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WO1999056865A1 - Dispositif et procede pour produire un ensemble de molecules lineaires sur un materiau support - Google Patents

Dispositif et procede pour produire un ensemble de molecules lineaires sur un materiau support Download PDF

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Publication number
WO1999056865A1
WO1999056865A1 PCT/EP1999/002958 EP9902958W WO9956865A1 WO 1999056865 A1 WO1999056865 A1 WO 1999056865A1 EP 9902958 W EP9902958 W EP 9902958W WO 9956865 A1 WO9956865 A1 WO 9956865A1
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Prior art keywords
synthesis
chain
run
reaction cell
building blocks
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PCT/EP1999/002958
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German (de)
English (en)
Inventor
Stephan Burkhardt
Jonathan Hall
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Novartis Ag
Novartis-Erfindungen Verwaltungsgesellschaft Mbh
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Application filed by Novartis Ag, Novartis-Erfindungen Verwaltungsgesellschaft Mbh filed Critical Novartis Ag
Priority to AU38258/99A priority Critical patent/AU3825899A/en
Priority to JP2000546874A priority patent/JP2002513549A/ja
Priority to EP99920827A priority patent/EP1079920A1/fr
Publication of WO1999056865A1 publication Critical patent/WO1999056865A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00427—Means for dispensing and evacuation of reagents using masks
    • B01J2219/0043—Means for dispensing and evacuation of reagents using masks for direct application of reagents, e.g. through openings in a shutter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00457—Dispensing or evacuation of the solid phase support
    • B01J2219/00475—Sheets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00513—Essentially linear supports
    • B01J2219/00518—Essentially linear supports in the shape of tapes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00533—Sheets essentially rectangular
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00612—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports the surface being inorganic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00614—Delimitation of the attachment areas
    • B01J2219/00621—Delimitation of the attachment areas by physical means, e.g. trenches, raised areas
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00605—Making arrays on substantially continuous surfaces the compounds being directly bound or immobilised to solid supports
    • B01J2219/00623—Immobilisation or binding
    • B01J2219/00626—Covalent
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00657—One-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00689—Automatic using computers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00695—Synthesis control routines, e.g. using computer programs
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • the invention relates to a method for producing a carrier material with chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular oligonucleotides complementary to at least parts of sections of genetic information carriers, in which at least one reaction cell is relative in one synthesis cycle in a sequence of synthesis cycles in one cycle direction positioned to the carrier material with partially overlapping synthesis areas and chain building blocks of a basic type series intended for chain extension are passed through the reaction cell, so that chain molecules with sequences of the same sequences of chain building blocks are synthesized in several synthesis cycles in synthesis fields repeatedly covered by synthesis areas of a reaction cell.
  • the invention further relates to a device, in particular for producing a carrier material with chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular oligonucleotides complementary to at least parts of sections of genetic information carriers, with a carrier material holder to which the carrier material can be applied, with a storage unit, with at least one reaction cell connected to the supply unit, through which chain components provided for chain extension when the or each reaction cell is placed on the support material in a sealing manner can be passed through in contact with the support material in a synthesis area, with a positioning unit with which the or each reaction cell can be passed through in a synthesis process in a sequence of synthesis cycles, each by a displacement path relative to the carrier material with synthesis areas partially overlapping in synthesis fields is possible, and with a control unit for controlling the supply unit and the positioning unit, so that chain molecules with the same sequences of chain components can be synthesized in several synthesis cycles in synthesis fields repeatedly covered by synthesis areas of a reaction cell.
  • a control unit for
  • the invention further relates to the use of a device for producing a carrier material with chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular at least in part of - 2 -
  • Sections of genetic information carriers of complementary oligonucleotides in particular for carrying out a method according to one of claims 1 to 4, with a carrier material holder to which the carrier material is applied, with a supply unit, with at least one reaction cell connected to the supply unit, by means of which the or each reaction cell on the support material for chain extension intended chain blocks are passed through in contact with the support material in a synthesis area, with a positioning unit with which the or each reaction cell in a synthesis run in a sequence of synthesis cycles each by a displacement path relative to the support material with partially in Positioned synthesis field overlapping synthesis areas, and with a control unit for controlling the storage unit and the positioning unit, so that in several synthesis cycles in the synthesis area repeatedly In a reaction cell covered by synthesis cells, chain molecules with the same sequences of chain building blocks are synthesized.
  • Such a method and such a device are known from the article "Arrays of complementary oligonucleotides for analyzing the hybridization behavior of nucleic acids" by E.M. Southern, S.C. Case-Green, J.K. Eider et al., Pages 1368 to 1373, Nucleic Acid Research, 1994, vol. 22, no. 8, known.
  • a reaction cell with a diamond-shaped or circular synthesis area is offset in one synthesis step in a sequence of synthesis cycles in one passage direction relative to a carrier material with partially overlapping synthesis areas, with a reaction solution with one provided for chain extension in each synthesis cycle Chain building block is passed through the reaction cell in contact with the carrier material.
  • Chain molecules with a predetermined sequence of chain building blocks in particular oligonucleotides complementary to sections of genetic information carriers, can be synthesized by passing a reaction solution with a predetermined chain building block in the synthesis area over the carrier material in each case during the synthesis cycles, so that a reaction cell is repeated from synthesis areas covered synthetic fields, chain molecules with sections of the same sequence of chain building blocks are synthesized.
  • the chain molecules are preferably designed as oligonucleotides.
  • the oligonucleotides serve to analyze the hybridization behavior of nucleic acids.
  • the target nucleic acid when inhibiting the expression of a protein encoded by a target nucleic acid by interaction (hybridization) of relatively short oligonucleotides complementary to the base sequence of the target nucleic acid with the target nucleic acid or a target nucleic acid - protein complex, the target nucleic acid in particular a single-stranded target nucleic acid with a secondary and tertiary structure, in particular a "messenger RNA" (mRNA) (so-called “antisense technology”), there is the problem of inhibiting translation due to the complicated secondary and tertiary structure of the mRNA to identify available binding sites for a specific mRNA for an oligonucleotide, the localization of which is difficult to predict.
  • mRNA messenger RNA
  • a carrier material on which the above-mentioned arrangement of oligonucleotides is located can be used in particular in an assay with which it is determined whether or how strongly a target nucleic acid, such as such an mRNA molecule, is able to hybridize one or more of the oligonucleotides on the support material.
  • the hybridization conditions are chosen as far as possible so that the secondary or tertiary structure of the target nucleic acid is largely preserved and matches the corresponding in vivo structure of the RNA.
  • the strength of the hybridization can serve as a measure of the accessibility of a specific region of the target nucleic acid by the oligonucleotides.
  • a single oligonucleotide, identified in this way and capable of hybridizing with the target nucleic acid can then be produced by known methods and used in antisense technology for medical-therapeutic or diagnostic purposes.
  • oligonucleotides that can be hybridized to an mRNA on a solid support as so-called “antisense” to identify binding sites that can be used for inhibition
  • the oligonucleotides each consisting entirely of a single defined type of chain building block, for example of the deoxyribonucleotide -Type, of the deoxyribophosphorothioate nucleotide type or of the 2'-O-methyl-ribonucleotide type (ie in each case from chain building blocks of a single basic type series).
  • oligonucleotides on the support which are composed entirely of chain molecules of a single specific basic type series.
  • Such oligonucleotides often do not have the multiple properties desired in antisense technology, such as, for example, high binding affinity for the target nucleic acid and or high resistance to endogenous nucleases combined with the ability to effectively inhibit expression of the target protein at the level of the target nucleic acid.
  • oligonucleotides are those which combine several properties, for example a high binding affinity for the target nucleic acid / or resistance to degradation by endonucleases combined with the property that the target nucleic acid is cleaved efficiently by the endogenous one To cause RNAse H. It has been possible to combine a large number of such properties in one and the same molecule in oligonucleotides, in that such an oligonucleotide is constructed in sections from chain building blocks of a specific basic type series, each section being responsible for a specific property or function of the oligonucleotide. Such oligonucleotides are often referred to as "chimeric" oligonucleotides.
  • Chimeric oligonucleotides consist of chain building blocks of at least two different basic type series, which are synthesized in at least two successive or adjacent sections, often also in three or more such sections (see, for example, ST Crooke et al., J. of Pharmacology and Experimental Therapeutics 277 (1996) , Pp. 923-937).
  • the invention has for its object to provide a method and a device of the type mentioned, in which on the carrier material an efficient synthesis of an arrangement of chain molecules with the same sequence of sections of chain building blocks of different basic type series, in particular oligonucleotides with a chimeric structure, but otherwise largely the same characteristic characteristics such as base recognition characteristics can be carried out on oligonucleotides.
  • the invention is based in particular on the object of providing a method and a device of the type mentioned at the beginning for chain molecules which are at least partially complementary to oligonucleotides of genetic information carriers, the oligonucleotides having a chimeric structure, as a result of which, for example, a high binding affinity and or a high resistance against such oligonucleotides - 5 -
  • chain components of at least two different basic type series are stored in the supply unit, that the or each reaction cell can be positioned with the control unit in at least two synthesis cycles in the same direction in such a way that synthesis cycles of a synthesis cycle the associated synthesis area is overlaid in sections with a synthesis field hitherto uncovered in this synthesis run, in which chain molecules with a maximum chain length synthesized in the or each previous synthesis run are present, and that, controlled by the control unit, chain building blocks of different basic type series can be passed through the or each reaction cell in two successive synthesis runs .
  • a device can comprise a corresponding supply unit, which in each case contains chain components of a single basic type series, and in which the supply unit is designed to be interchangeable, so that after each synthesis cycle the supply unit is replaced by a another supply unit with chain components of another basic type series can be exchanged.
  • chain components of at least two different basic type series are stored in the supply unit, that with the control unit the or each reaction cell can be positioned in at least two synthesis cycles in the same direction in such a way that one during synthesis cycles
  • the associated synthesis area is partially sectioned with a synthesis field hitherto uncovered in this synthesis process, in which chain molecules with a maximum chain length synthesized in the or each previous synthesis process are present, and that, controlled by the control unit, chain building blocks of different basic type series are passed through the or each reaction cell in two successive synthesis processes become.
  • the present invention thus furthermore relates to the use of a device for producing a carrier material comprising an arrangement of chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular of oligonucleotides complementary to at least parts of sections (20) of genetic information carriers, adjacent synthesis fields Chain molecules with sections of the same sequence of chain building blocks of different basic type series include.
  • Such an arrangement thus comprises chain molecules that comprise at least two sections, each with different basic type series of chain building blocks.
  • Another object of the invention is a carrier material, comprising an arrangement of chain molecules synthesized in synthesis fields with a predetermined sequence of chain building blocks, in particular of oligonucleotides complementary to at least parts of sections (20) of genetic information carriers, adjacent synthesis fields being chain molecules with the same sequence of chain building blocks of different basic type series include.
  • Such an arrangement thus comprises chain molecules that comprise at least two sections, each with different basic type series of chain building blocks.
  • the present invention is directed to a carrier material which can be produced by a method according to the present invention. - 7 -
  • chain molecules with blocks of chain building blocks of different basic type series, but otherwise the same characteristics as base recognition characteristics can be synthesized, whereby, for example, different sequences of basic type series and / or differently designed synthesis areas of reaction cells used in the various synthesis runs can achieve high varieties in the synthesized chain molecules with high efficiency.
  • the synthesis areas of each reaction cell are expediently rectangular in shape, each having the same transverse sides, the length of each long side of a reaction cell corresponding to an integral multiple of a displacement path in the longitudinal direction.
  • the reaction cells are shifted from one synthesis cycle to the next by the displacement distance corresponding to the size of a synthesis field in the longitudinal direction.
  • the reaction cells are to be positioned in a first synthesis cycle of each synthesis run in such a way that the synthesis areas of the reaction cells are flush with one another in the passage direction.
  • the chain molecules are preferably oligonucleotides.
  • Such oligonucleotides are capable of pairing with a complementary nucleic acid strand, in particular an mRNA, and consist of individual chain building blocks, namely nucleosides or nucleoside analogues, which as a rule carry a nucleic acid base or a base analog, which mediate the properties of the oligonucleotides for base pairing .
  • a chain building block also includes an intemucleosidic bridge bond to the next building block, for example of the phosphodiester, phosphorothioate or amide type, as described, for example, in A. de Mesmaeker et al., Acc. Chem. Res. 28 (1995), pp. 366-374.
  • a basic type series of the chain molecules is in each case chain building blocks of the same basic structure, members of a first basic type series being members of a second basic type series in particular by the properties to be achieved, for example those by the part of the chain molecule under consideration - 8th -
  • oligonucleotides such properties of the chain molecules are, for example, the affinity for a target nucleic acid, the nuclease resistance, the sensitivity to RNAse H or the toxic properties.
  • An advantageous oligonucleotide which is used as a drug in the context of antisense technology, combines several advantageous properties.
  • the basic types of building blocks are natural nucleosides, such as 2'-deoxyribonucleosides or ribonucleosides; or by 2'-0-alkyl-modified ribonucleosides, the 2'-O-alkyl group, which is preferably 2'-OC 1 -C 5 alkyl, in particular 2'-O-methyl or 2'-O-ethyl, unsubstituted or, in the case of 2'-O-ethyl at the ⁇ -position, can be substituted with -OH, -F or alkoxy, preferably with dC 5 -alkoxy, in particular methoxy, where 2'-O- (2-methoxyethyl ) -modified ribonucle
  • building blocks of a basic type series are synthesized in one section in an oligonucleotide, an oligonucleotide consisting of at least 2, for example 2 or 3 sections.
  • Chimeric oligonucleotides which consist of two sections are preferred as chain molecules, one section consisting of 2'-O- (2-methoxyethyl) ribonucleosides which are linked to one another via phosphodiester bridges or phosphorothioate bridges, and the other section 2'-deoxyribonucleosides, which are linked together via phosphorothioate bridges.
  • Chain molecules are chimeric oligonucleotides which consist of three sections, the first section consisting of 2'-O- (2-methoxyethyl) ribonucleosides, viewed from 5'- in 3'-direction, via phosphodiester bridges or phosphorothioate bridges are linked together, the adjoining second section consists of 2'-deoxyribonucleosides which are linked together via phosphorothioate bridges, and the adjoining third section in turn consists of 2'-O- (2-methoxyethyl) ribonucleosides , which are linked together via phosphodiester bridges or phosphorothioate bridges.
  • two individual sections are preferably linked to one another via a phosphorothioate bridge if the nucleoside at the 3 'end of a section is one 2'-deoxyribonucleoside; or alternatively, two individual sections are linked to one another via a phosphodiester bridge or via a phosphorothioate bridge if the nucleoside at the 3 'end of a section is a 2'-O- (2-methoxyethyl) ribonucleoside.
  • Suitable, preferred chimeric oligonucleotides have a total length of about 15 to about 25, for example 20, nucleoside building blocks, the section consisting of 2'-deoxyribonucleoside building blocks comprising at least about 5 such building blocks.
  • FIG. 1 shows a schematic representation in one exemplary embodiment of a carrier material strip with a reaction cell which has been placed on and has a synthesis area covering four synthesis fields, after a first synthesis - 10 -
  • FIG. 2 shows the arrangement according to FIG. 1 after the last synthesis cycle of the first synthesis run has been carried out
  • FIG. 3 shows the arrangement according to FIG. 2 after carrying out a first synthesis cycle of a further second synthesis run with chaining of a chain building block of a different basic type series used in relation to or in the first synthesis run,
  • FIG. 5 in a schematic representation in a further embodiment
  • FIG. 6 shows the arrangement according to FIG. 5 with a reaction cell placed on top of it, which has a synthesis area covering five synthesis fields, after a second synthesis cycle of a further second synthesis run, FIG.
  • FIG. 7 shows the arrangement according to FIG. 6 after the last synthesis cycle of the second synthesis run has been carried out
  • FIG. 8 shows the arrangement of FIG. 7 with a covering two synthesis fields
  • Reaction cell having synthesis area after carrying out the last synthesis cycle of a further third synthesis pass Reaction cell having synthesis area after carrying out the last synthesis cycle of a further third synthesis pass.
  • FIG. 9 shows a device for producing a carrier material with chain molecules synthesized in synthesis fields according to the invention in a schematic perspective view. - 11 -
  • Fig. 10 shows the lifting unit of the device schematically in a perspective enlarged view.
  • FIG. 11 shows the lifting unit according to FIG. 10 in a top view.
  • Fig. 12 shows a cross section through the support beam of the lifting unit with a
  • reaction cell 13 is a plan view of a reaction cell
  • FIG. 1 shows, in a schematic representation in an exemplary embodiment of a method and a device according to the invention, a support material strip 17 which can be occupied with a total of 16 synthesis fields 1 to 16 and which is made of polypropylene (PP), for example chemically treated for the synthesis of oligonucleotides, as the support material.
  • the carrier material strip 17 is placed on a carrier material holder, not shown in FIG. 1, for example in the form of a metal plate provided with an elastic layer.
  • a displaceable reaction cell 18, shown in its outline is placed on the carrier material strip 17 and is connected to a storage unit, not shown in FIG. 1.
  • reaction cells 18 are provided which, for example for carrying out a parallel synthesis, are arranged next to one another on a reaction cell carrier and can be moved together.
  • reaction fluids for example reaction solutions, which are provided for a synthesis of chain molecules with chain extension with a predetermined, specific sequence of chain components, are stored or are successively stored, which are controlled by the control unit or can be fed to each reaction cell 18.
  • the reaction cell 18 can be placed on the carrier material strips 17 with a seal.
  • the reaction cell 18 has a recess with a rectangle-shaped border which is open on one side and delimits a synthesis region 19, the dimensions of which in the illustrated exemplary embodiment correspond to the size of four adjacent synthesis fields 1 to 16. 1 is the means of a - 12 -
  • oligonucleotides complementary to at least part of a section 20 of a genetic information carrier are synthesized with the above-described device in a method.
  • the individual bases of section 20 carrying the genetic information are symbolically identified in FIG. 1 with B1 to B17 and each stand for one of the bases A, C, G and U or T according to the Watson-Crick model.
  • oligonucleotides 21 each assigned to a synthesis field 1 to 16 as chain molecules. Each oligonucleotide 21 is attached to a residual group R of the substrate strip 17 prepared for binding.
  • the reaction cell 18 is arranged in the position of a first synthesis cycle of a first synthesis run in which a reaction fluid intended for chain extension with a natural nucleotide N1 complementary to the base B1 of the section 20 has been passed through the reaction cell 18 is.
  • the reaction fluid has washed over the synthesis area 19, so that the nucleotide N1 has been added to the residual groups R of the first four synthesis fields 1 to 4 on the left in the illustration in FIG. 1.
  • FIG. 2 shows the arrangement according to FIG. 1 with the reaction cell 18 in one of the four synthesis fields 13 to 16 located on the left in the selected illustration and in the passage direction. - 13 -
  • each four natural nucleotides N1 to N13-containing oligonucleotides 21 have been synthesized, each oligonucleotide 21 synthesized in one of the synthesis fields 4 to 13, as represented by the same sequence of atomic numbers, being complementary to a coherent part of section 20 of bases B1 to B13 of the genetic information carrier.
  • FIG. 3 shows the arrangement according to FIG. 2 with the reaction cell 18 having the synthesis area 19 covering the four synthesis fields 1 to 16 in the positioning of a first synthesis cycle of a further second synthesis run.
  • the reaction cell 18 is brought into the same position as in the first synthesis cycle of the first synthesis run, covering the first four synthesis fields 1 to 4 on the left in the illustration in FIG. 3.
  • a reaction fluid with a now modified nucleotide n5 as a chain building block of a different basic type series compared to the one used in the first synthesis run is then flushed out, the base of the modified nucleotide n5 being complementary to the base B5 of section 20 of the genetic information carrier.
  • an oligonucleotide 21 composed of five chain building blocks in the sequence N1-N2-N3-N4-n5 is synthesized in the fourth synthesis field 4 counted from the left, which oligonucleotide 21 forms the first five bases B1 to B5 of section 20 of the genetic information carrier is complementary and has natural nucleotides N1 to N4 of the basic type series used in the first synthesis run and the modified nucleotide n5 of the further basic type series used in the second synthesis run.
  • edge-side synthesis fields 1 to 3 oligonucleotides 21 that are not or not completely complementary to the beginning of section 20 of the genetic information carrier have been synthesized.
  • reaction fluids with modified nucleotides n6 to n17 are then passed through after shifting the reaction cell 18 by a displacement path corresponding to the size of an adjacent synthesis field 5 to 16, whereby the sequence follows - 14 -
  • the sequence of the modified nucleotides n6 to n17 complementary to the bases B6 to B17 is predetermined.
  • FIG. 4 shows in the arrangement according to FIG. 3 the reaction cell 18 in the position in the last synthesis cycle of the second synthesis run after flushing with a reaction fluid containing the modified nucleotide n17 complementary to the last base B17 of section 20 of the genetic information carrier. From Fig. 4 it can be seen that now except for the three edge-side synthesis fields 1, 2, 3, 14, 15, 16 in the middle synthesis fields 4 to 13 sequence homologues, each with a part of section 20 of the genetic information carrier complementary oligonucleotides 21 with a Length of eight nucleotides have been synthesized.
  • Each oligonucleotide 21 has four natural nucleotides N1 to N13 in blocks and then four modified nucleotides n5 to n17, each following the natural nucleotides N4 to N13 added last in the first synthesis run in one of the sequences of bases B1 to B17 of section 20 of the Genetic information carrier corresponding order.
  • the carrier material strip is now, in particular with regard to the oligonucleotides 21 synthesized in the middle synthesis fields 4 to 13, as chain molecules with eight chain building blocks arranged in two blocks or sections with different basic type series, for use in an assay for examining parts of section 20 of the genetic information carrier with regard to accessible Binding sites for so-called "antisense 'oligonucleotides are available, as mentioned above.
  • FIGS. 1 to 4 shows schematically similar to FIGS. 1 to 4 in a further exemplary embodiment of a method and a device according to the invention the carrier material strip 17 according to the aforementioned exemplary embodiment.
  • oligonucleotides 21 which are complementary to connected parts of section 20 of the genetic information carrier and have a chain length which is greater than the exemplary embodiment explained with reference to FIGS. 1 to 4 using natural and modified nucleotides in three differently long blocks of pairs of different basic type series are intended be synthesized.
  • a reaction cell 22 with a synthesis area 23 covering three synthesis fields 1 to 16 is arranged in the arrangement with a tenth synthesis cycle - 15 -
  • the reaction cell 22 was arranged in such a way that the synthesis area 23 covered the third to fifth synthesis fields 3, 4, 5 counted from the left in the illustration in FIG. 5. Then, in the subsequent synthesis cycles of the first synthesis run, the reaction cell 22 was placed in each case on the next neighboring synthesis field 5 to 14 and flushed with a reaction fluid containing a complementary natural nucleotide N2 to N10 corresponding to the sequence of bases B2 to B10, so that up to the in 5, the tenth synthesis cycle shown have built up the complementary oligonucleotides 21 shown.
  • FIG. 6 shows the arrangement according to FIG. 5 after a second synthesis cycle of a further second synthesis run.
  • a reaction cell 24 is used, with the synthesis area 25 of which five synthesis fields 1 to 16 can be covered.
  • the reaction cell 24 is arranged such that the only synthesis field 5 to 12 with a chain of three natural nucleotides N1 to N12 is the fifth synthesis field 5 counted from the left in the illustration in FIG. 6 together with the left side outer four synthesis fields 1 to 4 has been covered for the first time by the synthesis area 25.
  • the first two synthesis fields 1, 2 located on the left outside in the illustration in FIG. 6 were first flushed with a reaction fluid.
  • a reaction fluid which contains the modified nucleotide n4 which is complementary to the fourth base B4 of section 20, while in the second synthesis cycle of the second synthesis run shown in FIG. 6 that to the fifth base B5 of section 20 of the genetic information carrier complementary, modified nucleotide n5 was contained in the reaction fluid.
  • reaction cell 24 is in each case displaced by a displacement path corresponding to the size of a synthesis field 1 to 16, and reaction fluids with complementary modified nucleotides n6 to n15 in the corresponding sequence of the bases B6 to B15 of section 20 of the genetic information carrier are passed through . - 16 -
  • FIG. 7 shows in the illustration in FIG. 6 the positioning of the reaction cell 24 used in the second synthesis run after the last synthesis cycle of the second synthesis run has been carried out. From Fig. 7 it can be seen that apart from the four synthesis fields 1 to 4, 13 to 16 located on the right and left edges in the middle synthesis fields 5 to 12 oligonucleotides with eight chain components N1 to N10, n4 to n15 have been synthesized, the in the first synthesis step, three nucleotides N1 to N10 synthesized block by block of a natural basic type series and the five nucleotides n4 to n15 linked in the second synthesis cycle are assigned in blocks to a modified basic type series.
  • FIG. 8 shows the arrangement according to FIG. 7 after carrying out the last synthesis cycle of a further third synthesis run, in which, as chain building blocks, a basic type series different from the previous synthesis run in turn complements the natural nucleotides N9 to N17 to the bases B9 to B17 to those in the first two Synthesis runs synthesized oligonucleotides 21 have been linked.
  • a reaction cell 26 is used, with the synthesis region 27 of which two adjacent synthesis fields 1 to 16 can be covered.
  • a reaction fluid with a natural nucleotide N9 complementary to the ninth base B9 of section 20 of the genetic information carrier was passed through the reaction cell 26.
  • reaction fluids containing complementary natural nucleotides N10 to N17 predetermined by the sequence of the bases B10 to B17 were flushed through the reaction cell 26 to cover for the first time the next neighboring synthesis field 6 to 16. - 17 -
  • 5 to 12 sequence homologous oligonucleotides 21 with a length of ten chain building blocks, which are each complementary to part of section 20 of the genetic information carrier and consist of a block of three, are now synthesized in the middle synthesis fields 5 to 12 after completion of the third synthesis run natural nucleotides N1 to N10, a block of five modified nucleotides n4 to n15 and a further block of two natural nucleotides N9 to N17 are constructed.
  • Each block of natural nucleotides N1 to N10 or N9 to N17 and of modified nucleotides n4 to n15 has a length which corresponds to the number of synthesis fields covered by the synthesis areas 23, 25, 27 of the reaction cells 22, 24, 26 used in the corresponding synthesis runs Corresponds to 1 to 16.
  • longer-chain chain molecules can be synthesized by using reaction cells having larger synthesis areas and by carrying out a plurality of synthesis cycles having a greater number of synthesis cycles than in the exemplary embodiments explained in detail above.
  • reaction cells with differently sized synthesis areas, diverse variations in the block-wise sequence of basic type series can be achieved, it being expedient that the rectangular sides of the synthesis areas have the transverse sides of the reaction cells of the same size and the size of the long sides a full-page multiple of that due to a displacement path in the passage direction correspond to the specified dimension of the synthesis fields and the reaction cells are displaced in the longitudinal direction by the corresponding dimension of the synthesis fields.
  • a cut polypropylene film (PP: Mobil 40MB400, 400 mm x 300 mm) is placed in a cylindrical glass drum (diameter: 170 mm, length: 400 mm) in a solution (180 ml) of 0.9 g chromium (VI) oxide (Fluka), dissolved in a 1: 1 mixture of acetic anhydride: acetic acid (Fluka), immersed.
  • the reaction drum is quickly rotated for 1 hour using a mechanical rolling device.
  • the film is then removed, immersed in a bath of 1 M aqueous acetic acid for 30 minutes and then rinsed with methanol (Fluka) for 30 minutes.
  • the film is dried in a high vacuum, being located between 2 glass plates for protection.
  • the film is then immersed in a corresponding glass container in a 1 M solution of BH 3 (Aldrich Chemical Co.) in tetrahydrofuran (180 ml), which is diluted to 0.2M with additional tetrahydrofuran.
  • the glass vessel is rotated quickly for 1 hour.
  • the film is removed, immersed in 1 M aqueous acetic acid, rinsed in methanol and dried under vacuum.
  • the film treated in this way is used without further treatment as a carrier material in a process according to the invention.
  • FIG. 9 shows an automated device (so-called "array maker") which is used for the synthesis of a carrier material according to the invention with chain molecules synthesized in synthesis fields.
  • the device comprises an automatic cell positioning device or cell positioning unit 36 and a DNA synthesizer (ABI 394/4) 31.
  • the DNA synthesizer 31 is connected to the automatic cell positioning device 36 by means of hose connections for the reagents (not particularly reaction solutions) which are not shown in the drawing , Purge liquids and purge gases).
  • the inlet tubing connections that are normally used to deliver the reagents from the DNA synthesizer to the 4 reaction columns, which comprise a solid support and are standard for the synthesis of oligonucleotides, are instead with the inlet openings of 4 reaction cells 32, 33, 34, 35 connected.
  • the outlet tube connections that normally connect the DNA synthesizer to the respective outlet openings of the 4 reaction columns mentioned are instead to the outlet openings of the 4 reaction cells - 19 -
  • the cell positioning machine 36 is connected via a trigger line (not shown in the drawing) to an electronic control unit 37, with the aid of which a trigger signal ("advance fraction collector signal") for the respective next cell position of the cell positioning machine 36 from the DNA synthesizer 31 the cell positioning machine 36 is transmitted.
  • the automatic cell positioning device 36 and the DNA synthesizer 31 stand on a stable base 38 below an extractor hood 39 with a rear wall 40.
  • the automatic cell positioning device 36 has its own protective hood 41, which is only shown in parts in the drawing, and which has a vertical sliding door is provided, which are moved by means of a handle 42 and via pulling ropes (not shown in the drawing), which are connected to a counterweight (not shown in the drawing) and are deflected via rollers 43, into an indicated upper position and a likewise indicated lower position can.
  • the rollers 43 are connected via brackets and struts to the profile frame 45, which consists of several profile tubes 44 and is supported on the base 38.
  • the automatic cell positioning device (36) is moved by an electrically operated unit 46, which, controlled by the control unit 37, moves a pressure plate 47 vertically in stages between an illustrated upper position and an indicated lower position.
  • the PP film 49 is fastened on the pressure plate 47 with the aid of 5 vertical metal holding bars 48 by means of screws located at the upper and lower ends of the holding bars.
  • a thin piece of rubber 90 (see FIG. 11) is located between the PP film 49 and the pressure plate 47.
  • the pressure plate 47 is moved vertically to preprogrammed positions.
  • the reaction cells 32, 33, 34 and 35 are horizontally next to each other on a support beam
  • the lifting unit 51 is shown separately and enlarged in FIG. 10. 11 shows the lifting unit
  • the lifting unit 51 in plan view together with the reaction cells 32, 33, 34 and 35 and with the pressure plate 47.
  • the lifting unit 51 has a left-hand guide 52 and a right-hand guide 53, each of which is designed as a hollow cylinder.
  • a left push rod 54 and a right push rod 55 each extend through the guides 52 and 53.
  • Both push rods 54, 55 are prestressed with the aid of pressure springs (not shown in the drawing) arranged in the guides 52, 53 such that the reaction cells 32, 33, 34, 35 are pressed onto the pressure plate 47.
  • eccentric disks not shown in the drawing, are provided, which cooperate with the end faces 56 and 57 of the pressure rods 54, 55, around the - 20 -
  • the eccentric discs are adjusted with the aid of an electric drive, which is also connected to the control electronics 37.
  • the push rods 53, 54 are detachably connected to the support beam 50 by means of knurled nuts 58. In this way, the reaction cells 32, 33, 34, 35 can be removed together with the support bar 50. After dismantling the support bar 50, it is possible to detach each individual reaction cell 32, 33, 34, 35 from the support bar 50 and, if necessary, to replace it with another reaction cell 32, 33, 34, 35.
  • the connection of the reaction cell 32 is shown in FIG. 12, for example.
  • the reaction cell 32 is fastened to a pin 59 which extends through a bore 60 in the support beam 50.
  • the pin 59 On the side of the support beam 50 pointing away from the reaction cell 32, the pin 59 is provided with a thread onto which a knurled nut 61 is screwed.
  • the pin 59 with the screwed-on knurled nut 61 is prestressed with the aid of a compression spring 62, so that the knurled nut 61 is pressed against the back of the support beam 50.
  • the compression spring 62 is designed to be significantly weaker than the compression springs provided in the guides 52, 53, so that when the reaction cells 32, 33, 34, 35 are placed on the plastic film 49 and the pressure plate 47, the compression spring 52 is compressed and the pressure force of the reaction cell 32, 33, 34, 35.
  • the optimal pressure force can be set quickly and easily by changing the pressure spring 62.
  • a pressure spring 62 with 42N is used for the PP film 40MB400 used as carrier material in this example.
  • the compression spring 62 ensures a uniform pressure of the reaction cells 32, 33, 34, 35 against the plastic film 49 (see FIG. 11), which either directly on the pressure plate 47 or with the interposition of an intermediate carrier film 90 (see FIG.
  • the intermediate carrier film 90 which is preferably up to 1 mm thick (see FIG. 11), consists, for example, of a polymer film made of Neoprene 60 ° Shore A or natural rubber (for example Paragummi 40 ° Shore A), both from Richterich + Zeller AG, Basel, Switzerland are distributed.
  • reaction cells 32, 33, 34, 35 which are open on one side, is explained in more detail below with reference to FIGS. 12 and 13. - 21 -
  • the reaction cells 32, 33, 34, 35 consist of a block with a rectangular cross section made of a deformable material or a plastic.
  • PTFE is used.
  • the rear side 63 facing the support beam 50 and the outer sides 64, 65, 66, 67 and 68 are each flat.
  • a peripheral edge strip 70 is provided, which encloses a flat space 71 (corresponding to 19, 23, 25, 27 in FIGS. 1 to 8).
  • the height of this flat space 71 and the height of the edge strip 70 are one millimeter.
  • the four edge strip sections circumscribing a rectangle are each 40 mm x 10 mm in size. In the area of the corners 72, 73, 74 and 75, the edge strip sections run in an arc shape, the inner radius being approximately 0.5 millimeters.
  • bores 76 and 77 running at right angles to the upper side, which in the exemplary embodiment shown likewise have a radius of 0.5 millimeters.
  • the bore 76 opens into an outlet channel 78.
  • the lower bore 77 opens into an inlet channel 79.
  • Each inlet channel 79 and each outlet channel 78 of the reaction cells 32, 33, 34, 35 is connected to the synthesizer 1 via a hose connection, not shown in the drawing.
  • the support bar 50 is moved horizontally in order to press the reaction cells 32-35 against the PP film.
  • the control unit 37 sends a signal to the cell positioner 36.
  • the support bar 50 is then moved horizontally to remove the reaction cells 32-35 from the surface of the PP film .
  • the pressure plate 47 is then moved vertically into a new position.
  • the carrier bar 50 is again moved horizontally, and the reaction cells 32-35 are pressed again onto the surface of the PP film.
  • the preparation of an arrangement of chimeric oligonucleotides on the solid support material comprises the following steps: - 22 -
  • a PP film 49 preactivated as in Example 1 is fastened in the cell positioning device 36 using a rubber piece 90 in order to guarantee a good seal.
  • the vertical support bars 48 are attached in place to ensure that the film 49 holds well.
  • the selected reaction cells (between 1 and 4) 32, 33, 34, 35, with corresponding compression springs 62 are attached to the support bar 50, which is installed on the cell positioning machine.
  • the selected reaction cells 32, 33, 34, 35 are connected to the inlets and outlets of the DNA synthesizer via hose connections.
  • the coordinates of the starting position and the length of the displacement path are programmed into the control unit 37, the pressure plate 47 is moved into the starting position, and the reaction cells 32, 33, 34, 35 are brought into contact with the PP film 49.
  • the DNA synthesizer is programmed to synthesize the desired oligonucleotide sequences in the desired order and the synthesis begins.
  • the lifting unit 51 lifts the reaction cells away from the surface of the PP film, the pressure plate moves vertically upwards by the selected displacement length, and moves the lifting unit 51 forwards the support beam 50, whereby the contact between the reaction cells and the film is established.
  • a reaction cycle is then carried out by means of the DNA synthesizer in order to synthesize a first building block of an oligonucleotide on the surface of the film or in order to carry out a chain extension reaction of an oligonucleotide.
  • Step 7 is repeated as many times as necessary for a synthesis run.
  • the PP film 49 is pretreated as described herein.
  • a sheet of natural rubber (Paragummi 40 ° Shore, Richterich & Zeller, Basel, Switzerland, 0.5 mm thick, 400 x 300 mm) 90 is used as described.
  • 4 rectangular reaction cells 32, 33, 34, 35 made of Teflon are used, each reaction cell being held by two compression springs 62 (42N).
  • Each reaction cell is connected to the inlet and outlet tubing connections of an ABI 394/4 DNA synthesizer (Perkin Elmer Corp., Foster City, CA, USA).
  • ABI 394/4 DNA synthesizer Perkin Elmer Corp., Foster City, CA, USA.
  • the starting position of the beam is set to 5 mm below the top edge of the PP film. 5 mm are programmed as the length of the displacement path of the reaction cells after each synthesis cycle. Due to the length of the reaction cells of 40 mm, an arrangement of oligonucleotides is generated in one synthesis run, which have a maximum length of 8 building blocks.
  • chimeric oligonucleotides total length of a chimeric oligonucleotide: 16 nucleotide building blocks, of which (i) building blocks 1 to 8: deoxyribonucleosides, which are linked to one another via phosphorothioate bridges, and (ii) building blocks 9 to 16 : 2'-0- (2-methoxyethyl) ribonucleosides which are linked to one another via phosphodiester bridges).
  • building blocks 1 to 8 deoxyribonucleosides, which are linked to one another via phosphorothioate bridges
  • building blocks 9 to 16 2'-0- (2-methoxyethyl) ribonucleosides which are linked to one another via phosphodiester bridges.
  • the supply unit comprises, among other things, 8 bottles with nucleoside building blocks, 4 bottles each containing one of the four deoxyribonucleotides (corresponds to chain building blocks of a first basic type series), and the 4 remaining bottles each to one of the four 2'-0- (2-methoxyethyl) -ribonucleosides (corresponding to Chain components of a second basic type series) included.
  • the corresponding bottles with the building blocks of the first basic type series are activated in the synthesis of the 1st to 8th building blocks of the oligonucleotides
  • the corresponding bottles with the building blocks of the second basic type series are activated for the synthesis of the 9th to 16th building blocks.
  • the synthesis of the DNA phosphorothioate portion of the oligonucleotides is accomplished by programming the DNA synthesizer to use one of the four possible reaction cells, for example cell 32, to provide the desired arrangement of oligonucleotides - 24 -
  • reaction cells for example 33, 34 and 35, are freely available in order to simultaneously synthesize adjacent arrays of oligonucleotides on the same PP film which correspond to other target sequences.
  • Commercially available phosphoramidites are used as nucleotide building blocks (Cruachem, A: 208120-21; G: 208190-2; C: 208130-21; T: 208100-21).
  • the synthesis machine dilutes 2 g of the phosphoramidites with 50 ml of acetonitrile (MWG-Biotech). In the synthesis, tetrazole (Cruachem.
  • this sequence is complementary to the sequence of the target nucleic acid. This sequence is programmed into the synthesizer, the synthesis taking place from 3 'to 5'.
  • the synthesis protocol reproduced below for coupling a nucleotide building block to the film or to a nucleotide chain is a protocol of the DNA synthesizer ABI 394/4, which is based on the specific circumstances of the production of an arrangement of oligonucleotides has been adapted on a PP film.
  • the protocol is reproduced in English, since the machine must be programmed in English: -25-
  • Bottle 18 contains acteonitrile, bottle 14 the detritylation solution, bottle 10 the phenylacetyl disulfide reagent and bottle B the respective phosphoramidite solution.
  • the described protocol is repeated correspondingly often until the desired arrangement of the first part of chimeric oligonucleotides has been completed, the first part, based on a single oligonucleotide, each comprising a maximum of the first 8 building blocks, and the arrangement as a whole includes the sequence given above.
  • the reaction cell is reset to the original starting position.
  • the DNA synthesizer is programmed so that 2'-0- (2-methoxyethyl) ribonucleoside phosphoramidite building blocks (2g) are used for the 9th to 16th building blocks, which according to P. Martin, Helv. Chim. Acta 78 (1995), pp. 486-504 and dissolved in 50 ml of acetonitrile.
  • the tetrazole (Cruachem. No. 17112044) used in the further course of the synthesis is used as obtained from the manufacturer.
  • Trichloroacetic acid in 1, 1, 1 trichloroethane (2.5% w / v) is used for demetilation.
  • a mixture of iodine / lutidine water / acetonitrile (0.65 g (Fluka) / 50 ml (Fluka) / 2.5 ml / 450 ml) is used for the oxidation.
  • the synthesis protocol is modified according to the use of the modified phosphoramidite building blocks.
  • the first base (3 'end) in each sequence is coupled to the PP film at the starting position of the reaction cell.
  • the starting position corresponds to the starting position at the beginning of the first synthesis run (5mm below the top edge of the PP film).
  • Subsequent bases are added while the reaction cell is moved vertically along the carrier film (displacement length 5 mm).
  • the synthesis sequence is based on the following base sequence:
  • This sequence is programmed into the synthesizer; the synthesis again takes place in the 3'-5 'direction.
  • the first base "g" at the 3 'end of SEQ ID No 2 corresponds to the 9th base "G” counted from the 3' end of SEQ ID NO 1, since the maximum length of the oligonucleotides synthesized in the 1st synthesis step is 8 chain building blocks .
  • Bottle 18 contains acteonitrile
  • bottle 14 contains the detritylation solution
  • bottle 15 contains the oxidation reagent (l 2 / lutidine / H 2 0)
  • bottle B contains the respective phosphoramidite solution.
  • the PP film is removed from the device and rinsed in isopropanol.
  • the film is then washed in 32% ammonia at room temperature in a reaction drum (see Example 1).
  • the chamber is rotated rapidly for 16 hours to ensure good reagent mixing.
  • the film is removed from the chamber, rinsed with water and methanol and briefly dried under vacuum.
  • the film with the desired arrangement of chimeric oligonucleotides can now be used in a standard hybridization assay, as mentioned above.

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Abstract

L'invention concerne un procédé, un dispositif et une utilisation dudit dispositif pour produire un matériau support présentant des molécules linéaires synthétisées dans des zones de synthèse, notamment des oligonucléotides complémentaires de segments de supports d'informations génétiques. Selon l'invention, après un passage de synthèse pour synthétiser des molécules linaires (21) dans une succession de zones de synthèse (1 à 16), on effectue au moins un autre passage de synthèse au cours duquel des éléments linéaires (n4 n15) d'une série de types de base sont rattachés aux molécules linéaires (21) qui sont synthétisées lors du passage de synthèse précédent ou lors de chaque passage de synthèse précédent et comportent d'autres éléments linéaires (N1 à N12 ; N9 à N17) d'une autre série de types de base. L'apport d'éléments linéaires (N1 à N17 ; n4 à n15) aux zones de synthèse (1 à 16) dans les passages de synthèse correspond à la succession prévue des éléments linéaires (N1 à N17 ; n4 à n15) dans les molécules linéaires (21) à synthétiser. Les groupes des régions de synthèse (27) associées aux zones de synthèse (1 à 16) sont décalés d'une zone de synthèse (1 à 16) à chaque cycle de synthèse dans la même direction de passage. Il est ainsi possible de synthétiser de manière efficace par exemple des molécules linéaires (21) complémentaires d'au moins des parties des segments (20) de supports d'informations génétiques, avec des groupes d'éléments linéaires (N1 à N17 ; n4 à n15) pour obtenir différentes séries de types de base.
PCT/EP1999/002958 1998-05-02 1999-04-30 Dispositif et procede pour produire un ensemble de molecules lineaires sur un materiau support WO1999056865A1 (fr)

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AU38258/99A AU3825899A (en) 1998-05-02 1999-04-30 Device and method for producing a system of chain molecules on a support material
JP2000546874A JP2002513549A (ja) 1998-05-02 1999-04-30 支持物質上に鎖状分子のアレイを製造するための装置および方法
EP99920827A EP1079920A1 (fr) 1998-05-02 1999-04-30 Dispositif et procede pour produire un ensemble de molecules lineaires sur un materiau support

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