WO2002083292A2 - Chemical libraries based on coded particles - Google Patents

Chemical libraries based on coded particles Download PDF

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Publication number
WO2002083292A2
WO2002083292A2 PCT/GB2002/001788 GB0201788W WO02083292A2 WO 2002083292 A2 WO2002083292 A2 WO 2002083292A2 GB 0201788 W GB0201788 W GB 0201788W WO 02083292 A2 WO02083292 A2 WO 02083292A2
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library
chemical
code
holder
microparticle
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PCT/GB2002/001788
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French (fr)
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WO2002083292A3 (en
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Susan Louise Watson
Amit Kumar Som
Nigel Guy Skinner
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3 D Molecular Sciences Limited
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Publication of WO2002083292A3 publication Critical patent/WO2002083292A3/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00457Dispensing or evacuation of the solid phase support
    • B01J2219/00459Beads
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00457Dispensing or evacuation of the solid phase support
    • B01J2219/00459Beads
    • B01J2219/00461Beads and reaction vessel together
    • B01J2219/00463Directed sorting
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00457Dispensing or evacuation of the solid phase support
    • B01J2219/00459Beads
    • B01J2219/00468Beads by manipulation of individual beads
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00497Features relating to the solid phase supports
    • B01J2219/005Beads
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/00497Features relating to the solid phase supports
    • B01J2219/00502Particles of irregular geometry
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/0054Means for coding or tagging the apparatus or the reagents
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/0054Means for coding or tagging the apparatus or the reagents
    • B01J2219/00547Bar codes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277Apparatus
    • B01J2219/0054Means for coding or tagging the apparatus or the reagents
    • B01J2219/00554Physical means
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00585Parallel processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/0059Sequential processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00592Split-and-pool, mix-and-divide processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583Features relative to the processes being carried out
    • B01J2219/00596Solid-phase processes
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068Means for controlling the apparatus of the process
    • B01J2219/00686Automatic
    • B01J2219/00689Automatic using computers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • B01J2219/00722Nucleotides
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718Type of compounds synthesised
    • B01J2219/0072Organic compounds
    • B01J2219/00725Peptides
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07BGENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11Compounds covalently bound to a solid support
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/06Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00Libraries per se, e.g. arrays, mixtures
    • C40B40/04Libraries containing only organic compounds
    • C40B40/10Libraries containing peptides or polypeptides, or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C40COMBINATORIAL TECHNOLOGY
    • C40BCOMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes

Definitions

  • the present invention relates to a method of fabricating chemical libraries on coded particles.
  • oligomers formed from monomer units of a similar type to one another but differing in detailed structure .
  • the aim may be to discover with which sequence of monomer units forming an oligomer a particular chemical entity will react. In some cases this may be for identifying the sequence of an unknown oligomer.
  • the oligomers may be formed from nucleotides and their analogues (e.g. RNA, DNA or PNA) , amino acids (peptides and proteins), sugars or any other oligomerisable chemical compound.
  • oligonucleotides With regard to oligonucleotides, one approach is to place an unknown (analyte) strand in the presence of all possible (target) strands, typically of some shorter length. A small number of complementary target strands will bind to the analyte strand and this binding event may be identified by any suitable means, for example fluorescence, electro- chemiluminescence, che iluminescence, biochemiluminescence or phosphorescence. In an existing technology, the target strands are spatially distributed on a surface. The sequence of each target strand is encoded in its location on the surface. The identity of the analyte strand can, therefore, be deduced from the physical location of the binding event in relation to the surface.
  • peptide sequences are built-up on particles (one sequence per particle) and interaction between each peptide sequence and an active molecule such as a peptide cleaving enzyme is looked for.
  • the substrate sequence for the enzyme may be discovered and inhibitors for it may be developed.
  • the beads upon which a binding event occurred may then be separated from the bulk of the beads and the sequence of the analyte strand deduced from the sequence of the bound target strand that is identifiable by a number of means .
  • One approach to placing the members of the library on the coded particles is to have the particles fixed on a substrate and to synthesise or place the library compounds on the particles in a spatially directed manner. Knowledge of the location at which each coded particle is present combined with knowledge of the location at which each library member was synthesised or placed provides knowledge of which particle code is associated with which library member. Each library of coded particles has to be created separately. Each synthesis step is effectively conducted separately for each bead.
  • An alternative approach to building the required library is disclosed in W096/36436.
  • coded particles linked to a memory device are provided with individual identifier codes. They are mixed together and split into a number of separate reaction containers based on the number of choices for the first component of the molecules of the library, e.g. four reaction containers each for one DNA nucleotide reactant .
  • the codes of each particle going into each container are read and recorded. After synthesis of the first component, they are re-mixed and then split again into the reaction containers with the codes being read and recorded again.
  • each particle is given a multitude of identical molecules of the library and all the desired library members are present on at least one particle. This process is referred to as
  • a process of this type is also used in WO 97/15390 to build a library on physically encoded silicon microparticles in such a way that there is only one microparticle per library member.
  • the code of each particle has to be read in each iteration. So, to make a single library containing say 10 10 compounds of say 10 sub-units in length, one has to read 10 10 particles on each of at least 10 occasions. To make a second library which is the same as the first one, one has to go through the whole procedure again.
  • the present invention now provides a chemical library (e.g.
  • a combinatorial chemical library comprising a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) and bearing a chemical or combination of chemicals within the library which is the same as a chemical or combination of chemicals borne by each other microparticle of the respective individual holder, such that each microparticle code maps to a unique chemical or combination of chemicals within the library and each holder code maps to a unique chemical or combination of chemicals within the library.
  • Such a library may be prepared by taking a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) , in a first round of library synthesis splitting the plurality of holders into at least two groups (group la and group Ila and optionally N-2 further groups Ilia to Na) and conducting a first chemical library synthesis iteration on each group to add to each microparticle of each group of holders a first respective chemical library member component, in a second round of library synthesis re-splitting the plurality of holders into at least two new groups (group lb and group lib and optionally N-2 further groups Illb to Nb) and conducting a second chemical library synthesis iteration on each group to add to each microparticle of each group of holders a second respective chemical library member component, and in further rounds of library synthesis repeating said mixing, re-splitting and
  • each holder may hold a multiplicity of coded microparticles
  • the library produced can be further processed by removing the microparticles from the holders in such a way as either to produce multiple identical libraries or to produce one or more libraries in which each library member is present on a multiplicity of preferably identical microparticles .
  • each microparticle code of each microparticle bearing a particular library member may be the same, but this is not essential. Accordingly, whilst each microparticle code maps to a single library member, it is not necessary that each library member maps to a single microparticle code.
  • any chemical library according to the invention is associated with a database which contains for each microparticle code the identity of the chemical or chemicals mapped thereto.
  • Said database may further contain for each holder code the identity of the chemical or chemicals mapped thereto.
  • each chemical or combination of chemicals on a microparticle within the library maps to a unique holder code.
  • more than one holder may hold microparticles having a particular library member thereon, and each said holder may if desired be differently encoded.
  • each chemical or combination of chemicals on a microparticle within the library maps to a unique micro-particle code. This will imply that each microparticle associated with a particular holder has the same micro-particle code.
  • microparticles associated with a single holder may have different micro-particle codes.
  • the microparticle code may be provided by the shape of each microparticle.
  • GB 0009723 describes particles formed in plastics. Each generally rod shaped particle has a series of notches along each long side which form a machine readable bar code. Optionally, one end of each particle is marked with a notch serving to differentiate that end from the other end of the particle.
  • Microparticles of this type are preferred for use according to the invention. However, other shaped microparticles may be used including those described in WO97/15390, which describes shaped microparticles made in silicon and encoded by pits, holes, hollows, grooves, or notches or combinations thereof. Any such morphological features may be used according to the present invention. However, the microparticle code does not need to be produced by shaping the microparticles.
  • microparticle code may be a bar code printed on the particle.
  • each microparticle has a maximum dimension of less than 5mm, preferably less than 1mm, more preferably less than 500 ⁇ m, e.g. about 250 ⁇ m.
  • the beads may be of any shape, e.g. shaped as cubes, spheres, rods, plates, discs or blocks. Preferably, they are generally rod shaped and have a lesser dimension of from 10 to 100 ⁇ m, e.g. about 25 ⁇ m.
  • the holder code may be provided by any of the methods described above.
  • the holder may be a container in which the micro-particles are free. Such a container will need to allow reagents to pass in and out whilst the microparticles are retained.
  • the container has porous walls. It may be in the form of a bag of flexible material but is more preferably of rigid construction, having one or more walls of mesh or other permeable material .
  • each said oligomer is an oligonucleic acid, an oligonucleic acid analogue, a peptide, a peptide containing one or more non-peptide linking groups, or a sugar.
  • Oligonucleic acids will include DNA and RNA oligomers, preferably of from 5 to 20, e.g. from 8 to 14 nucleotides in length.
  • Nucleic acid analogues are compounds which are different from DNA or RNA but which mimic the ability of DNA or RNA to hybridise to DNA or RNA strands of complementary sequence. These analogues typically have a modified backbone designed to resist proteolytic degradation by nucleases . This may be for instance a peptide backbone as in PNAs or hybrid PNA-DNAs.
  • the invention includes a method of forming a combinatorial chemical library, which method comprises taking a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) , in a first round of library synthesis splitting the plurality of holders into at least two groups (group la and group Ila and optionally N-2 further groups Ilia to Na) and conducting a first chemical library synthesis iteration on each group to add to each micro- particle of each group of holders a first respective chemical library member component, in a second round of library synthesis re-splitting the plurality of holders into at least two new groups (group lb and group lib and optionally N-2 further groups Illb to Nb) and conducting a second chemical library synthesis iteration on each group to add to each microparticle of each group of holders a second respective chemical library member component, and in further rounds of library
  • the method may further comprise removing the microparticles from said holders.
  • all of the holders may be returned to the master storage chamber prior to re-splitting into new groups.
  • the invention includes a support unit for use in synthesising a chemical library comprising a holder and a multitude of microparticles held by the holder, each microparticle being encoded with an identical code and each being suitable as a substrate on which to synthesise a chemical library, the holder being such as to provide access to the microparticles for reagents applied to the holder during library synthesis.
  • a support unit for use in synthesising a chemical library comprising a holder and a multitude of microparticles held by the holder, each microparticle being encoded with an identical code and each being suitable as a substrate on which to synthesise a chemical library, the holder being such as to provide access to the microparticles for reagents applied to the holder during library synthesis.
  • the invention further includes apparatus for use in synthesising a chemical library comprising a conduit connecting a master storage chamber to a plurality of reaction chambers branching off said conduit at respective branch points, at or before each branch point a code reader suitable for reading codes present on microparticle holders passing along said conduit in use and a valve associated with each reaction chamber and operable in response to the reading of codes by a respective said code reader to open temporarily to allow a selected said holder to pass into the respective reaction chamber.
  • Figure 1 shows in side view a holder for use in the invention
  • Figure 2 shows apparatus for use in synthesising a combinatorial chemical library.
  • Figure 1 shows a holder for use in the method of library synthesis of the invention. It comprises a small pot 2 having a solid base and rigid mesh sides 4 and a removable lid 6. Each holder is marked with a machine readable bar code. However, other methods of coding and the holders may be employed.
  • the lid may contain a microchip containing a remotely readable code uniquely identifying the holder.
  • Within the holder there are contained a multitude of microparticles each of which is shaped by a series of bars and grooves along at least one side which constitute a bar code. Each microparticle within the holder has the same bar code.
  • the code on the microparticles within each holder will be the same and will differ from the code of the microparticles in each other holder.
  • microparticles may be subjected to chemical combinatorial library synthesis using conventional and accepted chemical methodology in the apparatus shown in
  • a master chamber 10 is connected to one end of a conduit 12. From conduit 12 there are four branch conduits 14, 16, 18, 20 which join the conduit 12 at respective branch points. Each branch conduit leads to a respective reaction chamber 11, 13, 15, 17. At each branch point a respective holder code reader (not shown) is provided at the locations 22, 24, 26, 28. Each reader is designed to read the code associated with a holder of the kind shown in Figure 1 as such a holder passes down the conduit 12. A valve (not shown) is provided at each branch point . Each code reader is adapted to communicate with the adjacent valve via a computer (not shown) to cause the valve to open when a desired code is read.
  • each microparticle within each holder will bear numerous molecules of a particular library member. In any single holder each microparticle will bear the same library member. At this point one may either produce multiple identical libraries by opening the holders and allocating one or more of the microbeads from each holder to a respective library or one may produce a library in which each library member is represented by numerous microparticles simply by opening each holder and mixing all of the contents together.
  • Methods of combinatorial chemical synthesis suitable for use in the context of this invention include those already known generally in the art and do not require detailed description here. Examples of suitable synthesis techniques are for instance given in WO 96/36436 and WO 96/24061. Code readers of the kind described in those specifications may be employed in this invention also.

Abstract

The invention provides a chemical library, and a method for making said library, wherein said library comprises a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) and bearing a chemical or combination of chemicals within the library which is the same as a chemical or combination of chemicals borne by each other microparticle of the respective individual holder, such that each microparticle code maps to a unique chemical or combination of chemicals within the library and each holder code maps to a unique chemical or combination of chemicals within the library.

Description

Chemical Libraries based on Coded Particles
The present invention relates to a method of fabricating chemical libraries on coded particles. In a wide range of biochemical and chemical procedures there is a requirement for working with oligomers formed from monomer units of a similar type to one another but differing in detailed structure . The aim may be to discover with which sequence of monomer units forming an oligomer a particular chemical entity will react. In some cases this may be for identifying the sequence of an unknown oligomer. The oligomers may be formed from nucleotides and their analogues (e.g. RNA, DNA or PNA) , amino acids (peptides and proteins), sugars or any other oligomerisable chemical compound. With regard to oligonucleotides, one approach is to place an unknown (analyte) strand in the presence of all possible (target) strands, typically of some shorter length. A small number of complementary target strands will bind to the analyte strand and this binding event may be identified by any suitable means, for example fluorescence, electro- chemiluminescence, che iluminescence, biochemiluminescence or phosphorescence. In an existing technology, the target strands are spatially distributed on a surface. The sequence of each target strand is encoded in its location on the surface. The identity of the analyte strand can, therefore, be deduced from the physical location of the binding event in relation to the surface.
In another chemical combinatorial procedure, peptide sequences are built-up on particles (one sequence per particle) and interaction between each peptide sequence and an active molecule such as a peptide cleaving enzyme is looked for. The substrate sequence for the enzyme may be discovered and inhibitors for it may be developed.
Beads or other particles which are identical to one another may be used to support the library. An alternative technology is based on physically differentiable beads or other particles . Target strands of known sequence are attached to beads in such a way that all of the attached strands on any one bead have the identical sequence. In this way, the sequence of a particular strand may be identifiable from the particular bead to which the strand is attached. The beads are then exposed to the analyte strand and binding between the analyte strand and any target strands may be detected using any suitable detection means, for example fluorescence. The beads upon which a binding event occurred may then be separated from the bulk of the beads and the sequence of the analyte strand deduced from the sequence of the bound target strand that is identifiable by a number of means . One approach to placing the members of the library on the coded particles is to have the particles fixed on a substrate and to synthesise or place the library compounds on the particles in a spatially directed manner. Knowledge of the location at which each coded particle is present combined with knowledge of the location at which each library member was synthesised or placed provides knowledge of which particle code is associated with which library member. Each library of coded particles has to be created separately. Each synthesis step is effectively conducted separately for each bead. An alternative approach to building the required library is disclosed in W096/36436. Here, coded particles linked to a memory device such as a microchip are provided with individual identifier codes. They are mixed together and split into a number of separate reaction containers based on the number of choices for the first component of the molecules of the library, e.g. four reaction containers each for one DNA nucleotide reactant . The codes of each particle going into each container are read and recorded. After synthesis of the first component, they are re-mixed and then split again into the reaction containers with the codes being read and recorded again. By reiteration of this procedure, each particle is given a multitude of identical molecules of the library and all the desired library members are present on at least one particle. This process is referred to as
'split and mix' . A process of this type is also used in WO 97/15390 to build a library on physically encoded silicon microparticles in such a way that there is only one microparticle per library member. In the split and mix process, the code of each particle has to be read in each iteration. So, to make a single library containing say 1010 compounds of say 10 sub-units in length, one has to read 1010 particles on each of at least 10 occasions. To make a second library which is the same as the first one, one has to go through the whole procedure again.
It would be desirable to develop a more efficient method of producing multiple copies of identical libraries. It would also be desirable to develop more efficient methods of producing libraries in which each library member is present on multiple identically coded particles whilst minimising the number of code reading steps and synthesis steps involved. The present invention now provides a chemical library (e.g. a combinatorial chemical library), wherein said library comprises a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) and bearing a chemical or combination of chemicals within the library which is the same as a chemical or combination of chemicals borne by each other microparticle of the respective individual holder, such that each microparticle code maps to a unique chemical or combination of chemicals within the library and each holder code maps to a unique chemical or combination of chemicals within the library.
Such a library may be prepared by taking a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) , in a first round of library synthesis splitting the plurality of holders into at least two groups (group la and group Ila and optionally N-2 further groups Ilia to Na) and conducting a first chemical library synthesis iteration on each group to add to each microparticle of each group of holders a first respective chemical library member component, in a second round of library synthesis re-splitting the plurality of holders into at least two new groups (group lb and group lib and optionally N-2 further groups Illb to Nb) and conducting a second chemical library synthesis iteration on each group to add to each microparticle of each group of holders a second respective chemical library member component, and in further rounds of library synthesis repeating said mixing, re-splitting and synthesis steps through as many further synthesis iterations as desired to form said library. The result of this is that for each microparticle code there is only one chemical or combination of chemicals within the library carried by microparticles with that microparticle code. Reading the microparticle code of a microparticle enables one immediately to tell what member of the library is present on the particle.
Since each holder may hold a multiplicity of coded microparticles, the library produced can be further processed by removing the microparticles from the holders in such a way as either to produce multiple identical libraries or to produce one or more libraries in which each library member is present on a multiplicity of preferably identical microparticles . Thus, one can first isolate a chosen holder, remove the microparticles bearing a library member therefrom and split these into respective storage vessels (placing one microparticle or several into each vessel) , repeating the operation for each holder in turn to produce multiple identical libraries. Alternatively, one can simply remove all the microparticles from their holders and mix them together to produce a library in which each library member is present on numerous microparticles. For convenience the microparticle code of each microparticle bearing a particular library member may be the same, but this is not essential. Accordingly, whilst each microparticle code maps to a single library member, it is not necessary that each library member maps to a single microparticle code.
Preferably, any chemical library according to the invention is associated with a database which contains for each microparticle code the identity of the chemical or chemicals mapped thereto.
Said database may further contain for each holder code the identity of the chemical or chemicals mapped thereto. Optionally, in a chemical library of the invention, each chemical or combination of chemicals on a microparticle within the library maps to a unique holder code. However, if desired, more than one holder may hold microparticles having a particular library member thereon, and each said holder may if desired be differently encoded.
Optionally, in any chemical library of the invention, each chemical or combination of chemicals on a microparticle within the library maps to a unique micro-particle code. This will imply that each microparticle associated with a particular holder has the same micro-particle code.
Alternatively however, the microparticles associated with a single holder may have different micro-particle codes.
The microparticle code may be provided by the shape of each microparticle. GB 0009723 describes particles formed in plastics. Each generally rod shaped particle has a series of notches along each long side which form a machine readable bar code. Optionally, one end of each particle is marked with a notch serving to differentiate that end from the other end of the particle. Microparticles of this type are preferred for use according to the invention. However, other shaped microparticles may be used including those described in WO97/15390, which describes shaped microparticles made in silicon and encoded by pits, holes, hollows, grooves, or notches or combinations thereof. Any such morphological features may be used according to the present invention. However, the microparticle code does not need to be produced by shaping the microparticles. It can be constituted by any of the encoding means described in WO 96/36436 including a microchip associated with each microparticle on which a code may be recorded electronically or magnetically. It may be constituted by multiple chemical entities, the presence of which is machine readable. For instance, multiple fluorescent molecules may be used having different fluorescence characteristics. The microparticle code may be a bar code printed on the particle.
The microparticle code may be modified or assembled progressively during the process of synthesising the respective library member on the microparticle.
Preferably, each microparticle has a maximum dimension of less than 5mm, preferably less than 1mm, more preferably less than 500 μm, e.g. about 250 μm. The beads may be of any shape, e.g. shaped as cubes, spheres, rods, plates, discs or blocks. Preferably, they are generally rod shaped and have a lesser dimension of from 10 to 100 μm, e.g. about 25 μm. The holder code may be provided by any of the methods described above.
The holder may be a container in which the micro-particles are free. Such a container will need to allow reagents to pass in and out whilst the microparticles are retained. Advantageously, therefore the container has porous walls. It may be in the form of a bag of flexible material but is more preferably of rigid construction, having one or more walls of mesh or other permeable material .
Alternatively, the holder may be in the nature of the surface of a member on which the microparticles are releaseably or temporarily retained. The holder may for instance be a plate member, block, sphere, cylinder or other shape having the microparticles temporarily adhered to its surface. The nature of the chemicals constituting the library is not restricted in any way but preferably each microparticle bears as said chemical an oligomer comprising a sequence of linked differing sub-unit molecule residues, said oligomer differing from similar oligomers on other microparticles by the sequence of said sub-unit molecule residues.
These may include libraries in which each said oligomer is an oligonucleic acid, an oligonucleic acid analogue, a peptide, a peptide containing one or more non-peptide linking groups, or a sugar. Oligonucleic acids will include DNA and RNA oligomers, preferably of from 5 to 20, e.g. from 8 to 14 nucleotides in length. Nucleic acid analogues are compounds which are different from DNA or RNA but which mimic the ability of DNA or RNA to hybridise to DNA or RNA strands of complementary sequence. These analogues typically have a modified backbone designed to resist proteolytic degradation by nucleases . This may be for instance a peptide backbone as in PNAs or hybrid PNA-DNAs.
Modified peptide libraries will include peptides containing a non-peptide linkage such as a phosphate group. These may for instance be designed to resist the action of peptidase enzymes whilst mimicking the binding site of a substrate for the enzyme. A library of such peptide analogues may be used in developing protease inhibitors . As used in the present invention, a combinatorial chemical library may be one wherein each microparticle bears as said chemical a member of a family of chemical molecules having a structural characteristic in common and differing from other members of said family. This may be the case where the members of the family may be synthesised in part in situ on the microparticles with different combinations of for instance substituent groups being introduced during said synthesis iterations.
As indicated above, the invention includes a method of forming a combinatorial chemical library, which method comprises taking a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) , in a first round of library synthesis splitting the plurality of holders into at least two groups (group la and group Ila and optionally N-2 further groups Ilia to Na) and conducting a first chemical library synthesis iteration on each group to add to each micro- particle of each group of holders a first respective chemical library member component, in a second round of library synthesis re-splitting the plurality of holders into at least two new groups (group lb and group lib and optionally N-2 further groups Illb to Nb) and conducting a second chemical library synthesis iteration on each group to add to each microparticle of each group of holders a second respective chemical library member component, and in further rounds of library synthesis repeating said re-splitting and synthesis steps through as many further synthesis iterations as desired to form said library.
The method may further comprise removing the microparticles from said holders.
Preferably, the holders are split into groups in each round of library synthesis by passage along a conduit from a master storage chamber to a plurality of reaction chambers branching off said conduit at respective branch points, the holder code of each holder being read prior to each branch point and selected holders being diverted from said conduit into said reaction chambers to split said holders into said groups .
After each iteration of library synthesis, all of the holders may be returned to the master storage chamber prior to re-splitting into new groups.
The invention includes a support unit for use in synthesising a chemical library comprising a holder and a multitude of microparticles held by the holder, each microparticle being encoded with an identical code and each being suitable as a substrate on which to synthesise a chemical library, the holder being such as to provide access to the microparticles for reagents applied to the holder during library synthesis. There may be a multitude of such holders, each containing a set of micro-particles differing in code from those of other holders .
The invention further includes apparatus for use in synthesising a chemical library comprising a conduit connecting a master storage chamber to a plurality of reaction chambers branching off said conduit at respective branch points, at or before each branch point a code reader suitable for reading codes present on microparticle holders passing along said conduit in use and a valve associated with each reaction chamber and operable in response to the reading of codes by a respective said code reader to open temporarily to allow a selected said holder to pass into the respective reaction chamber. The invention will be further described and illustrated with reference to the accompanying drawings, in which:
Figure 1 shows in side view a holder for use in the invention; and
Figure 2 shows apparatus for use in synthesising a combinatorial chemical library.
Figure 1 shows a holder for use in the method of library synthesis of the invention. It comprises a small pot 2 having a solid base and rigid mesh sides 4 and a removable lid 6. Each holder is marked with a machine readable bar code. However, other methods of coding and the holders may be employed. For instance, the lid may contain a microchip containing a remotely readable code uniquely identifying the holder. Within the holder there are contained a multitude of microparticles each of which is shaped by a series of bars and grooves along at least one side which constitute a bar code. Each microparticle within the holder has the same bar code. For use in the invention, as many separate holders will be provided as there are desired members of the combinatorial library. The code on the microparticles within each holder will be the same and will differ from the code of the microparticles in each other holder.
The microparticles may be subjected to chemical combinatorial library synthesis using conventional and accepted chemical methodology in the apparatus shown in
Figure 2. In Figure 2, a master chamber 10 is connected to one end of a conduit 12. From conduit 12 there are four branch conduits 14, 16, 18, 20 which join the conduit 12 at respective branch points. Each branch conduit leads to a respective reaction chamber 11, 13, 15, 17. At each branch point a respective holder code reader (not shown) is provided at the locations 22, 24, 26, 28. Each reader is designed to read the code associated with a holder of the kind shown in Figure 1 as such a holder passes down the conduit 12. A valve (not shown) is provided at each branch point . Each code reader is adapted to communicate with the adjacent valve via a computer (not shown) to cause the valve to open when a desired code is read.
Entry ports 30, 32, 34, 36 are provided in the branch conduits 14-20 for the introduction of synthesis reagents. In use, holders of the kind shown in Figure 1 (marked in Figure 2 as A, B, C, D,E, and F) pass one at a time from the master chamber 10 down the conduit 12 past each code reader in turn. The computer, previously programmed with the codes of the holders A-F and the reaction chambers into which each of those holders is desired to pass, reacts to the code read by each code reader and operates the valves as required. As shown in the Figure, holder F has just been read by reader 26 and the respective valve of branch conduit 18 has been opened to allow holder F to fall into reaction chamber 17. Of course, this is for the purposes of illustration only and in reality there would be a far greater number of holders. The arrangement of four reaction chambers is suitable for the production of oligonucleotides where each synthesis iteration reaction in respect of any particular holder is with one of the four nucleotide bases. For the synthesis of peptides, one would provide a larger number of reaction chambers.
To cause the holders to move down the conduit 12 from the master chamber 10, one may employ gravity assisted by vibration of the conduit, or a fluid containing the holders may be pumped.
After the first round of synthesis, all of the holders may be returned to the master chamber. This may be achieved simply by decoupling each reaction chamber from its conduit and pouring its contents into the master chamber or by a process of aspirating the contents of the reaction chambers using a suitable pump arrangement. The process of sorting the holders into the reaction chambers may then be repeated with the computer selecting different holders to go into the respective reaction chambers.
When the synthesis is complete, each microparticle within each holder will bear numerous molecules of a particular library member. In any single holder each microparticle will bear the same library member. At this point one may either produce multiple identical libraries by opening the holders and allocating one or more of the microbeads from each holder to a respective library or one may produce a library in which each library member is represented by numerous microparticles simply by opening each holder and mixing all of the contents together. Methods of combinatorial chemical synthesis suitable for use in the context of this invention include those already known generally in the art and do not require detailed description here. Examples of suitable synthesis techniques are for instance given in WO 96/36436 and WO 96/24061. Code readers of the kind described in those specifications may be employed in this invention also.
While the invention has been described with reference to the preferred embodiment illustrated in the accompanying drawings, it will be appreciated that many modifications and variations of the invention are possible.

Claims

1. A chemical library, wherein said library comprises a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) and bearing a chemical or combination of chemicals within the library which is the same as a chemical or combination of chemicals borne by each other microparticle of the respective individual holder, such that each microparticle code maps to a unique chemical or combination of chemicals within the library and each holder code maps to a unique chemical or combination of chemicals within the library.
2. A chemical library as claimed in claim 1, in combination with a database wherein said database contains for each microparticle code the identity of the chemical or chemicals mapped thereto.
3. A chemical library as claimed in claim 2, wherein said database further contains for each holder code the identity of the chemical or chemicals mapped thereto.
4. A chemical library as claimed in any preceding claim, wherein each chemical or combination of chemicals on a microparticle within the library maps to a unique holder code.
5. A chemical library as claimed in any preceding claim, wherein each chemical or combination of chemicals on a microparticle within the library maps to a unique microparticle code.
6. A chemical library as claimed in any preceding claim, wherein the microparticle code is provided by the shape of each microparticle.
7. A chemical library as claimed in any preceding claim, wherein the holder code is provided by a bar code .
8. A chemical library as claimed in any preceding claim, wherein the library is a combinatorial chemical library and each microparticle bears as said chemical an oligomer comprising a sequence of linked differing sub- unit molecule residues, said oligomer differing from similar oligomers on other microparticles by the sequence of said sub-unit molecule residues.
9. A combinatorial chemical library as claimed in claim 8, wherein each said oligomer is an oligonucleic acid, an oligonucleic acid analogue, a peptide, or a peptide containing one or more non-peptide linking groups.
10. A chemical library as claimed in any one of claims 1 to 7, wherein the library is a combinatorial chemical library and each microparticle bears as said chemical a member of a family of chemical molecules having a structural characteristic in common and differing from other members of said family.
11. A method of forming a chemical library, which method comprises taking a plurality of holders, each holder having a distinguishing machine readable code (the holder code) and each holding a multiplicity of microparticles, each microparticle of each individual holder bearing a machine readable code (the microparticle code) , in a first round of library synthesis splitting the plurality of holders into at least two groups (group la and group Ila and optionally N-2 further groups Ilia to Na) and conducting a first chemical library synthesis iteration on each group to add to each microparticle of each group of holders a first respective chemical library member component, in a second round of library synthesis re-splitting the plurality of holders into at least two new groups (group lb and group lib and optionally N-2 further groups Illb to Nb) and conducting a second chemical library synthesis iteration on each group to add to each microparticle of each group of holders a second respective chemical library member component, and in further rounds of library synthesis repeating said re- splitting and synthesis steps through as many further synthesis iterations as desired to form said library.
12. A method as claimed in claim 11, further comprising removing the microparticles from said holders .
13. A method as claimed in claim 11 or claim 12, wherein said holders are split into groups in each round of library synthesis by passage along a conduit from a master storage chamber to a plurality of reaction chambers branching off said conduit at respective branch points, the holder code of each holder being read prior to each branch point and selected holders being diverted from said conduit into said reaction chambers to split said holders into said groups .
14. A method as claimed in claim 13 , wherein after each iteration of library synthesis, all of the holders are returned to the master storage chamber prior to re- splitting into new groups.
15. A support unit for use in synthesising a chemical library comprising a holder and a multitude of microparticles held by the holder, each microparticle being encoded with an identical code and each being suitable as a substrate on which to synthesise a chemical library, the holder being such as to provide access to the microparticles for reagents applied to the holder during library synthesis.
16. Apparatus for use in synthesising a chemical library comprising a conduit connecting a master storage chamber to a plurality of reaction chambers branching off said conduit at respective branch points, at or before each branch point a code reader suitable for reading codes present on microparticle holders passing along said conduit in use and a valve associated with each reaction chamber and operable in response to the reading of codes by a respective said code reader to open temporarily to allow a selected said holder to pass into the respective reaction chamber.
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