphorescence activated sorting. In a preferred embodiment, flow cytometry is employed to
`sort genetic elements, for example, light scattering (Kerker, 1983) and fluorescence polarisation (Rolland et al., 1985)
`can be used to triggerflow sorting. In a highly preferred embodiment genetic elements are sorted using a fluorescence
`activated cell sorter (FACS) sorter (Norman, 1980; Mackenzie and Pinder, 1986).
`[0026] Changes in optical properties may be direct or indirect. Thus, the change may result in the alteration of an
`optical property in the genetic element itself, or may lead indirectly to such a change. For example, modification of a
`genetic element may alter its ability to bind an optically active ligand, thus indirectly altering its optical properties.
`[0027] Alternatively, imaging techniques can be used to screen thin films of genetic elements to allow enrichment
`for a genetic element with desirable properties, for example by physical isolation of the region where a genetic element
`with desirable properties is situated, or ablation of non-desired genetic elements. The genetic elements can be detected
`by luminescence, phosphorescence or fluorescence.
`[0028] According to a preferred embodiment of the first aspect ofthe present invention, the sorting of genetic elements
`may be performed in one of essentially two techniques.
`
`(I) In a first embodiment, the genetic elements are sorted following pooling ofthe microcapsules into one or more
`common compartments. In this embodiment, a gene product having the desired activity modifies the genetic ele—
`ment which encoded it (and which resides in the same microcapsule) so as to make it selectable as a result of its
`modified optical properties in a subsequent step. The reactions are stopped and the microcapsules are then broken
`so that all the contents of the individual microcapsules are pooled. The modification of the genetic element in the
`microcapsule may result directly in the modification of the optical properties of the genetic element. Alternatively,
`the modification may allow the genetic elements to be further modified outside the microcapsules so as to induce
`a change in their optical properties. Selection for the genetic elements with modified optical properties enables
`enrichment of the genetic elements encoding the gene product(s) having the desired activity. Accordingly, the
`invention provides a method according to the first aspect of the invention, wherein in step (b) the gene product
`having the desired activity modifies the genetic element encoding it to enable the isolation of the genetic element
`as a result in a change in the optical properties of the genetic element.
`It is to be understood, of course, that
`modification may be direct, in that it is caused by the direct action of the gene product on the genetic element, or
`indirect, in which a series of reactions, one or more of which involve the gene product having the desired activity,
`leads to modification of the genetic element.
`
`(II) In a second embodiment, the genetic elements may be sorted by a multi-step procedure, which involves at
`least two steps, for example, in order to allow the exposure of the genetic elements to conditions which permit at
`least two separate reactions to occur. As will be apparent to persons skilled in the art, the first microencapsulation
`step of the invention advantageously results in conditions which permit the expression of the genetic elements -
`be it transcription, transcription and/or translation, replication or the like. Under these conditions, it may not be
`possible to select for a particular gene product activity, for example because the gene product may not be active
`under these conditions, or because the expression system contains an interfering activity. The invention therefore
`provides a method according to the first aspect of the present invention, wherein step (b) comprises expressing
`the genetic elements to produce their respective gene products within the microcapsules, linking the gene products
`to the genetic elements encoding them and isolating the complexes thereby formed. This allows for the genetic
`elements and their associated gene products to be isolated from the capsules before sorting according to gene
`product activity takes place. In a preferred embodiment, the complexes are subjected to a further compartmental-
`isation step priorto isolating the genetic elements encoding a gene product having the desired activity. This further
`compartmentalisation step, which advantageously takes place in microcapsules, permits the performance of further
`reactions, under different conditions,
`in an environment where the genetic elements and their respective gene
`products are physically linked. Eventual sorting of genetic elements may be performed according to embodiment
`(I) above.
`
`[0029] The "secondary encapsulation" may also be performed with genetic elements linked to gene products by other
`means, such as by phage display, polysome display, RNA—peptide fusion or lac repressor peptide fusion.
`[0030] The selected genetic element(s) may also be subjected to subsequent, optionally more stringent rounds of
`sorting in iteratively repeated steps, reapplying the method of the invention either in its entirety or in selected steps
`only. By tailoring the conditions appropriately, genetic elements encoding gene products having a better optimised
`activity may be isolated after each round of selection.
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`[0031] Additionally, the genetic elements isolated after a first round of sorting may be subjected to mutagenesis
`before repeating the sorting by iterative repetition of the steps of the method of the invention as set out above. After
`each round of mutagenesis, some genetic elements will have been modified in such a way that the activity of the gene
`products is enhanced.
`[0032] Moreover, the selected genetic elements can be cloned into an expression vectorto allowfurther character—
`isation of the genetic elements and their products.
`[0033]
`In a second aspect, the invention provides a product when selected according to the first aspect of the inven-
`tion. As used in this context, a "product" may refer to a gene product, selectable according to the invention, or the
`genetic element (or genetic information comprised therein).
`[0034]
`In a third aspect, the invention provides a method for preparing a gene product, the expression of which may
`result, directly or indirectly, in the modification the optical properties of a genetic element encoding it, comprising the
`steps of:
`
`a) preparing a genetic element encoding the gene product;
`b) compartmentalising genetic elements into microcapsules;
`c) expressing the genetic elements to produce their respective gene products within the microcapsules;
`d) sorting the genetic elements which produce the gene product(s) having the desired activity using the changed
`optical properties of the genetic elements; and
`(e) expressing the gene product having the desired activity.
`
`( ( ( (
`
`In accordance with the third aspect, step (a) preferably comprises preparing a repertoire of genetic elements,
`[0035]
`wherein each genetic element encodes a potentially differing gene product. Repertoires may be generated by conven-
`tional techniques, such as those employed for the generation of libraries intended for selection by methods such as
`phage display. Gene products having the desired activity may be selected from the repertoire, according to the present
`invention, according to their ability to modify the optical properties of the genetic elements in a manner which differs
`from that of other gene products. For example, desired gene products may modify the optical properties to a greater
`extent than other gene products, orto a lesser extent, including not at all.
`[0036]
`In a fourth aspect, the invention provides a method for screening a compound or compounds capable of
`modulation the activity of a gene product, the expression of which may result, directly or indirectly, in the modification
`of the optical properties of a genetic element encoding it, comprising the steps of:
`
`a) preparing a repertoire of genetic elements encoding gene product;
`b) compartmentalising genetic elements into microcapsules;
`c) expressing the genetic elements to produce their respective gene products within the microcapsules;
`d) sorting the genetic elements which produce the gene product(s) having the desired activity using the changed
`optical properties of the genetic elements; and
`(e) contacting a gene product having the desired activity with the compound or compounds and monitoring the
`modulation of an activity of the gene product by the compound or compounds.
`
`( ( ( (
`
`[0037] Advantageously, the method further comprises the step of:
`
`(g) identifying the compound or compounds capable of modulating the activity of the gene product and synthesising
`said compound or compounds.
`
`[0038] This selection system can be configured to select for RNA, DNA or protein molecules with catalytic, regulatory
`or binding activity.
`
`Brief Description of the Figures
`
`[0039]
`
`Figure 1 . Dihydrofolate reductase can be expressed from genes in vitro translated in solution and genes attached
`to paramagnetic beads with identical efficiency. The DHFR activity resulting from in vitro translation of folA genes
`in solution or folA genes attached to paramagnetic microbeads is determined by monitoring the oxidation of NADPH
`to NADP spectrophotometrically at 340 nm and activity is calculated by initial velocities under So>>KM conditions
`(omax). (o), translated from genes in solution; (I), translated from genes attached to microbeads.2.
`
`Figure 2. Epifluorescence microscopy of water-in-oil emulsions demonstrating that GFP can be translated in vitro
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`EP 1 522 582 A2
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`from genes attached to single microbeads encapsulated in the aqueous compartments of the emulsions and the
`translated gene-product bound back the microbeads making them fluorescent.
`
`Figure 3. Flow cytometric analysis of GFP expression in microcapsules and in situ binding to the genetic element
`(microbeads). A: The light scattering characteristics of the beads before reaction. 75% of beads run as single
`beads. B: The light scattering characteristics of the beads after in vitro translation reaction. About 50% of beads
`fall into the gate for single beads. C: Fluorescence from microbeads (gated for single beads only) coated with
`T7-GFP gene and anti-GFP polyclonal antibody is significantly higher than the signal from the beads where either
`the GFP gene or the anti-GFP antibody were omitted.
`
`Fi gu re 4. Synthesis of Biotin-GS—DNP by the h uman glutathione S-transferase M2-2 ( GST M2-2) catalysed reaction
`of 1-chloro-2,4—dinitrobenzene (CDNB; Sigma) with reduced biotinylated—gluta thione (Biotin-68H).
`
`Figure 5. Detecting paramagnetic beads coated with the product of an enzyme catalysed reaction by flow cytom-
`etry Sera-MagTM streptavidin-coated magnetic microparticles incubated with Biotin-GS-DNP made by the GST
`M2-2 catalysed reaction of Biotin-GSH and CDNB. The captured Biotin-GS-DNP was detected by incubation of
`the microparticles with a mouse anti-dinitrophenol antibody followed by a (FlTC)-conjugated F(ab')2 fragment goat
`anti-mouse lgG, F(ab')2 fragment. After washing, 2 x 105 microparticles were analysed by flow cytometry. All
`reagents, no reagents omitted from the enzymatic synthesis of with Biotin-GS-DN P; minus GST, the enzyme GST
`M2—2 was omitted from the synthesis; minus biotin—GSH, biotin—GSH was omitted from the synthesis; minus CDNB,
`CDNB was omitted from the synthesis.
`
`Figure 6. Synthesis of MeNPO-CO—Biotin-B-Ala-GSH (caged-biotin-BaIa—GSH). Acetyl chloride (5 ml) was added
`to anhydrous methanol (80 ml). The stirred solution was allowed to cool down and d-biotin (4 g) was added. After
`over-night stirring the solvents were evaporated in vacuum to afford a white solid. The solid was triturated with
`ether, filtered and dried under vacuum (in the presence of phosphorus pentoxide) and stored at -20°C.
`
`Figure 7. Reaction of caged-biotin-Bala-GSH with 1-chloro-2,4-dinitrobenzene (CDNB) andphotochemical uncag-
`ing of the biotin group. Figure 8. Reaction of caged-biotin-BaIa-GSH with 4-chloro-3-nitrobenzoate (CNB) and
`photochemical uncaging of the biotin group
`
`Figure 9. Human GST M2—2 catalyses the reaction of caged biotin-BaIa-GSH with CDNB and CNB in solution and
`the reaction products can be uncaged by UV irradiation, captured on beads and detected using fluorescent/y la-
`belled anti—product antibodies and flow cytometry.
`Panel A: light scattering characteristics of beads and gate for single beads (R1). Panel B: fluorescence from mi-
`crobeads (gated through R1) from reactions with CDNB. Panel C: fluorescence from microbeads (gated through
`R1) from reactions with CNB. Signals from microbeads from reactions with and without GST M2-2 are annotated
`+enz and -enz respectively. Signals from microbeads from reactions which were UV irradiated and those which
`were not are annotated +UV and -UV respectively.
`
`Fi gu re 10. Flow cytometry can be used to distinguish beads from aqueous compartments ofan emulsion containing
`GST M2—2 from beads from compartments without GST M2—2 by using caged biotinylated—BAla—GSH and CNB as
`substrates.
`
`Panel A: light scattering characteristics of a mixture of a mixture of 1.0 pm diameter nonfluorescent neutravidin
`labelled microspheres (Molecular Probes, F-87T7) or 0.93 pm diameter streptavidin-coated polystyrene beads
`(Bangs Laboratories) and gates set for single Bangs beads (R1) and single Molecular Probes beads (R2). Panel
`B: fluorescence from microbeads taken from a non-emulsified mixture of 98% Bangs beads (without GST) and
`2% Molecular Probes beads (with GST). Panel C: fluorescence from microbeads taken from a mixture of two
`emulsions in a ratio of 98% emulsion containing Bangs beads (without GST) and an emulsion containing 2%
`Molecular Probes beads (with GST). Panel D: fluorescence from microbeads taken from a non-emulsified mixture
`of 98% Molecular Probes beads (without GST) and 2% Bangs beads (with GST). Panel E: fluorescence from
`microbeads taken from a mixture of two emulsions in a ratio of 98% emulsion containing Molecular Probes beads
`(without GST) and an emulsion containing 2% Bangs beads (with GST). Fluorescence of ungated beads (No gate),
`beads gated through R1 (R1) and beads gated through R2 (R2) are overlayed.
`
`Figure 11. Human GST M2—2 transcribed and translated in vitro in the aqueous compartments of a water—in oil
`emulsion catalyses a reaction which gives rise to a change in the fluorescence properties of co-compartmentalised
`microspheres.
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`EP 1 522 582 A2
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`Panel A: light scattering characteristics of beads and gate for single beads (R1). Panel B: fluorescence from mi-
`crobeads (gated through R1) from non-emulsified reactions. Panel C: fluorescence from microbeads (gated
`through R1) emulsified reactions. Signals from microbeads from reactions with and without GSTM2-2.LMB2-3
`DNA are annotated +DNA and -DNA respectively. Signals from microbeads from reactions with and without re-
`combinant GST M2—2 are annotated +GST and —GST respectively.
`
`Figure 12. Synthesis of the caged-biotinylated substrate EtNP-BzGlu-cagedBiotin (17).
`
`Figure 13. Hydrolysis of the PTE substrate EtNP-B7—Glu-cagedBiotin (17) to yield the product Et-B7—Glu-caged—
`Biotin, and uncaging of both substrate and product to yield the corresponding biotinylated substrate (EtNP-Bz-Glu-
`Biotin) and product (EtNP-Bz-GIu-Biotin)
`
`Figure 14. Preparation of protein conjugates of a PTE substrate and product for immunisation and ELISA.
`
`Figure 15. PTE catalyses the reaction of EtNP—Bz-Glu-cagedBiotin in the presence of streptavldin-coated beads,
`and the reaction products uncaged by UV irradiation, are captured on beads and detected using fluorescent/y
`labelled anti-product antibodies and flow cytometry.
`Panel A: light scattering characteristics of the beads and gate selected for single beads (R2). Panel B: fluorescence
`from beads (gated through R2) from reactions with 10 uM EtNP-Bz-Glu-cagedBiotin in the presence of in vitro
`translated OPD.LMB3—2biotin DNA fragments (0 PD) or M.Haelll.LMB3—2biotin DNA fragments (M.Haelll) Panel
`C: As B but with 20 (M EtNP-Bz-Glu-cagedBiotin. Panel D: As B but with 50 uM EtNP-Bz-Glu-cagedBiotin.
`
`Figure 16. Reaction of EtNP—BZ—Glu—cagedBiotin in the presence of beads to which genetic elements encoding
`the phosphotriesterase tagged with the Flag peptide (N-Flag—OPD.LMB3-2biotin) or another enzyme (N—Flag—M.
`Haelll.LMBS-2biotin) were attached alongside with an antibody thatbinds the Flag peptide. The beads were reacted
`and subsequently analysed by tlow—cytometry as described in the text.
`Panel A: light scattering characteristics of beads and gate for single beads (R1). Panel B: fluorescence from mi—
`crobeads (gated through R1 ) to which were attached N-Flag-OPD.LMBB-2biotin DNAfragments (OPD) or M.Haelll.
`LMBS-Zbiotin DNAfragments (M.Haelll) from reactions with 12.5 uM EtNP-Bz-Glu-cagedBiotin. Panel C: As B but
`with 25 uM EtN P-BZ-Glu-caged-Biotin.
`
`Figure 17. E. coli BirA transcribed and translated in vitro catalyses a reaction which gives rise to a change in the
`fluorescence properties ofsubstra te-labelled microspheres in the aqueous compartments of a water-in oil emulsion
`and in bulk solution.
`
`Figure 18. Flow cytometric analysis of samples prepared for the sorting experiment.
`
`Figure 19. Fluorescence-activated flow cytometric sorting of the genetic elements.
`Panel A: Samples #1 to #4 before sorting and after sorting. Panel B: Genes recovered from individual beads sorted
`from sample #3 sorted into a 96-well plate. Panel C: Genes recovered from individual beads sorted from sample
`#4 sorted into a 96—well plate. DNA markers (M) are ¢X174—Haelll digest.
`
`(A) GENERAL DESCRIPTION
`
`[0040] The microcapsules of the present invention require appropriate physical properties to allow the working of
`the invention.
`
`First, to ensure that the genetic elements and gene products may not diffuse between microcapsules, the
`[0041]
`contents of each microcapsule are preferably isolated from the contents of the surrounding microcapsules, so that
`there is no or little exchange of the genetic elements and gene products between the microcapsules overthe timescale
`of the experiment.
`[0042]
`Second, the method ofthe present invention requires that there are only a limited number of genetic elements
`per microcapsule. This ensures that the gene product of an individual genetic elementwill be isolated from other genetic
`elements. Thus, coupling between genetic element and gene product will be highly