WO2004024950A1 - Procede de selection d'acides nucleiques ligands - Google Patents

Procede de selection d'acides nucleiques ligands Download PDF

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Publication number
WO2004024950A1
WO2004024950A1 PCT/EP2003/010053 EP0310053W WO2004024950A1 WO 2004024950 A1 WO2004024950 A1 WO 2004024950A1 EP 0310053 W EP0310053 W EP 0310053W WO 2004024950 A1 WO2004024950 A1 WO 2004024950A1
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Prior art keywords
target molecule
nucleic acid
mixture
bind
higher affinity
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PCT/EP2003/010053
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German (de)
English (en)
Inventor
Dirk Eulberg
Sven Klussmann
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Noxxon Pharma Ag
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Priority to AU2003271598A priority Critical patent/AU2003271598A1/en
Publication of WO2004024950A1 publication Critical patent/WO2004024950A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/16—Feed pretreatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1048—SELEX
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2311/00—Details relating to membrane separation process operations and control
    • B01D2311/04—Specific process operations in the feed stream; Feed pretreatment
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6811—Selection methods for production or design of target specific oligonucleotides or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00465—Separating and mixing arrangements
    • G01N2035/00475—Filters
    • G01N2035/00485—Filters combined with sample carriers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/028—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having reaction cells in the form of microtitration plates

Definitions

  • the present invention relates to a method for selecting one or more nucleic acid ligands which bind to a target molecule, the use of filter columns in such a method and a vacuum chamber for use in such a method.
  • nucleic acids The understanding of the importance of nucleic acids has fundamentally developed in recent years. If nucleic acids were used at the beginning of molecular biological research as the carrier of a cell's genetic information, a structural function was soon assigned to different nucleic acid molecules. The fact that nucleic acids basically have the ability to interact specifically with other molecules was confirmed to the extent that there were nucleic acid-binding proteins of the transcription and translation complex.
  • nucleic acid ligands The basic suitability of nucleic acids to bind to any target molecule was disclosed in international patent application WO 91/19813 dated June 10, 1991. There, a method for the selection of one or more nucleic acid ligands from a mixture of candidate nucleic acids is described. On the basis of the considerations set out in WO 91/19813, it is assumed that in a mixture of candidate nucleic acids, i. H. a library of nucleic acids that differ, at least in part, in their primary sequence and thus also in their secondary and tertiary structure, a nucleic acid ligand, i. H. a nucleic acid binding to the target molecule will be included if the library is only large enough to cover a suitably large space of different binding pockets forming nucleic acids which are necessary for the binding of a certain target molecule.
  • Enantiomer of the actual target molecule which typically does not occur in nature as such, can be selected using SELEX technology and, after receiving one or more corresponding nucleic acid ligands, these using L-
  • nucleotide consisting of nucleic acid ligand (s) binds to the actual target molecule and no longer to the enantiomer of the target molecule.
  • the advantage of such Spiegelmers can be seen in the fact that due to their construction from L-nucleotides in biological systems there is no enzymatic activity that would be able to degrade this type of nucleic acid.
  • nucleic acid ligands Although the generation of corresponding nucleic acid ligands is still associated with experimentally considerable difficulties, and to this extent the predictability of whether a suitable nucleic acid ligand can actually be isolated seems to be a matter of luck, the method for producing such nucleic acid ligands is fundamentally automation accessible at least in some areas. This is due to the fact that in the various selection rounds that are generally required in order to isolate a nucleic acid ligand that binds to a target molecule with a sufficiently high affinity, a number of steps must be carried out which as such appear to be fundamentally accessible to automation and must be repeated in their entirety in the various selection rounds.
  • the method for selecting nucleic acid ligands that bind to a specific target molecule can be developed in a wide variety of ways.
  • the different forms of the method are based on the fact that the nucleic acid ligands can in principle be isolated either as double-stranded or single-stranded nucleic acids and more precisely as single- or double-stranded RNA or single- or double-stranded DNA.
  • the single-stranded RNA and the single-stranded DNA are generally preferred. Further variants in the execution of the method can be justified by the type of the target molecule and in particular its handling within the scope of the method for the selection of nucleic acid ligands.
  • nucleic acids or nucleic acid ligands amplified. The separation of those with a higher
  • Affinity to the target molecule binding nucleic acids from the rest of the candidate mixture is typically in the form that the complex of target molecule and
  • Nucleic acid ligand is separated from the rest of the candidate mixture. In a next one
  • Step is the nucleic acid ligand (s) binding or binding to the target molecule from the
  • Deoxyribonucleic acid is then again transcribed into ribonucleic acid.
  • the ribonucleic acid obtained in this way can be used in a further selection round. It is obvious that the proportion of those that specifically bind to a target molecule
  • each round of selection provides one with regard to one or more nucleic acid ligands.
  • Nucleic acids are separated, their sequence determined and then chemically synthesized on a larger scale or prepared using other suitable techniques.
  • the object of the present invention was to further develop the SELEX process in such a way that, due to the process steps which were always the same, this was carried out in automated form, i. H. using a robot, preferably a computer-controlled robot.
  • the object is achieved in a first aspect by a method for selecting one or more nucleic acid ligands from a mixture of candidate nucleic acids, the nucleic acid ligands being able to bind to a target molecule, comprising the steps
  • nucleic acid (s) which bind to the target molecule with a higher affinity from the rest of the candidate mixture, the nucleic acid (s) which bind to the target molecule with a higher affinity being present in a complex with the target molecule,
  • nucleic acid (s) optionally amplifying the nucleic acid (s) which bind with higher affinity, resulting in an enriched nucleic acid product
  • the target molecule is preferably immobilized on a surface and the method comprises an ultrafiltration step.
  • the object is achieved in a second aspect by a method for selecting one or more nucleic acid ligands from a mixture of candidate nucleic acids, the nucleic acid ligands being able to bind to a target molecule, comprising the steps
  • nucleic acid (s) which bind to the target molecule with a higher affinity from the rest of the candidate mixture, the nucleic acid (s) which bind to the target molecule with a higher affinity being present in a complex with the target molecule
  • nucleic acid s) binding to the target molecule with a higher affinity from the target molecule, and e) optionally amplifying the nucleic acids which bind with higher affinity), resulting in an enriched nucleic acid product,
  • the target molecule is preferably immobilized on a surface and the method comprises an ultrafiltration step.
  • the ultrafiltration step takes place during or after the elution.
  • the ultrafiltration step takes place during or after the amplification.
  • the object is achieved in a third aspect by a method for selecting one or more nucleic acid ligands from a mixture of candidate nucleic acids, the nucleic acid ligands being able to bind to a target molecule, comprising the steps
  • nucleic acid (s) which bind to the target molecule with a higher affinity from the rest of the candidate mixture, the nucleic acid (s) which bind to the target molecule with a higher affinity being present in a complex with the target molecule
  • step b) optionally amplifying the nucleic acid (s) which bind with higher affinity, resulting in an enriched nucleic acid product, the target molecule being immobilized on a surface and the separation according to step b) being carried out by means of a filtration, preferably a microfiltration, using a vacuum chamber comprising a base part (10) and a cover part (11), the base part (10) and the cover part (11) is preferably separated from one another by a seal, and the cover part (11) has at least one complete bore (13, 14) extending through the cover part, the bore having a minimum length, so that contamination occurs during the filtration step two side-by-side filtrations do not occur.
  • a filtration preferably a microfiltration
  • the separation in step b) takes place by means of microfiltration.
  • One embodiment of the method according to the invention provides that the method is an automated method.
  • the ultrafiltration takes place on an ultrafiltration membrane with a molecular exclusion size of approximately 1,000 to 100,000, preferably approximately 10,000 to 50,000 Da.
  • the microfiltration takes place on a microfiltration membrane or frit with an average pore size of about 0.1 to 100 ⁇ m, preferably about 1 to 35 ⁇ m and preferably most about 10 ⁇ m.
  • the surface is provided by particles, the particles preferably being selected from the group comprising magnetic and non-magnetic particles.
  • the denaturing agent is selected from the group comprising urea, EDTA, guanidinium-HCL, guanidinium isothiocyanate, detergent, combinations of urea and EDTA.
  • the competitor is a peptide or a fragment of the target molecule.
  • the ultrafilter and / or the microfilter is contained in a filter column, the filter column consisting of a receiving piece and an outlet piece, both of which are connected to one another by a conical intermediate piece and the ultrafilter or the microfilter on Transition between the conical intermediate piece and the outlet piece is arranged.
  • the filter column is held in a device consisting of a base part and a cover part, the cover part having one or more means for receiving the filter column.
  • the means for receiving the filter column is a bore, the bore having a minimum length.
  • the object is achieved in a fourth aspect by using a filter column in a method for selecting nucleic acid ligands, the frit comprising a first receiving piece (1) which merges into a conical intermediate piece (2) and the conical intermediate piece in turn into an outlet piece (3) passes, a filter being attached at the transition between the intermediate piece (2) and the outlet piece (3).
  • the filter column is used in one of the methods according to the invention.
  • the filter is a microfilter or an ultrafilter.
  • the receiving piece (1) has a fastening means.
  • the fastening means is attached to the opposite end of the adapter piece (1), where it merges into the conical intermediate piece (2).
  • the filter column has a broadening means on the outer wall, preferably on the outer wall of the receiving piece.
  • the widening means is designed as a ring attached to the outer wall, the ring preferably being arranged perpendicular to the longitudinal axis of the receiving piece (1) and / or the outlet piece (3).
  • a vacuum chamber for use in a method for selecting nucleic acid ligands, comprising a base part (10) and a cover part (11), the cover part (11) having at least one bore extending completely through the cover part (12), the bore having a minimum length.
  • the vacuum chamber is used in one of the methods according to the invention.
  • a vacuum chamber for use in a method for selecting nucleic acid ligands, comprising a base part (10) and a cover part (11), the cover part (11) at least two bores extending completely through the cover part (12), the bores having a minimum length which ensures that there is no contamination of the filtration process and / or solution to be filtered by a filtrate from the second filtration.
  • the vacuum chamber is used in one of the methods according to the invention.
  • vacuum chamber in a further embodiment of the vacuum chamber according to the invention it is provided that it comprises a filter column according to the invention or a filter column as used according to the invention herein.
  • the object is achieved in a seventh aspect by a device with a sequence control unit for controlling a pickup for a liquid and / or solid substance, the pickup being suitable for a sequence comprising picking up the stuff, transporting the stuff and dispensing the stuff in a container, the transducer being controlled
  • nucleic acid (s) binding to the target molecule can be separated from the rest of the candidate mixture with a higher affinity
  • nucleic acid (s) which bind to the target molecule with a higher affinity
  • an ultrafiltration step being carried out during or after the elution.
  • an ultrafiltration step is carried out during or after the amplification.
  • the object is achieved in an eighth aspect by a device with a sequence control unit for controlling a sensor for liquid and / or solid substances, the sensor being suitable for a sequence of taking up the substance, transporting the substance and dispensing the substance into a container perform, the transducer so is controlled in order to carry out one of the methods according to the invention by means of multiple sequences.
  • the object is achieved in a ninth aspect by a method for controlling a sensor for selecting one or more nucleic acid ligands, the sensor being suitable for a sequence comprising taking up a liquid and / or solid substance, transporting the substance and dispensing the substance To carry the substance into a container, the pickup being controlled in such a way
  • nucleic acid (s) binding to the target molecule can be separated from the rest of the candidate mixture with a higher affinity
  • nucleic acid (s) which bind to the target molecule with a higher affinity from the target molecule
  • nucleic acid (s) which bind to the target molecule with a higher affinity
  • the object is achieved in a tenth aspect by using a device with a sequence control unit for controlling a sensor for a liquid and / or solid substance, the sensor being suitable for a sequence comprising taking the substance, transporting the substance and a Dispensing the substance into a container for the selection of one or more nucleic acid ligands from a mixture of candidate nucleic acids, in particular for one of the methods according to the invention.
  • the present invention is based on the surprising finding that with the
  • Partial steps are used to remove from the respective reaction batch, and by the
  • Ultrafiltration creates the prerequisites for making the SELEX process accessible to automation with the unrestricted possibility of realizing the optimal reaction conditions, which are mainly mediated by low molecular weight compounds.
  • ultrafiltration therefore presents new features, particularly in its automated embodiment
  • the embodiments of the SELEX process according to the invention offer compared to the automated SELEX processes known in the prior art, as described, for example, by Cox et al. (Cox et al. 1998, Biotechnol. Prog. 14: 845 to 850 or from Cox & Ellington, 2000, Bioorg. Med. Chem. 9: 2525 to 2531) have a number of advantages.
  • the automated processes for the SELEX method described in the prior art do not allow, or at least only to a very limited extent, the selection of aptamers which can be used under physiological conditions and thus ultimately not Generation of therapeutically applicable aptamers. Nevertheless, it will have to be recognized that they are, however, at least to a certain extent suitable for providing diagnostically usable aptamers, the reaction conditions under which these are used essentially correspond to those used for the selection. This represents a very considerable restriction with regard to the range of applications of the nucleic acids produced in this way, which bind to the target molecule, in particular when they are used therapeutically and when physiological tests are carried out
  • Reaction conditions oriented detection tests can be a significant limitation.
  • an ultrafiltration step can be carried out after each sub-step of the SELEX process, in particular when the reaction parameters have to be changed, the reaction parameters preferably being the composition of the respective reaction batch, and in particular the influencing of the reaction batch with regard to the type and scope of the low molecular weight substances or compounds contained therein, as mentioned above.
  • An ultrafiltration step is preferably carried out in the case of elution of the nucleic acids bound to the target molecule. It is further preferred that, in addition or as an alternative to the use of ultrafiltration following the elution step, this is carried out after the amplification, again with the aim of providing the suitable reaction conditions for the reaction step following the amplification.
  • the nucleic acid present in the reaction mixture is retained by the ultrafilter, while the low molecular weight components, ie the low molecular weight compounds, of the reaction mixture are removed from the latter.
  • the nucleic acids can then be removed or detached from the surface of the ultrafilter, for example by adding an aqueous solution. It is particularly preferred if the solution is one that is used as a reaction batch in the step following the ultrafiltration, preferably one or more of the components of the reaction batch ultimately used not yet being included. Components of this type, which are not yet included in the reaction batch, are in particular those which have only a low stability and / or are expensive to obtain.
  • the component is the enzymatic component of the substep of the SELEX process carried out after ultrafiltration.
  • the use of an ultrafiltration step in the context of the SELEX process ensures effective retention of the nucleic acid eluted from the target molecules and thus the use of immobilized agents described in the prior art
  • the use of an ultrafiltration step is also advantageous compared to the use of purification systems with magnetic particles or chromatography columns, among other things in view of the increased speed with which the individual selection round can be carried out with it.
  • Another advantage that is decisive for the success of the SELEX process is the avoidance of cross-contamination, which is otherwise difficult to avoid in the case of highly parallel approaches, or the increased yield compared to the use of magnetic particles.
  • those nucleic acids that are potential binders to the target molecule remain on the filter and are not taken up in the filtrate or another volume of liquid, which in turn could be contaminated by other volumes of liquid.
  • nucleic acids from the complex of nucleic acid (s) and target molecule especially if that
  • Target molecule is immobilized on a surface, can be accomplished in various ways that were previously not possible at least with an automated form of the SELEX process.
  • denaturing agents or inhibitors of the binding between nucleic acid (s) and target molecule can in principle be used within the scope of the invention.
  • the inhibitors are preferably the target molecule or molecules or parts or fragments thereof.
  • a fragment may be a domain or part of it.
  • Suitable denaturing agents are those which are able to dissolve the complex of nucleic acid and target molecule, preferably with denaturation of one or both components of the complex.
  • Suitable denaturing agents are, for example, urea, EDTA, mixtures of urea and EDTA, dimethylformamide, guanidinium HC1, guanidinium isothiocyanate, various salts, such as e.g. NaCl, in high concentration, and pH changes. Because the denaturing agents are removed from the reaction mixture by ultrafiltration, all suitable agents can ultimately be used, since these do not have to be matched to the needs of the one-pot reactions described in the prior art in the automated SELEX processes.
  • a special embodiment of the SELEX process which is carried out using an ultrafiltration step, in particular following the elution of the nucleic acid (s) bound to the target molecule, is one in which heat is used as the elution principle.
  • the application of heat does not directly introduce any compound into the reaction mixture which, as in the case of using a denaturing agent or an inhibitor, has to be removed from it again so that the subsequent reactions can take place, but it has been observed that when used from heat from components of the reaction mixture, in particular from matrices on which the target molecule is mobilized, unknown substances can be released, which can adversely affect the subsequent reaction (s).
  • the SELEX process according to the invention represents a possibility of also using such matrices which give off compounds when exposed to heat or other treatment, which could adversely affect the subsequent reaction steps of the SELEX process and are therefore not used in SELEX processes other than the invention could.
  • Elution using heat is done in suitable buffers known to those skilled in the art. Preferred buffers are those that can be used in subsequent sub-steps of the SELEX process.
  • Another surprising advantage of the present invention is that the elution of the nucleic acid ligands from the nucleic acid ligand-target molecule complex can also be achieved with an excess of the target molecule or an excess of a part of the target molecule.
  • the excess of the target molecule or the excess of the part of the target molecule is used as an inhibitor or as a competitor with the aim of eluting the nucleic acid which binds to the target molecule from the target molecule which is preferably immobilized on a surface. Additional competitors to release the nucleic acid ligands bound to the target molecule can easily be identified within the knowledge of those of ordinary skill in the art.
  • the use of such competitors for subsequent process steps and in particular subsequent selection rounds is not a problem insofar as their size can be removed from the reaction mixture by the ultrafiltration step, only the nucleic acid ligands remaining in the reaction mixture and then in the course of the further steps or selection rounds can be used.
  • the selection of the size of the competitor or inhibitor is determined from the size characteristics of the ultrafilter used, which in turn is largely determined by the type of nucleic acid to be retained. In any case, it is necessary that the compound used as an inhibitor is filtered through the filter as part of the ultrafiltration and that the nucleic acid is not filtered through the filter.
  • the ultrafiltration can be carried out following the elution of the nucleic acid (s) bound to the target molecule.
  • an ultrafitration step is carried out after the amplification step of the SELEX process. It can, but does not have to be provided that an ultrafiltration step is carried out after the elution step. Purification of the products of the amplification reaction, also referred to herein as
  • Amplification products referred to by ultrafiltration especially if this by
  • the SELEX process according to the invention can in principle comprise any form of the amplification reaction known in the prior art.
  • the RT-PCR used for the amplification of RNA can be carried out as a two-step RT-PCR, as a one-step RT-PCR, but also as a continuous or isothermal amplification.
  • the RT reverse transcription
  • This first step of reverse transcription is followed by the second step consisting of the polymerase chain reaction, PCR, e.g. B. performed in 100 ul, d. H.
  • the reverse transcriptase reaction is diluted by a factor of 5. Both primers and Taq polymerase are used.
  • the RTase is inactivated at the high denaturation temperatures for e.g. 2-10 min at 95 ° C.
  • RT and PCR run in the same approach. Accordingly, all the reagents required for both the RT and the PCR are contained in the reaction mixture.
  • the reactions are not spatially separated, but only in terms of time, since a modified thermostable polymerase is used, which takes longer Incubation at 95 ° C is activated. By controlling the activity of the enzymes involved, the reactions are completely separated from one another.
  • the RT is first carried out at 50 ° C., where the PCR polymerase is not yet active. After heating to 95 ° C for
  • the RTase is inactivated for 15 min and the PCR polymerase is activated.
  • the following profiles are used in the subsequent thermal cycling: To activate the thermostable polymerase and to inactivate the Rtases: 20 min 50 °, then 10 min 60 °, finally 15 min 95 °; Typical thermal profile for PCR is 30 s denaturation at 95 °, 30 s
  • Annealing at a temperature of usually between 50 ° and 72 ° depending on the primers as known to those skilled in the art and 30 s polymerisation at 72 ° C.
  • nucleic acid ligands are RNA
  • the continuous or isothermal amplification which is also referred to as NASBA (nucleic acid sequence based amplification) represents a further method for the amplification of RNA which can be used in principle within the scope of the present invention.
  • Reverse transcription takes place by means of an RTase, whereby the RNA is converted into a hybrid of RNA and DNA.
  • the reaction mixture also contains an RNaseH, which degrades the RNA in this hybrid and the RTase also synthesizes the second strand from DNA.
  • the T7 RNA polymerase then transcribes the dsDNA to the RNA. All of these processes run simultaneously, i.e. H. continuously at a temperature between 37 ° C and 41 ° C and result in an accumulation of RNA.
  • the ultrafiltration is preferably designed as a vacuum-driven ultrafiltration.
  • the nominal molecular size exclusion limit can be about 3,000 to 300,000, preferably about 10,000 to 50,000 Da.
  • the materials used for the ultrafilter are, in particular, those selected from the group which include, for example, polyether sulfone or regenerated cellulose membranes. Such ultrafiltration materials are known to those skilled in the field of separating biomolecules such as nucleic acids and proteins and can be used according to the invention.
  • a microfiltration step After contacting the mixture of candidate nucleic acids with the preferably immobilized target molecule, the separation of the nucleic acids not bound to the target molecule from the Complex of target molecule and nucleic acid (s), the so-called partitioning, is carried out by a microfiltration step.
  • This microfiltration step can be carried out in any SELEX process according to the prior art as well as in any embodiment of the SELEX processes disclosed herein, which are also referred to herein as SELEX processes.
  • the selection of the specific filter, ie the exclusion size of the filter material used, depends on the type of implementation of the method.
  • the filter materials are selected such that the respective carrier material is retained.
  • Preferred pore sizes for microfiltration are approximately 0.1 to 100 ⁇ m, preferably approximately 1 to 35 ⁇ m and most preferably approximately 10 ⁇ m.
  • the vacuum chamber has a specific cover construction which is designed to accommodate commercially available filtration units, for example for ultrafiltration and / or microfiltration.
  • the lid construction can also be designed such that it accommodates or can accommodate one or more of the filter columns described herein.
  • This configuration which is typically in the form of bores in the lid construction, is designed in such a way that protection against cross-contamination is provided during the respective filtration process, ie during ultrafiltration and / or microfiltration.
  • the individual bore into which the filtration units are introduced into the lid of the vacuum chamber is so long that the filtrate that drips from the lid into the vacuum chamber is practically not in contact with other filtration units or the one obtained therefrom Eluate is coming.
  • the freedom from cross-contamination can be ensured, for example, by ensuring that the individual filter only moves together with the lid of the vacuum chamber, which, in particular in vacuum operation or after the vacuum chamber has been vented, prevents liquid bridges from forming between the individual holes in the lid construction.
  • the filters are firmly inserted into the cover construction, for example by the cover construction and the filters being injection molded from plastic.
  • a further embodiment in order to ensure freedom from contamination is that in the event that the filter columns described herein are introduced into the lid structure of the vacuum chamber and a seal, preferably an O-ring seal, is arranged between the bore in the lid structure and the individual filter column so no capillary forces arise as a result of which filtrate passes the filters or filter columns in the area of the liquid to be filtered and thus a cross contamination of the various seals, preferably an O-ring seal, is arranged between the bore in the lid structure and the individual filter column so no capillary forces arise as a result of which filtrate passes the filters or filter columns in the area of the liquid to be filtered and thus a cross contamination of the various
  • a further apparatus-technical embodiment as can be used in the method according to the invention, consists in that the individual reaction vessels in which the various reaction components used in the method according to the invention are stored can be closed again. This also reduces the risk of cross-contamination and considerably improves the storage of the reagents. a. the reason for this is that enzymes must be stored refrigerated, for example for medium-term storage for up to 48 hours at at least 10 ° C., preferably 4 ° C. If stored open, condensation would form after only a few hours and the reagents would be watered down. Protection from cross-contamination would also be achieved by means of these, in particular, automatically reclosable containers. Finally, the said closable vessels are also advantageous in that they provide protection against evaporation, which, at least in theory, could allow the reaction batches to be incubated for as long as desired.
  • the methods according to the invention are particularly suitable for high throughput formats, in particular if the methods according to the invention are carried out automatically, ie using a robot or automaton.
  • the methods can be carried out, for example, using modified microtite slats with, for example, 96 or 384 wells. These modified microtitre plates have a filter membrane instead of the solid plastic base, the pore size corresponding to that as generally described herein in connection with microfiltration.
  • the surfaces typically used in the process according to the invention for immobilizing the target molecule, preferably particles, are retained as the unbound nucleic acid molecules pass through the membrane.
  • This filtration represents a very efficient way of separating the matrix and thus the nucleic acid ligand in turn bound to the target molecule bound to the matrix.
  • the use of non-magnetic particles is particularly preferred.
  • the use of a filtration step for separating the matrix from the reaction mixture, ie separating off the the target molecule and thus nucleic acid ligands bound to the matrix are thus relieved to the extent that when magnetic particles are used and a magnetic field is used to separate them, the only one on the wall of the reaction vessel is the only one
  • FIG. 5 shows a basic flow diagram of an automated RNA selection
  • Fig. 6 is a schematic representation of the implementation of the invention
  • FIG. 8A is a top view of the vacuum chamber according to the invention.
  • FIG. 8B shows a cross section of the vacuum chamber shown in FIG. 8A;
  • 8C shows a cross section through the cover part of the vacuum chamber according to the invention.
  • 9 shows the scheme of automatic in vitro selection against D-CGRP;
  • Fig. 12 u. 13 possible secondary structures of the isolated RNA Spiegelmers from clone Hl, G2 and HIV;
  • 19A u. 19B shows the result of the sequence analysis of the automated
  • 20A u. 20B the measurement of the binding of mirror bucket PL-D8 to rat (A) or human (B) ⁇ -L-CGRP at 37 ° C. by means of isothermal titration calorimetry.
  • the buffers are adjusted to suitable reaction conditions in a first step. These are removed from a reagent rack, which is typically kept at 4 ° C, and brought to a work platform, where they are heated to room temperature or 37 ° C.
  • the nucleic acid library used for selection is also pretreated in this way. Which in the present case
  • RNA existing nucleic acid library is then denatured using a thermal cycler.
  • a temperature of 50-100 ° C., preferably 60-90 ° for a period of 1-10 min, preferably 2-7 min is typically achieved.
  • a precolumn is used to bind nucleic acids from the substrate that are non-specific to the carrier material on which the target molecule is or is to be immobilized
  • nucleic acid library Remove nucleic acid library, upstream and treated the reaction mixture using a shaker at room temperature or 37 ° C on the work platform.
  • the matrix is placed in the wells of the microtiter plate on the 4 ° work station.
  • the matrix is transferred by resuspension in the nucleic acid preparation which is to be incubated therewith; By pipetting up and down several times, all particles are in suspension and can be broken down into new ones
  • Pre-column treatment turns the supernatant, which contains nucleic acid ligands that do not interact non-specifically with the carrier material of the target molecule, into a new one
  • the reaction vessel transfers the reaction vessel and incubate there with the target molecule. Incubation is again carried out at room temperature or at 37 ° C.
  • the target molecule can be in the new
  • reaction vessel is either freely in solution or is already bound to a suitable surface or support material.
  • the complexes have to be made
  • nucleic acid ligand (s) and target molecule are immobilized on a suitable matrix
  • Ratios ie the number of bound RNA molecules per target molecule, and on the other hand in the type of nucleic acids that have bound to the target molecule, which is due to the fact that those are usually within the framework of the SELEX method nucleic acid libraries used contain a number typically from 10 12 to 10 16 different nucleic acid species.
  • the unbound nucleic acids i.e. the nucleic acids of the library that are not bound to the target molecule are removed from the complexes of nucleic acid and target molecule.
  • This is typically done at room temperature or 37 ° C. by subjecting the reaction batch to filtration, preferably by subjecting it to vacuum-operated filtration.
  • the surface or the carrier material on which the target molecule and thus also the nucleic acids bound to it are immobilized is filterable surfaces such as magnetic or non-magnetic particles
  • the filtration and in particular the selection of the Filter material and Filte ⁇ or diameter determined by the size of the particles, ie the filter must be designed so that the carrier material is retained in the reaction vessel.
  • the previous incubation of the nucleic acid with the target molecule has already been carried out in the filtration unit, and consequently this represents the reaction vessel, or from the corresponding reaction batch the reaction batch or an aliquot thereof, or at least that Target molecule and thus also the nucleic acid-bound carrier is transferred into the filtration unit.
  • this represents the reaction vessel, or from the corresponding reaction batch the reaction batch or an aliquot thereof, or at least that Target molecule and thus also the nucleic acid-bound carrier is transferred into the filtration unit.
  • the filtration column or the vacuum chamber is used.
  • the vacuum chamber preferably comprises at least two bores into which the suitable filtration units are preferably introduced.
  • the design of the hole in the cover plate and, if necessary, the relative distance between the cover plate and the base part ensure that the filtrate from the filtration carried out in a first hole does not come into contact with the filtrate from a filtration in a second hole, the the two bores are typically arranged next to one another in order to avoid contamination of the different reaction mixtures with one another.
  • the specific design of the bore length depends on several factors, such as the design of the column, the amount of liquid to be filtered, the amount of filtrate, the amount of applied vacuum and the like from. The appropriate parameters can be determined by those skilled in the art through routine testing in light of the disclosure made herein.
  • the nucleic acid bound to the target molecule is eluted from the complex using suitable elution conditions.
  • the elution can again take place in the vacuum chamber or the reaction batch, more precisely the connection batch, can be transferred to a new reaction vessel beforehand.
  • the elution is carried out by heating using the thermal cycler.
  • the reaction mixture is preferably incubated at 95 ° C. for 3 minutes
  • the RT-PCR begins in the subsequent step.
  • the necessary reagents from the storage vessels kept at 4 ° C. are added to the reaction mixture and preferably incubated at 50 ° C. If, in one embodiment, the RT-PCR takes place in the presence of the matrix with bound nucleic acid molecules, the matrix is resuspended in the RT-PCR buffer in the column which is in the vacuum chamber (part A) and then in a fresh bowl pipetted the 96-well plate. Thereafter, the heat-eluting of the bound nucleic acid is heated, for example, at 70-95 ° C. for 3 minutes in the resuspended matrix.
  • the supernatant from the matrix incubated with Denarurans (which contains the eluted nucleic acids) is placed in an ultrafiltration tube in the vacuum chamber (Part B) and freed from the Denarurans there.
  • the RT reaction is started hot on the 50 ° work station and incubated there for 20 min.
  • the reactions in the plate are placed in the thermal cycler, where they are incubated for a further 10 min at 60 ° C before denaturing for 15 min at 95 ° C and the thermocycling program begins (30 s 95 ° C; 30 s annealing temperature ; 30 s 72 ° C).
  • the result of the PCR is checked.
  • the control can take place during the PCR and or after its completion.
  • the result of the PCR is typically checked by determining the fluorescence of the reaction mixture using a suitable fluorescent dye and a fluorescence reading device.
  • the RT-PCR reactions are temporarily stored in the 4 ° working platform, in which the required amount of DNA has already been generated during the PCR and which are therefore already ready for transcription.
  • the plate in which the PCR takes place is placed in the 50 ° work station in order to take the samples for fluorescence measurement, since it is not possible to pipette in the thermal cycler itself. The The plate is kept hot in order to minimize the risk of mispriming (which is particularly great if the batch cools down considerably before being heated again).
  • the DNA obtained in this way is then used as a template or template for a transcription reaction.
  • the necessary reagents such as ribonucleotides and T7 RNA polymerase, are removed from the corresponding storage vessels stored at 4 ° C., added to the reaction mixture on the work platform and incubated at 37 ° C. for 1 to 24 h, preferably 1.5 to 4 h ,
  • the T7 transcription reaction is then carried out according to the standard protocol, as described, for example, in Cox et al., 1998. Biotechnol. Prog. 14: 845-850 and then the RNA obtained was purified by vacuum-driven ultrafiltration.
  • T7 RNA polymerase other RNA polymerases can also be used, such as T3 or SP6 RNA polymerase or thermostable RNA polymerases such as those from Thermus thermophilus.
  • the purification of the RNA is preferably carried out using an ultrafiltration, the ultrafiltration being a vacuum-driven ultrafiltration under the conditions as disclosed here.
  • the reaction mixture which has provided the RNA as part of the transcription reaction is inserted into the filtration unit, more precisely there into chamber B, the chamber, which is also referred to herein as the reaction chamber ultrafiltration is allowed.
  • the configuration of the ultrafiltration direction is in turn preferably such that contamination of the reaction batches by filtration steps carried out in parallel or sequentially is avoided.
  • the RNA purified in this way is free of low-molecular substances such as NTPs, DTT, salts, EDTA or detergents (i.e.
  • RNA contains macromolecules (proteins that were used in the amplification reactions; double-stranded DNA that was used as a template for transcription from the RT-PCR was not included in the scheme due to DNasel digestion - degraded and the pyrophosphate precipitate formed during the T7 transcription was dissolved by adding EDTA).
  • the RNA can then be used as the starting material for a further round of selection.
  • the presence of proteins is not as critical as that of buffer salts or EDTA, as long as the proteins do not have any disruptive activities.
  • the Polymerases from RT, PCR and transcription do not interfere (and are mostly used in the
  • the DNase on the other hand, can be problematic
  • Heat and EDTA can be inactivated.
  • these macromolecules can essentially only be removed from the reaction mixture during partitioning, ie during the washing process.
  • FIG. 2 shows the arrangement of the various workstations which have to be controlled by a robot when the selection round described in FIG. 1 is carried out.
  • FIG. 3 A further embodiment of the automated RNA selection is described in FIG. 3, wherein the immobilization of the target molecule does not take place on particles as the carrier surface, but on membranes, such as a nitrocellulose membrane.
  • membranes such as a nitrocellulose membrane.
  • suitable membranes are mixed cellulose ester membranes or other membranes, provided that they bind the target molecules and allow the nucleic acids to pass through.
  • Corresponding membranes are known to those skilled in the art and can be tested for their suitability for use in the methods according to the invention as part of routine tests.
  • the steps of setting the buffer conditions and denaturing the nucleic acid, specifically the ribonucleic acid take place in the same way as for the method described in FIG. 1.
  • the precolumn differs in that membrane portions are introduced into the reaction vessel to serve as a precolumn and the non-specific binding RNA molecules are removed from the nucleic acid library.
  • the nucleic acid is then incubated with the target molecule in a manner similar to that described for the method according to FIG. 1.
  • the complexes from RNA and the target molecule are obtained on a workstation at room temperature or at 37 ° C.
  • a filtration unit specifically at a first station A, on which a filtration and in particular a microfiltration is provided .
  • inserts from the nitrocellulose membrane or another of the membranes disclosed for this purpose are included. This is followed by the usual washing step, in which the nitrocellulose membrane binds the target molecules together with the nucleic acid molecules specifically bound to them.
  • RNA molecules that do not bind or that do not bind specifically under the washing conditions are removed, the elution of the RNA molecules that bind specifically to the target molecule can be carried out. Molecules take place. In the present case, this cannot be done by temperature, since the
  • Vacuum chamber with the ultrafiltration inserts can not be tempered, but through
  • Competitors or by denaturing reagents are Competitors or by denaturing reagents.
  • the elution takes place at ambient temperature on the vacuum chamber.
  • the target molecule binds essentially only to the membrane surface and that
  • Target molecule with a solution that dissolves the complex of nucleic acid and target molecule
  • agents such as urea or guanidinium salt, is dissolved from this surface. In the specific embodiment shown, elution takes place or
  • RNA and thus releasing it from the target molecule preferably not by heating, but rather by using the agents disclosed in principle, such as competitors or denaturing agents.
  • the step of specifically removing the denaturing agent is now carried out.
  • a filtration is carried out on the vacuum station at position B, which is preferably carried out as ultrafiltration, as described here.
  • the reaction takes place at room temperature or 37 ° C.
  • the membrane itself is not transferred.
  • the target molecules should bind to the surface of the membrane, where they can be detached by denaturing agents.
  • the supernatant will then contain the corresponding molecules, which is simply pipetted off and transferred to the ultrafiltration units in station B.
  • the automated DNA selection shown in FIG. 4 is a further embodiment of the method according to the invention.
  • the main difference from the method shown in FIG. 1 is that DNA ligands are to be isolated and that the nucleic acid library also consists of DNA and not RNA.
  • the RT-PCR which is replaced by a PCR, is therefore not required in the specific embodiment.
  • the steps of transcribing the DNA into RNA are also eliminated. Rather, after the PCR reaction, single-stranded DNA can be isolated directly on the work platform at room temperature or 37 ° C.
  • a magnetic separator is introduced for cleaning, the separation of the DNA strands by means of magnetic particles at high pH is made. The unwanted (-) strand was synthesized during the PCR with oligonucleotide containing biotin.
  • the alkaline supernatant contains the free (+) strands and can be pipetted off after magnetic separation of the particles and into a new one
  • DNA is otherwise carried out in a manner analogous to that described for RNA in the method shown in FIG. 1.
  • the steps differ from the sequence of steps shown in FIG. 1 only in that the elution or denaturation of the RNA can not only take place in the thermal cycler but also on the 50 ° C work platform or at ambient temperature conditions. Elution is carried out with urea and EDTA at 20 - 100 ° C, preferably at 37 - 95 ° C on the 50 ° C workstation or in the thermal cycler. As a result of the combination of elevated temperature and denaturing agents, nucleic acids bound to the target molecule can be eluted from it again.
  • the denaturing agent such as, for example, urea / EDTA
  • the reaction mixture is incubated at elevated temperature. Similar to the step of purifying the RNA, the denaturing agent is then removed from the reaction mixture using ultrafiltration at room temperature or 37 ° C. as an additional step. For this purpose, the urea / EDTA eluate is pipetted into position B of the filtration unit.
  • the method described for denaturing elution can also be used in the embodiment in which the elution is carried out by competitive elution.
  • a high concentration of competitor as described herein, would be added and this would then be separated off by means of ultrafiltration. In this case, however, the elution would not be carried out at 50 ° C. as in the case of denaturing elution, but at room temperature or 37 ° C.
  • Fig. 6 shows a device for automatically performing the invention
  • the device has a work area (21) on which various modules
  • the modules can contain containers for carrying out reactions
  • the modules can be heated containers or containers with a
  • Shaker are coupled to keep the added substance in motion.
  • the modules can have filter devices in order to filter the added substance.
  • the modules and their possible arrangement are shown in FIG. 2.
  • a movable arm (23) is arranged above the work area (21) so that it extends over the entire work area.
  • This arm is held by one or two supports (24) at the same height above the work area (21).
  • the supports (24) are movably held in rails (not shown) so that the arm (23) can be moved across the working area (21).
  • At least one of the supports (24) has a drive unit (25) with which the arm (23) can be moved to a specific position across the working area (21).
  • the drive unit (25) is controlled by a control unit (26).
  • the carriage (27) On the arm (23) there is a carriage (27) movable in the longitudinal direction of the arm (23), which can be moved along the arm via a further drive unit (not shown), so that one is controlled by the control unit (26) with the carriage certain position along the arm (23) can be approached.
  • the carriage (27) has a sensor for a liquid and or solid substance, e.g. B. a pipette or the like, which can be lowered to take up or deliver a substance.
  • the lowering and raising of the transducer is also controlled by the control unit (26).
  • the control unit (26) controls the arm, the slide and the pickup in such a way that a certain module is first approached with the slide and the arm so that the pickup (28) is located above the module and by lowering a substance can be absorbed or released by the transducer.
  • the process according to the invention can be achieved by a suitable combination of taking up and dispensing substances into the reaction containers provided for this purpose, as disclosed herein carry out. Since after every contact with one of the provided materials the
  • the senor (28) is contaminated with the respective substance, the sensor is preferably provided so that the part that comes into contact with the substance can be repelled and can be replaced by a new, not yet used part.
  • further modules are provided, in which on the one hand new number of pick-up parts are made available and another module in which the parts of the pick-up already used can be handed over in order to dispose of them.
  • the various modules described here correspond to the various workstations as described in the context of FIGS. 1 to 5.
  • the control unit (26) first contacts a mixture of candidate nucleic acids with a target molecule in a container.
  • the nucleic acid ligands can then bind to the target molecule.
  • This contacted candidate mixture is optionally contacted with a matrix on which the target molecules are immobilized and then transported to a separation device, preferably a filter device, in which nucleic acid binding to the target molecule can be separated from the rest of the candidate mixture with a higher affinity, whereby it It is also within the scope of the present invention that the target molecules are immobilized on a matrix from the outset.
  • the transport of the candidate mixture into the separating device is preferably carried out using a pipette which is located on the receiver (28).
  • the carriage is moved over the container with the candidate mixture and the receiver is lowered so that the pipette can take up the candidate mixture.
  • the transducer is raised and the carriage is moved over the corresponding microfiltration device. There, the previously absorbed substance is released into the microfiltration device.
  • this process can be carried out several times in succession, so that a sufficiently large amount of the candidate mixture to be filtered is available in the microfiltration device.
  • the nucleic acid which binds to the target molecule with a higher affinity is then removed from the microfiltration device and amplified by suitable known methods. This requires a large number of repetitive steps, so the use of an automatically controlled device is very advantageous for reasons of reliability.
  • ultrafiltration can be carried out to purify the amplification products formed in the amplification process.
  • amplification process amplification process, according to the invention, ultrafiltration can be carried out to purify the amplification products formed in the amplification process.
  • amphfication process To the mixture obtained as a result of the amphfication process becomes a
  • the method according to the invention is carried out several times in parallel.
  • a number of containers can be provided in the modules, which can be started separately by the pickup, the arm and the slide, controlled by the control unit (26). This means that waiting times caused by process steps can be bridged. You also have the advantage of getting comparable results.
  • control unit controls the various reactions to be carried out by the method according to the invention.
  • FIG. 7 shows the filter column according to the invention, the column comprising a first receiving piece (1) which merges into a conical intermediate piece (2) and the conical intermediate piece in turn merges into an outlet piece (3), the transition between the intermediate piece (2) and a filter is attached to the outlet piece (3).
  • the filter can be both a microfilter and an ultrafilter, preferably those as disclosed herein. Such a frit can be carried out in the course of any filtration step, as it is in connection with the method according to the invention, as described, for example, in FIGS. 1 and 2.
  • Similar filter columns that can also be used in the context of the present invention are commercially available, for example, as MicroCon filters from Millipore.
  • the receiving piece (1) can furthermore have a fastening means.
  • the fastening means is preferably arranged on the outer wall of the receiving piece and extends radially to the longitudinal axis of the filter column.
  • the fastening means can be arranged at one or more locations on the outer wall of the receiving piece.
  • the fastening means is preferably designed as a ring, the inside diameter of which corresponds to the outside diameter of the receiving piece. It is particularly preferred that the Fastening means is arranged at the end of the receiving piece (1) which corresponds to the end of the
  • Receiving piece (1) is opposite, which merges into the conical intermediate piece (2).
  • FIG. 8 shows a vacuum chamber as can be used in the method according to the invention.
  • 8A shows a top view of the vacuum chamber, as can be used, for example, at the vacuum station, as described in FIGS. 1 and 3 to 5.
  • Position A of the two-part vacuum chamber shown in FIG. 8 corresponds to position A, position B to position B of the vacuum chamber, as is used in the process described in FIGS. 1 and 3 to 5.
  • Both subchambers have a large number of bores (13) and (14), into which, for example, the frit, also referred to herein as a filtration column, can be introduced as shown in FIG. 7.
  • FIG. 8B shows a cross section through the vacuum chamber shown in FIG. 8A with two partial vacuum chambers.
  • the length of the bore (14) is designed as a multiple of the diameter of the bore. This ratio ensures that the filtration steps carried out in adjacent bores (13) can be carried out without mutual contamination. Contamination could occur, for example, if the filtrate of any filtration unit placed in a distinct bore (13) comes into contact with the filtrate of a further filtration unit and, in this respect, in particular by forming a liquid bridge, but actually remove the ingredients of the one filtrate, such as ions even nucleic acid species that do not bind to the target molecule under the selected conditions enter the reaction mixture or the filtrate of a filtration unit which is preferably used in a neighboring hole.
  • FIG. 8 A shows a further detailed view of a part of the cover construction.
  • an o-ring is provided as a seal in the one of the plurality of bores contained in the cover construction, which serves to receive a filter or a filter column, which is arranged between the bore receiving the filter column and the cover construction, so that no liquid bridge is formed, in particular not between different filtration approaches which are contained in the lid construction.
  • FIG. 8 C shows an embodiment of the vacuum chamber according to the invention.
  • the cover part (11) of the chamber provided with bores (13) or (14) sits on the bottom part (10) of the vacuum chamber (not shown), the cover part (11) and bottom part (10) being separated by a seal.
  • each bore (13) or (14) there is an O-ring (15) attached as a seal, which ensures that the filtration column (1), which is preferably designed, as shown in Fig. 7, is fixed in the holes and in particular that there is no cross-contamination between those carried out in the individual holes
  • RNA molecules specifically binding rat ⁇ ! -D-CGRP (calcitonin gene-related peptides; Amara et al., 1982, Nature 298, 240-244) from a starting pool is described.
  • NTPs were from Larova, Berlin; dNTPs obtained from Qiagen, Hilden.
  • the T7 RNA polymerase was from Stratagene, Heidelberg; DNase I from Sigma-Aldrich, Taufkirchen; Taq polymerase and RNase Out RNase inhibitor from Invitrogen.
  • Qiagen's OneStep RT-PCR Kit was used for RT-PCR.
  • Rat ⁇ -D-CGRP was synthesized by BACHEM, Heidelberg.
  • the peptide used for the selection carries a biotin group at the carboxyl terminus in order to enable the separation of unbound nucleic acids by means of the biotin-neutrAvidin interaction.
  • NeutrAvidin-Agarose and NeutrAvidin-UltraLink from Pierce were used for this.
  • the DNA for the start pool was synthesized in-house and is based on pool DE.40 with the sequence 5'-TCT AAT ACG ACT CAC TAT AGG AGC TCA GAC TTC ACT CGT GN 40 - CAC GTA CCA CTG TCG GTT CCA C-3 ' (SEQ.ID.No. 22).
  • All non-enzymatic steps of the selection were carried out in selection buffer (HEPES-KOH, pH 7.5; 150 mM NaCl; 1 mM MgCl 2 ; 1 mM CaCl 2 ; 0.1% [w / vol] Tween-20).
  • the denaturation was carried out for 5 minutes at 95 ° C. in selection buffer without CaCl 2 and MgCl 2 .
  • the RNA was cooled to room temperature, MgCl 2 and CaCl 2 were added and incubated for a further 5 minutes at room temperature.
  • the RNA was first incubated at room temperature for 15 minutes without peptide with the matrix (NeutrAvidin-Agarose or NeutrAvidin-UltraLink). This so-called preselection was used to remove potential matrix binders. After this incubation step, the matrix was separated from the RNA by sedimentation, mixed with the concentrations of biotinylated rat cD-CGRP shown in FIG. 9 and incubated for 1 h at room temperature. The biotin-binding matrix was then added to the binding mixture and incubated again with shaking for 10 minutes at room temperature. The matrix was then washed to remove non-binding RNA species from binding. The wash volume used here was 5 times the volume of the matrix in the first rounds (50 ⁇ l matrix for rounds 1 and 2, 10 ⁇ l for rounds 3-9), in later rounds up to 135 times the wash volume was used. elution of the matrix in the first rounds (50 ⁇ l matrix for rounds 1 and 2, 10 ⁇ l for rounds 3-9), in later rounds up to 1
  • RNA was eluted by resuspending the matrix particles with bound RNA in RT-PCR buffer and heating to 95 ° C. for 3 minutes. Enzymes were then added to the batches (reverse transcriptase and thermostable DNA polymerase from the OneStep RT-PCR Kit [Qiagen]) and the reverse transcription and the subsequent PCR were carried out in the presence of the matrix.
  • ssDNA In order to obtain double-stranded, in vitro transcribable DNA, 2 nmol synthesized ssDNA (Pool DE.40) were filled in enzymatically with the appropriate primer DE.40T7 to double-stranded DNA.
  • the reaction conditions were as follows: ssDNA pool, 2 ⁇ M; DE.40T7, 6 ⁇ M; dNTPs, 200 ⁇ M; Taq polymerase, 200 U / ml; 1 x reaction buffer as recommended by the manufacturer with 2.5 mM Mg 2+ .
  • the reaction volume was 1 ml, the incubation was carried out at 63 ° C. for 30 minutes.
  • T7 RNA polymerase and 40 U RNase Out RNase inhibitor were performed with 100 U T7 RNA polymerase and 40 U RNase Out RNase inhibitor in T7 reaction buffer (80 mM HEPES pH 7.5; 22 mM MgCl 2 ; 1 mM Spe ⁇ nidin; 10 mM dithiothreitol [DTT]; 4 mM GTP each , ATP, CTP and UTP; 80 ⁇ g / ml BSA) in a total volume of 100 ⁇ l. Between 100 and 30 ul RT-PCR reaction with the resulting double-stranded DNA was used as a transcription template per 100 ul reaction. The reactions were incubated for 3-12 hours at 37 ° C and then DNase I was added to digest the template DNA.
  • T7 reaction buffer 80 mM HEPES pH 7.5; 22 mM MgCl 2 ; 1 mM Spe ⁇ nidin; 10 mM dithiothreitol [DTT]; 4 mM
  • RNA produced was then separated from non-incorporated NTPs either under denaturing conditions using an 8% polyacrylamide gel with 8 M urea or alternatively using ultrafiltration with ultrafiltration units. Ultrafiltration-purified RNA was rinsed from the filter, gel-purified RNA from the cut out pieces of gel eluted, precipitated with ethanol, dried and taken up in water.
  • RNA molecules The reverse transcription of selected RNA molecules was carried out with the Qiagen OneStep RT-PCR Kit under buffer conditions recommended by the manufacturer in the presence of the NeutrAvidin-Agarose or -UltraLink matrix in 100 ⁇ l volume.
  • the batches were completely heated with matrix, RNA adhering to them and RT-PCR reaction buffer at 95 ° C. for 3 minutes, then equilibrated at 50 ° C. for 2 minutes before the enzymes were added.
  • the reactions were held at 50 ° C for 20 minutes, then at 60 ° C for 10 minutes. Inactivation of the RT enzymes and activation of the thermostable polymerase was achieved by heating to 95 ° C for 15 minutes.
  • the parameters of the thermal cycling were as follows: denaturing, 30 s at 95 ° C; Annealing, 30 s at 63 ° C; Polymerization, 30 s at 72 ° C.
  • the amount of double-stranded DNA generated during the PCR was monitored semi-quantitatively. The purpose of this was to keep the number of PCR cycles as small as possible. This means that only as many PCR cycles are carried out as are necessary to obtain enough templates for the T7 reaction. Aliquots were therefore taken from the PCR batches during the PCR from a certain number of cycles and added to 90 ⁇ l of a PicoGreen solution (diluted 1: 400 in TE [10 mM Tris-HCl, pH 8; 1 mM EDTA]). PicoGreen is a fluorescent dye that hardly free in solution, but strongly fluoresces when bound to double-stranded DNA (Ex: 485 nm; Em: 520 ran).
  • Double vacuum chamber with chamber A for the separation of bound and unbound RNA species and chamber B for the purification of transcription reactions.
  • hot start • 50 ° C work station, in order to be able to start enzymatic reactions directly at this temperature (“hot start”) or to not allow PCR reactions to cool down too much during sampling for fluorescence control.
  • the selection rounds 1 and 2 were carried out manually because the large amounts of matrix which are required for the binding of the D-CGRP in the (necessarily high) concentrations of these initial rounds can no longer be processed safely by pipetting machines. From round 3 onwards, selection was made fully automatically, and after two automatic selection rounds it was decided which selection strand was to be continued.
  • the selection strand is highlighted by thick bars, which ultimately also produced the sequences below.
  • the selected RNA of the most stringent strand i.e. the lowest peptide concentration or the highest wash volume, which gave a significant signal above the zero control during the amplification, used in the next round.
  • the number of cycles required to reach the threshold value was used as a measure of this signal.
  • a total of 9 preparative rounds, seven of which were automatic, and another analytical round with a total of seven different peptide concentrations were carried out.
  • the populations of dsDNA molecules from round 9 with 110 nM (approach 110) or round 9 with 12 nM D-CGRP (approach 12) were cloned and a total of 96 clones (48 each) were sequenced.
  • RNA molecules were examined with Seq.ID.No. 1 to 10. For this purpose, 7 pmol each of the labeled RNA was denatured for 3 minutes at 95 ° C. in selection buffer without Ca “ * ” * or Mg ⁇ 1 “1” , folded by adding 1 mM of these ions at room temperature and then folded incubated for 1 hour with biotmylated D-CGRP in concentrations of 0, 100 or 300 nM at room temperature. A constant amount of NeutrAvidin-Agarose particles was then added as a matrix and the RNA: peptide complex was immobilized for 10 minutes with shaking. The matrix was separated, the supernatant removed and the difference in bound / unbound RNA determined. The control (0 nM D-CGRP) as background. The percentage connections are shown in Fig. 11.
  • Molecule Hl differs from G2 in that it lacks two complementary bases that are part of an intramolecular helix (G25 and C59 in molecule G2). Since both molecules bind comparatively well to the target molecule, the sequence of the shorter molecule Hl (82mer) was assumed.
  • a corresponding ssDNA molecule for in vivo transcription of the molecule HIV (47-mer; removal of bases 1-17 and 65-82; exchange of C18 -> G and G64 -> C) was synthesized and filled in to form the dsDNA and generates the desired, radioactively labeled aptamer (SEQ.ID.No. 17) using T7 polymerase.
  • SEQ.ID.No. 17 radioactively labeled aptamer
  • Example 3 Inhibition of cAMP production by rat ⁇ -L-CGRP binding
  • cells of the human neuroblastoma line SK-N-MC were seeded in a number of 4 ⁇ 10 4 per well of a 96 well microtiter plate and at 37 ° C. and 5% CO 2 in DMEM (1,000 mg / 1 glucose) with 10% heat-inactivated fetal calf serum (FCS), 4 mM L-alanyl-L-glutamine (GLUTAMAX), 50 units / ml penicillin and 50 ⁇ g / ml streptomycin. 48 hours after sowing, the cells were 80-90% confluent and were used for the experiments.
  • DMEM 1,000 mg / 1 glucose
  • FCS heat-inactivated fetal calf serum
  • GLUTAMAX 4 mM L-alanyl-L-glutamine
  • 50 units / ml penicillin and 50 ⁇ g / ml streptomycin 48 hours after sowing, the cells were 80-90% confluent and were used for the experiments.
  • the Spiegelmers were together with 1 nM L-CGRP (Bachern) in Hank's balanced salt solution (HBSS) + 1 mg / ml BSA for 15 - 60 minutes at 37 ° C in a 0.2 ml "low profile 96-tube" - Incubated plate. Shortly before addition to the cells, 2 ⁇ l of a 50 mM IBMX solution (3-isobutyl-l-methylxanthine) were added. The cells were pretreated with 1 mM IBMX 20 minutes before adding the L-CGRP / Spiegelmer batches.
  • HBSS Hank's balanced salt solution
  • the medium was aspirated from the cells and the pre-incubated batches were added. After incubation for 30 minutes at 37 ° C., the supernatants were aspirated and the cells were lysed with 50 ⁇ l / well lysis buffer for 30 minutes at 37 ° C.
  • the lysis buffer is part of the "cAMP-Screen TM System” kit (Applied Biosystems), with which the cAMP content of the extracts is determined. 10 ⁇ l of the extracts are used in the test.
  • the test was carried out as described by the manufacturer. 10 ⁇ l / well of the lysate were added to 50 ⁇ l of the lysis buffer in an assay plate (coated with goat anti-rabbit IgG) and mixed with 30 ⁇ l / well of the cAMP-alkaline phosphatase conjugate diluted according to the manufacturer's instructions. Then 60 ⁇ l / well of the cAMP antibody supplied in the kit was added and incubated for one hour while shaking at room temperature. The solutions were then removed from the wells and washed 6 times with the washing buffer provided. For the detection, 100 ⁇ l / well CSPD / Sapphire-II RTU substrate were added, incubated for 30 minutes at room temperature and the luminescence was measured in a POLARstar Galaxy multi-detection plate reader (BMG).
  • BMG POLARstar Galaxy multi-detection plate reader
  • the biological effectiveness of the Spiegelmers was initially roughly estimated.
  • the molecules H1C, H1C 1, H1C 2 and H1C 1 + 2 were used only in a concentration (300 nM), the cells were stimulated, lysed and finally the cAMP content of the extracts was determined as described. Triplicate determinations were carried out and the amounts of cAMP formed were plotted as a percentage of the control (no mirror bucket in the pre-incubation batch) ⁇ standard deviation.
  • H1C inhibited cAMP production by 66%, H1C 1 by 37.8% and H1C 1 + 2 by 21.4%.
  • the activation of cAMP production observed for H1C 2 is most likely an artifact.
  • Example 4 Automatic preparation of aptamers using
  • the in vitro selection for the generation of aptamers usually starts from nucleic acid start libraries, in which the variable region is flanked by constant regions, which are normally approximately 20 nucleotides long. This enables problem-free duplication of single molecules by means of the polymerase chain reaction (PCR) in the course of the selection rounds.
  • PCR polymerase chain reaction
  • a shortening of the sequences, often 80-120 nucleotides long, while maintaining the affinity for the target molecule used, is very often not possible by simply omitting the constant regions.
  • the easy chemical representation of selected aptamers is only guaranteed for comparatively short molecules (if possible ⁇ 50 nucleotides). Aptamers that cannot be shortened to this length are therefore not suitable for use as therapeutic agents.
  • a selection process was developed in which the target peptide is linked to a population of short nucleic acids with randomized sequences, while the necessary constant regions for the steps of nucleic acid amplification are linked to the selected nucleic acid ligands by enzymatic ligation.
  • the ligands resulting from this method have a maximum length of the nucleic acids present in the binding step.
  • RNA molecules which specifically bind rat- ⁇ -D-CGRP from such a starting pool is described.
  • NTPs and dNTPs were obtained from Larova, Berlin.
  • the T7 RNA polymerase 50 U / ⁇ l was from Stratagene, Heidelberg; DNase I from Sigma-Aldrich, Taufkirchen; Taq polymerase (5 U / ⁇ l), SuperScript II reverse transcriptase (200 U / ⁇ l), and RNase Out RNase inhibitor (40 U / ⁇ l) from Invitrogen.
  • the T4 DNA ligase (30 U / ⁇ l) was obtained from Fermentas (St. Leon-Rot).
  • Rat ⁇ -D-CGRP was synthesized by BACHEM, Heidelberg.
  • the peptide used for the selection carries a biotin group at the carboxyl terminus in order to enable the separation of unbound nucleic acids by means of the biotin-neutrAvidin interaction.
  • NeutrAvidin-Agarose and NeutrAvidin-UltraLink from Pierce were used for this.
  • RNA and DNA nucleotides were all synthesized at NOXXON Pharma AG using standard phosphoramidite chemistry.
  • STAR-1 oligonucleotides used in this application example bold STAR-1 oligonucleotide library (RNA)
  • STAR-1 oligonucleotide library during ligation with a total of 5 different nucleic acid molecules (from left to right and top to bottom STAR-1 forward ligate, STAR-1 oligonucleotide library, STAR-1 reverse ligate, STAR-1 forward matrix, STAR -1 reverse matrix) are arranged as follows:
  • RNA 5'-gcgacuacuaauacgacucacuaua-3 '(25 nt) (SEQ. ID. NO. 27)
  • the selection was started with 1.67 nmol of synthesized single-stranded DNA (STAR-1 initial pool, reverse strand), corresponding to a complexity of approximately 10 15 molecules.
  • This start DNA was brought together with 2.5 nmol STAR-1 forward primer (1.5-fold excess) to a volume of 3.34 ml in in vitro transcription buffer, heated to 95 ° C. for 5 min and then slowly to 37 ° C cooled.
  • the DNA was transcribed into the corresponding RNA start pool. After the transcription reaction (for details see Section C - Enzymatic Reactions), the template DNA was digested with DNAse I, the RNA was gel purified and precipitated with ethanol.
  • RNA was first incubated at room temperature for 15 minutes without peptide with the matrix (NeutrAvidin-Agarose or UltraLink Plus nmobilized NeutrAvidin Gel; both matrices from Pierce). This so-called preselection was used to remove potential matrix binders. After this incubation step, the unbound RNA was removed from the matrix by filtration through Mobitec columns (Mobitec, Göttingen) with a pore size of 10 ⁇ m separated; in the case of automatic execution, the matrix was made simple
  • the remaining nucleic acid species were then mixed with various concentrations of biotmylated rat ⁇ -D-CGRP and incubated for 1-2 hours at room temperature or 37 ° C.
  • the biotin-binding matrix was then added to the mixture and incubated again with shaking for 10 minutes at room temperature.
  • the matrix was then washed to remove non-binding RNA species from binding.
  • the wash volume used was 5 to 10 times the volume of the matrix in the first two manual rounds, and 135 times the solid phase volume in later automatic rounds.
  • the bound RNA was eluted by incubating the matrix particles twice with bound RNA in 400 ⁇ l 8 M urea / 10 mM EDTA for 15 min at 65 ° C.
  • the eluted RNA was phenol extracted, ethanol precipitated and dried.
  • Elution was achieved by heating the matrix particles in 120 ul 8 M urea / 10 mM EDTA at 95 ° C for 10 min. After sedimentation of the matrix particles, the low molecular weight denaturants urea and EDTA were removed from the supernatant; this was transferred to MicroCon YM-10 ultrafiltration columns (Millipore, Schwalbach) and ultrafiltered using the vacuum chamber integrated in the robot. A vacuum of -700 to -900 mbar was applied for 20 min per 50 ⁇ l of solution to be filtered. In order to recover the expected small amounts of RNA (approx.
  • T7 RNA polymerase Transcriptions were carried out with 100 U T7 RNA polymerase and 40 U RNase Out RNase inhibitor in T7 reaction buffer (80 mM HEPES pH 7.5; 22 mM MgCl 2 ; 1 mM spermidine; 10 mM dithiothreitol [DTT]; 4 mM GTP each , ATP, CTP and UTP; 120 ⁇ g / ml BSA) per 100 ⁇ l volume.
  • GTP guanosine 5'-monophosphate
  • GTP guanosine 5'-monophosphate
  • RNA generated was then either separated manually from denatured NTPs under denaturing conditions using an 8% polyacrylamide gel with 8 M urea or automatically by means of ultrafiltration with ultrafiltration units. Ultrafiltration-purified RNA was rinsed from the filter, gel-purified RNA was eluted from the cut-out gel pieces, precipitated with ethanol, dried and taken up in water.
  • RNA to be ligated from the selection rounds was placed in 14 ⁇ l ligation buffer (40 mM Tris-HCl pH 7.8; 10 mM MgCl 2 ; 10 mM DTT; 10 ⁇ M EDTA; 0.5 mM ATP; 5% PEG 4000; 5 ⁇ M ds forward adapter [consisting of hybridized forward ligat RNA forward matrix]; 2.5 ⁇ M ds reverse adapter [consisting of hybridized reverse ligate / reverse primer ribol]; 2.5 ⁇ M ds reverse adapter N + l [consisting of hybridized N + l reverse ligate / reverse primer ribol) dissolved per pmol of eluted RNA (manual rounds 1 and 2) or in principle resuspended in 40 ⁇ l ligation buffer from the ultrafiltration membrane, of which 25 ⁇ l were used further (automatic rounds 3-15). The batches were then 5 min at 50 ° C and 5 min at 25 ° C and 5
  • RNA molecules were incubated at 51 ° C. for 20 min and then at 54 ° C. for 10 min.
  • the temperature profile was as follows: 20 min at 50 ° C.; 2 min 53.3 ° C; 2 min 56.6 ° C; 10 min 60 ° C.
  • the PCR was carried out with a maximum of 1 pmol template per 100 ⁇ l reaction volume.
  • 1/5 volume (20 ⁇ l) of each reverse transcription with 100 ⁇ l was used in order to serve as a template in four 100 ⁇ l batches (20 mM Tris-HCl pH 8.4; 50 mM KC1; 1.5 mM MgCl 2 ; 5 ⁇ M forward primer; 5 ⁇ M reverse primer ribol; 200 ⁇ M dNTPs; 50 U / ml Taq DNA polymerase).
  • the course of the PCR was monitored by means of fluorescence measurement of aliquots from the PCR reactions and the PCR was stopped after the required threshold value had been reached (see section D, checking the PCR progress).
  • the amount of double-stranded DNA generated during the PCR was monitored semi-quantitatively. It was intended to keep the number of PCR cycles as small as possible. For this reason, only as many PCR cycles were carried out as are necessary to obtain sufficient templates for the T7 reaction. Aliquots were therefore taken from the PCR batches during the PCR from a certain number of cycles and added to 90 ⁇ l of a PicoGreen solution (diluted 1: 400 in TE [10 mM Tris-HCl, pH 8; 1 mM EDTA]). PicoGreen is a fluorescent dye that hardly free in solution, but strongly fluoresces when bound to double-stranded DNA (Ex: 485 nm; Em: 520 ⁇ m).
  • Double vacuum chamber with chamber A for the separation of bound and unbound RNA species and chamber B for the removal of denaturants from the eluted RNA molecules after elution, the desalting of the PCR batches after alkaline digestion and the purification of transcription reactions before the start of the next round.
  • hot start • 50 ° C work station, in order to be able to start enzymatic reactions directly at this temperature (“hot start”) or to not allow PCR reactions to cool down too much during sampling for fluorescence control.
  • Rounds 1 and 2 were carried out manually because the large amounts of matrix which are required for the binding of the ⁇ -D-CGRP in the (necessarily high) concentrations of these initial rounds can no longer be processed safely by pipetting machines. From round 3, the selection was fully automatic.
  • the selected RNA of the most stringent strand i.e. the lowest peptide concentration or the highest wash volume, which gave a significant signal above the zero control during the amplification, used in the next round.
  • the number of cycles required to reach the threshold value was used as a measure of this signal.
  • FIGS. 19 A and B The result of the sequence analysis is shown in FIGS. 19 A and B, respectively. Of the total of 96 clones, both primers could be found in 88 clones. The individual clones were the following SEQ. ID. NOs. assigned.
  • Clone PL-D8 was synthesized as a mirror bucket from L-nucleotides for more detailed biophysical characterization.
  • the binding of mirror bucket PL-D8 to rat or human ⁇ -L-CGRP was determined using the method of isothermal titration calorimetry (ITC) with the VP ITC (Microcal).
  • the binding constant or dissociation constant K D is the reciprocal of the association constant K A (K in the enthalpy diagram) and was obtained for the mirror bucket PL-D8 when measuring with rats ⁇ -L-CGRP solution at 44 nM with an activity of 47% (Fig 20 A).
  • a dissociation constant of 171 nM at 64% activity was measured with this mirror bucket against human ⁇ -L-CGRP (FIG. 20 B).
  • Combination for realizing the invention in its various embodiments may be essential.

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Abstract

La présente invention concerne un procédé de sélection d'au moins un acide nucléique ligand à partir d'un mélange d'acides nucléiques candidats, les acides nucléiques ligands pouvant se fixer à une molécule cible. Ce procédé comprend les étapes suivantes: a) mise en contact du mélange avec la molécule cible immobilisée; b) séparation de l'acide ou des acides nucléiques se liant avec une affinité élevée à la molécule cible du reste du mélange de candidat, l'acide ou les acides nucléiques se fixant avec une affinité élevée à la molécule cible formant un complexe avec celle-ci; c) élution de la molécule cible de l'acide ou des acides nucléiques se fixant à celle-ci avec une affinité élevée; et d) amplification éventuelle de l'acide ou des acides nucléiques se fixant avec une affinité élevée, cette amplification permettant d'obtenir un acide ou des acides nucléiques enrichis. La molécule cible utilisée est immobilisée sur une surface et le procédé comprend une étape d'ultrafiltration.
PCT/EP2003/010053 2002-09-10 2003-09-10 Procede de selection d'acides nucleiques ligands WO2004024950A1 (fr)

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EP2336314A1 (fr) * 2007-07-17 2011-06-22 Somalogic, Inc. Selex et photoselex améliorés
US8409795B2 (en) 2007-07-17 2013-04-02 Somalogic, Inc. Selex and photoSELEX

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