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WO2003093504A1 - Procede d'amplification d'acides nucleiques - Google Patents

Procede d'amplification d'acides nucleiques Download PDF

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Publication number
WO2003093504A1
WO2003093504A1 PCT/EP2003/004747 EP0304747W WO03093504A1 WO 2003093504 A1 WO2003093504 A1 WO 2003093504A1 EP 0304747 W EP0304747 W EP 0304747W WO 03093504 A1 WO03093504 A1 WO 03093504A1
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Prior art keywords
nucleic acid
strand
amplified
partial sequence
sequence
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PCT/EP2003/004747
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German (de)
English (en)
Inventor
Axel Vater
Florian Jarosch
Axel Wettich
Sven Klussmann
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TME Pharma AG
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Noxxon Pharma AG
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Priority to AU2003233243A priority Critical patent/AU2003233243A1/en
Publication of WO2003093504A1 publication Critical patent/WO2003093504A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6865—Promoter-based amplification, e.g. nucleic acid sequence amplification [NASBA], self-sustained sequence replication [3SR] or transcription-based amplification system [TAS]
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
    • C12Q1/6855—Ligating adaptors
    • 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
    • C12Q2541/00—Reactions characterised by directed evolution
    • C12Q2541/10—Reactions characterised by directed evolution the purpose being the selection or design of target specific nucleic acid binding sequences
    • C12Q2541/101—Selex

Definitions

  • the present invention relates to a method for amplifying nucleic acids and the use of the method in a method for producing target molecule-binding nucleic acids.
  • nucleic acids The amplification of nucleic acids is a process that has long been known in biology and is essential for any self-replicating biological system. Amplifications are also used extensively in genetic engineering, in particular when nucleic acids that are only present in small amounts or low concentrations have to be amplified. Applications for this can be found in basic biological research as well as in the development of new drugs or forensics.
  • oligonucleotide ligands have recently become established as active pharmaceutical ingredients or precursors thereof.
  • these oligonucleotide ligands which include, among others, antisense and ribozyme oligonucleotides and RNAi / siRNA, the so-called aptamers are of particular interest insofar as they are nucleic acids, preferably single-stranded nucleic acids, that bind to a target molecule.
  • the technology with which these aptamers are produced is known as the SELEX process (systematic evolution of ligands by exponentialal enrichment) and is described, for example, in US Patents 5,270,163 and US 5,843,653.
  • the interaction of the aptamers with their target molecule is essentially not based on base hybridization, but on a combination of hydrophobic interactions, hydrogen bonds and Coulomb interactions.
  • the high affinity and highly specific interaction to a target molecule is mediated by complex and stable structures of the oligonucleotides.
  • oligonucleotides can form stable structures similar to those that have long been known for proteins.
  • SELEX process also referred to here as SELEX technology
  • a large number of aptamers - both on the basis of RNA and DNA - have been selected against a wide variety of targets.
  • the SELEX process basically provides for the following steps: providing a nucleic acid library, the different nucleic acids differing at least in a completely or partially randomized range, separating the nucleic acids that do not bind to the target molecule from the complexes of the target molecule and the nucleic acids that bind to it, and gaining the nucleic acids from the complex of nucleic acid and target molecule, reverse transcription in the event that the nucleic acid binding to the target molecule is a ribonucleic acid, carrying out the polymerase chain reaction, followed by transcription (if it is an RNA) and as a rule reusing the amplified nucleic acid thus obtained in a selection step.
  • the selection steps which are generally repeated several times, the nucleic acids, which typically bind to the target molecule with
  • the nucleic acid to be amplified also comprises at least one constant region in addition to the randomized region, which serves, for example, as a primer binding site or promoter for the various enzymes involved.
  • aptamers and Spiegelmers described in the literature ie aptamers consisting of L-RNA, (Leva S, Lichte et al. (2002) Chemistry & Biology 9: 351-9) contain part of the PCR primer binding regions as one for the Recognition of the respective target molecule essential part of the molecule again. Examples are: 2'-NH 2 RNA aptamer against IgE, in which the entire 5 'primer region with the exception of two 5'-terminal Gs occurs in the minimal binding motif (Wiegand, TW, PB Williams, et al.
  • the present invention is therefore based on the object of providing a method for the amplification of nucleic acids, which can preferably be used in a method for the selection of target molecule-binding nucleic acids, the nucleic acids selected in this way having only a small size compared to such selection methods in which Nucleic acid populations are used which consist of a randomized area and an approximately equally long constant area. Furthermore, it is an object of the present invention to provide a method which allows the method for the selection of nucleic acids binding to target molecules between the individual selection rounds to have a reduced number of cleaning steps compared to the prior art.
  • the object is achieved according to the invention by a method for amplifying a nucleic acid comprising the steps:
  • nucleic acid to be amplified, the nucleic acid preferably being a ribonucleic acid and comprising a first defined partial sequence at the 5 'end, a second defined partial sequence at the 3' end and an intermediate sequence between the first defined partial sequence and the second defined partial sequence .
  • first adapter molecule consisting of a double-stranded nucleic acid from a first and a second strand of nucleic acid and wherein the first strand of nucleic acid is preferably a ribonucleic acid and the second strand of nucleic acid is preferably a deoxyribonucleic acid and the 5 'end of the second nucleic acid provides an overhang, the overhang being at least partially complementary to the first defined partial sequence of the nucleic acid to be amplified or a part thereof,
  • the second adapter molecule consisting of a double-stranded nucleic acid from a first and a second strand of nucleic acid, the first strand of nucleic acid carrying a phosphate group at the 5 'position of the ribose or deoxyribose part of the 5'-terminal nucleotide and the second strand of nucleic acid is a deoxyribonucleic acid and the 3 'end of the second strand of nucleic acid is at least partially complementary to the second defined partial sequence of the amplifying nucleic acid or a part thereof and wherein the second nucleic acid strand has a cleavage site which, when cleaving the nucleic acid strand provides a first cleavage product and a second cleavage product, the first cleavage product being the 3 'end of the second nucleic acid strand of the second adapter molecule, which is at least partially complementary is to the second defined partial sequence of
  • step (g) optionally cleaving a strand of the reaction product from one of steps (d) to (f), the strand being that which is complementary to the nucleic acid to be amplified in order to obtain a transcription starting product, and
  • the object is achieved according to the invention by a method for amplifying a nucleic acid comprising the steps:
  • nucleic acid to be amplified, the nucleic acid preferably being a deoxyribonucleic acid and comprising a first defined partial sequence at the 5 'end, a second defined partial sequence at the 3' end and an intermediate sequence between the first defined partial sequence and the second defined partial sequence .
  • step (aa) phosphorylating the 5 'end of the nucleic acid to be amplified, provided that the nucleic acid to be amplified provided in step (a) has no phosphate at the 5' end
  • first adapter molecule (b) providing a first adapter molecule, wherein the first adapter molecule consists of a double-stranded nucleic acid from a first and a second strand of nucleic acid, and wherein the first strand of nucleic acid and the second strand of nucleic acid is a deoxyribonucleic acid and the 5 'end of the second nucleic acid strand provides an overhang, the overhang being at least partially complementary to the first defined partial sequence of the nucleic acid to be amplified or a part thereof,
  • step d) which ligates the first strand of the first adapter molecule at the 5 'end of the nucleic acid to be amplified and the first strand of the second at the 3' end of the nucleic acid to be amplified
  • Adapter molecule is ligated and at least the second strand of the second adapter molecule is hybridized at the 3 'end of the nucleic acid to be amplified and / or the first strand of the second adapter molecule ligated to the nucleic acid to be amplified
  • a primer molecule comprising a first region which is at least partially identical to the first defined partial sequence of the nucleic acid to be amplified and / or a second region adjoining it in the 5 'direction, which is at its 3' end has a cleavage site, and a third region adjoining it in the 5 ′ direction, the third region being partially identical to the sequence of the first strand of the first adapter molecule, and hybridizing the primer molecule to the transcription product,
  • step (i) optionally performing a cleavage at the cleavage site of the primer molecule.
  • the transcription starting product according to step (e) is the result of a second strand synthesis and the second strand is preferably produced by extending the second strand of nucleic acid of the second adapter molecule.
  • the transcription starting product according to step (e) is obtained by a polymerase chain reaction, a PCR primer molecule preferably being used for the ligation product in addition to the second nucleic acid strand of the second adapter molecule, the PCR Primer molecule is at least partially complementary to the first defined partial sequence of the nucleic acid to be amplified and / or to the first strand of nucleic acid of the first adapter molecule.
  • the reverse transaction product is subjected to an alkaline cleavage.
  • step (aa) is carried out by a kinization.
  • the second nucleic acid strand of the second adapter molecule contains a promoter or part of a promoter for an RNA polymerase, preferably T7, T3 or SP6 RNA polymerase.
  • the promoter is arranged in such a way that the resulting transcripts begin with the sequence which is complementary to the second defined partial sequence of the nucleic acid to be amplified.
  • the nucleic acid is selected from the group comprising double-stranded RNA, single-stranded RNA, double-stranded modified RNA and single-stranded modified RNA.
  • the nucleic acid is selected from the group comprising double-stranded DNA, single-stranded DNA, double-stranded modified DNA and single-stranded modified DNA.
  • the 5'-terminal nucleotide of the nucleic acid to be amplified carries a 5'-phosphate.
  • An embodiment of the method according to the invention in accordance with the first aspect provides that the first strand of the first adapter molecule is a ribonucleic acid and the nucleic acid to be amplified is a ribonucleic acid.
  • the first strand of the adapter molecule is a deoxyribonucleic acid and the nucleic acid to be amplified is a deoxyribonucleic acid.
  • the cleavage site is provided by a restriction enzyme interface and the cleavage is carried out by a restriction enzyme.
  • the cleavage site is formed by a ribonucleotide.
  • the nucleic acid to be amplified is a ribonucleic acid
  • the ribonucleotide in the second nucleic acid strand of the second adapter molecule is the first nucleotide 5 'of the region which is at least partially complementary to that second defined partial sequence of the nucleic acid to be amplified or a part thereof; and / or that if the nucleic acid to be amplified is a deoxyribonucleic acid, the ribonucleotide in the primer molecule according to step (g) of the method according to the second aspect is the first nucleotide 5 'of the first defined partial sequence.
  • the transcription product is selected from the group consisting of the RNA, 2'-F-RNA, 2'-NH 2 -RNA and base-modified ribonucleic acids and / or the reverse transcription product is selected from the group comprising DNA and base-modified deoxyribonucleic acids.
  • the transcription product is a nucleic acid, preferably a single-stranded nucleic acid, wherein
  • the nucleic acid is identical to the nucleic acid to be amplified, and / or
  • the nucleic acid is identical to the nucleic acid to be amplified and has an additional nucleotide at the 3 'end.
  • the transcription product has the nucleic acid to be amplified and additionally at the 3 'end at least one additional nucleotide, characterized in that a third adapter molecule is provided, the third adapter molecule being made from a double-stranded nucleic acid consists of a first and a second nucleic acid strand, the first nucleic acid strand carrying a 5'-phosphate at the 5 'end and the second nucleic acid strand being a deoxyribonucleic acid and the 3' end of the second nucleic acid strand being at least partially complementary to the second defined partial sequence of the nucleic acid to be amplified or a part thereof, and wherein the second strand of nucleic acid has a cleavage site which, when the strand of nucleic acid is cleaved, provides a first cleavage product and a second cleavage product, the first cleavage
  • the nucleic acid to be amplified is a deoxyribonucleic acid, optionally a base-modified deoxyribonucleic acid, and the first defined partial sequence of the nucleic acid to be amplified is a length of at least one nucleotide, preferably at least two nucleotides having.
  • the first defined partial sequence is selected from the group of nucleic acid sequences which comprises GG, CC and CG.
  • the nucleic acid to be amplified is selected from the group which modified ribonucleic acid, 2'-fluorine-modified ribonucleic acid, 2'-amino Ribonucleic acid and base-modified ribonucleic acid, and the first defined partial sequence has a length of at least one, preferably at least four nucleotides.
  • the first defined partial sequence is selected from the group of nucleic acid sequences which comprises GGAC.
  • the second defined partial sequence of the nucleic acid to be amplified has a length of at least one, preferably four to six, preferably six nucleotides.
  • the nucleic acid to be amplified is a ribonucleic acid and that the second defined partial sequence is selected from the group of nucleic acid sequences, which includes GACAGG and GGCCGG.
  • the nucleic acid to be amplified is a deoxyribonucleic acid and that the second defined partial sequence is selected from the group of nucleic acid sequences, which comprises CTGTCC or CCGGCC.
  • the region of the second strand of the first adapter molecule which is complementary to the nucleic acid to be amplified is at least partially composed of LNA.
  • At least one of the 3 'ends of at least one of the existing adapter molecules is blocked in order to avoid an extension of the adapter molecules in a second-strand synthesis.
  • the 3 'end of the second strand of nucleic acid of the first adapter molecule and of the first strand of nucleic acid of the second and third adapter molecules is blocked.
  • the blocking takes place in that the 3'-terminal nucleotide of the adapter molecule is a 3'-deoxynucleotide, preferably a 2 '-3'-dideoxynucleotide.
  • the second-strand synthesis is a reaction which is selected from the group comprising the polymerase chain reaction and filling reactions with DNA polymerases.
  • the first strand of the first adapter molecule or a part thereof at the 3 'end contains a promoter for an RNA polymerase.
  • additional bases are arranged in the 5 'direction of the promoter in order to increase the melting point of the primer used in the second-strand synthesis, using as a primer the first nucleic acid strand of the first adapter molecule is used and a DNA primer is added, the sequence of which corresponds to the sequence of the first strand of the first adapter and the 3 'end of which preferably additionally comprises a sequence which is identical to the first defined partial sequence of the one to be amplified Nucleic acid.
  • the second strand of the second adapter molecule and the second strand of the third adapter molecule or a part thereof is designed as a primer for reverse transcription and the nucleic acid to be amplified is a ribonucleic acid.
  • the second strand of the second adapter molecule or a part thereof is designed and used as a primer for the second strand synthesis, the nucleic acid to be amplified being a Is deoxyribonucleic acid.
  • the second strand of the second adapter molecule and the second strand of the third adapter molecule or a part thereof is designed and used as a primer for the polymerase chain reaction, the polymerase chain reaction being used as a second strand synthesis according to step (f) of the method according to claim 1 or according to step (e) of the method according to claim 2.
  • the first nucleic acid strand of the first adapter molecule and the second nucleic acid strand of the second adapter molecule and the second strand of the third adapter molecule are used as primers for the polymerase chain reaction, preferably on the first nucleic acid strand a further sequence is present at the respective 3 'end and the further sequence preferably corresponds to the first defined partial sequence of the nucleic acid to be amplified.
  • the second strand of nucleic acid of the third adapter molecule is used instead of the second strand of nucleic acid of the second adapter molecule, preferably in ligation, reverse transcription and / or in second strand synthesis.
  • the nucleic acid to be amplified is a ribonucleic acid and that the second strand synthesis takes place directly after the reverse transcription without prior purification.
  • the nucleic acid to be amplified is a deoxyribonucleic acid and that the second strand synthesis takes place directly after the ligation without prior purification.
  • the transcription product is a modified or unmodified ribonucleic acid
  • the 5'-monophosphate of the first nucleotide preferably guanosine 5 ' monophosphate is used as the starter nucleotide.
  • the starter nucleotide is in excess, preferably in a two to tenfold excess, is preferably used in a four- to eight-fold excess over the respective nucleotide 5'-triphosphate.
  • the nucleic acid to be amplified contains a randomized nucleotide sequence between the first defined partial sequence and the second defined partial sequence, the intermediate sequence preferably being a randomized nucleotide sequence.
  • the randomized nucleotide sequence has a length of 20 to 10,000 nucleotides, preferably of 30 to 60 nucleotides and preferably of 25 to 40 nucleotides.
  • the nucleic acid to be amplified is a single-stranded nucleic acid, the 5 'end and / or 3' end of which is / are part of a duplex structure, characterized in that the ligation step comprises the following substeps:
  • the object is achieved according to the invention by a method for amplifying a nucleic acid, preferably a single-stranded ribonucleic acid, comprising the steps:
  • steps (a) to (g) are carried out without a purification step, steps (a) - (h) preferably taking place in one and the same reaction vessel.
  • the object is achieved according to the invention by a method for amplifying a nucleic acid, preferably a single-stranded deoxyribonucleic acid, comprising the steps:
  • nucleic acid to be amplified (a) Provision of a nucleic acid to be amplified, the nucleic acid comprising a first defined partial sequence at the 5 'end and a second defined partial sequence at the 3' end and an intermediate sequence between the first defined partial sequence and the second defined partial sequence, the nucleotide comprising 5 'end of the nucleic acid to be amplified is or will be provided with a phosphate group,
  • step (g) providing a primer molecule with a cleavage site, in particular a primer molecule as described in step (g) of claim 2;
  • steps (a) to (f) being carried out without a purification step, steps (a) to (i) preferably being carried out in one and the same reaction vessel.
  • the method runs automatically.
  • the object is achieved according to the invention by a method for amplifying a nucleic acid, the nucleic acid to be amplified being a double-stranded ribonucleic acid and the double-stranded ribonucleic acid comprising a (+) strand and a (-) strand, the (+) Strand and the (-) strand are essentially complementary to each other, and
  • the (+) strand comprises a first defined partial sequence at the 5 'end, a second defined partial sequence at the 3' end and an intermediate sequence between the first defined partial sequence and the second defined partial sequence
  • the (-) strand comprises a first defined partial sequence at the 5 'end, a second defined partial sequence at the 3' end and an intermediate sequence between the first defined partial sequence and the second defined partial sequence
  • the (+) strand is amplified according to an inventive method for amplifying ribonucleic acid
  • the (-) strand is amplified according to an inventive method for amplifying ribonucleic acid.
  • the first nucleic acid strand of the first adapter molecule and / or the second strand synthesis primer used for the second strand synthesis, which is used in the amplification of the (+) strand comprises a promoter for a first RNA polymerase, and
  • the first nucleic acid strand of the first adapter molecule, and / or the second strand synthesis primer used for the second strand synthesis, which is used in the amplification of the (-) strand comprises a promoter for a second RNA polymerase.
  • the first and the second RNA polymerase are selected independently of one another from the group comprising 77-RNA polymerase, Ti-RNA polymerase and SP6-RNA polymerase ,
  • the first and the second polymerase are the same or different, preferably different.
  • the transcription with part of the reaction mixture obtained from the previous reaction steps with the first RNA polymerase and another part of the mixture obtained from the previous reaction steps with the second RNA Polymerase is carried out in separate reaction batches,
  • the transcription of the (+) strand and the (-) strand is carried out in one reaction mixture, the first and the second RNA polymerase being used.
  • the defined first partial sequence and the defined second partial sequence of the nucleic acid to be amplified each comprise at least one nucleotide, preferably at least two, preferably at least six nucleotides.
  • the arrangement of the promoter for the first and / or second RNA polymerase leads to a transcript, the beginning of which is equal to the first defined partial sequence of the (+) - or the ( -) - Strands of the double-stranded nucleic acid to be amplified.
  • the object is achieved according to the invention by a method for amplifying a nucleic acid, the nucleic acid to be amplified being a double-stranded deoxyribonucleic acid and the double-stranded deoxyribonucleic acid comprising a (+) strand and a (-) strand, the (+) Strand and the (-) strand are essentially complementary to each other, and
  • the (+) strand comprises a first defined partial sequence at the 5 'end, a second defined partial sequence at the 3' end and an intermediate sequence between the first defined partial sequence and the second defined partial sequence, and
  • the (-) strand comprises a first defined partial sequence at the 5 'end, a second defined partial sequence at the 3' end and an intermediate sequence between the first defined partial sequence and the second defined partial sequence, in which
  • the (+) strand is amplified by a method according to the invention for the amplification of deoxyribonucleic acid
  • the (-) strand is amplified by a method according to the invention for the amplification of deoxyribonucleic acid.
  • the second strand of nucleic acid of the second adapter molecule and / or the PCR primer molecule used for the second strand synthesis which is used in the amplification of the (+) strand, a promoter for comprises a first RNA polymerase, and
  • the second nucleic acid strand of the second adapter molecule, and / or the PCR primer molecule used for the second strand synthesis, which is used in the amplification of the (-) strand comprises a promoter for a second RNA polymerase.
  • the first and the second RNA polymerase are selected independently of one another from the group, the r7-RNA polymerase, r3-RNA polymerase and SP ⁇ - ' KNA polymerase includes.
  • the first and the second polymerase are different.
  • the transcription with part of the reaction mixture obtained from the previous reaction steps with the first RNA polymerase and another part of the mixture obtained from the previous reaction steps with the second RNA polymerase is carried out in separate reaction batches.
  • the transcription of the (+) ⁇ strand and the (-) - Stranges is carried out in a reaction mixture, the first and the second RNA polymerase being used and, before the reverse transcription is carried out, the ribonucleic acid present in the reaction mixture is denatured by an excess of the primer molecule according to step (g) of the method according to claim 2.
  • the separate reaction batches are combined before the alkaline cleavage of the ribonucleic acid present in the reaction batch.
  • the defined first partial sequence and the defined second partial sequence of the nucleic acid to be amplified each comprise at least one nucleotide, preferably 4 nucleotides each.
  • the arrangement of the promoter for the first and / or second RNA polymerase leads to a transcript, the beginning of which is complementary to the second defined partial sequence of the (+) - or the (- ) Strand of the double-stranded nucleic acid to be amplified.
  • the object is achieved according to the invention by a method for selecting a nucleic acid, in particular aptamers, which binds to a target molecule, comprising the steps:
  • nucleic acids in particular D-nucleic acids, each nucleic acid having a region with a randomized sequence and at least one constant sequence and the nucleic acids forming the population differing in the randomized sequence
  • step (d) separating the nucleic acid (s) which has / have interacted with the target molecule from the target molecule, (e) optionally repeating steps (a) to (d), the nucleic acid (s) from step (d) forming the heterogeneous population or being contained therein,
  • step (g) optionally repeating steps (a) to (f), the nucleic acid (s) from step (f) forming the heterogeneous population or being contained therein,
  • step (h) optionally sequencing the nucleic acid (s) obtained from step (d) or step (f),
  • step (f) wherein the amplification according to step (f) is carried out by a method according to one of the aspects one to six.
  • the object is achieved according to the invention by a method for producing L-nucleic acids which bind to a target molecule, which comprises the following steps:
  • step (b) contacting the population of step (a) with the target molecule, preferably with the optical antipode of the target molecule;
  • step (e) optionally repeating steps (b) to (d), the population of step (a) being replaced in whole or in part by the reaction product of steps (b) to (d).
  • the present invention is based on the surprising finding that, in contrast to the methods in the prior art, it is possible to allow an amplification reaction to take place sequentially or simultaneously using a suitable reaction procedure and suitable reactants without purification steps.
  • the nucleic acid to be amplified has short defined sequences at the 5 'end and at the 3' end, which are necessary for carrying out the amplification step, in particular for the base pairing of the adapter molecules.
  • a further sequence of nucleotides is arranged between these defined 5'-terminal and 3'-terminal defined partial sequences, which is also referred to herein as an intermediate sequence.
  • the cut sequence can be designed as a randomized sequence and thus the nucleic acid to be amplified is a nucleic acid that can be used in the context of the nucleic acid library and its use in the SELEX process, and thus the cut sequence is that sequence , which should ultimately deliver the nucleic acid binding to the target molecule.
  • first and second defined partial sequences are only very short, means that these defined partial sequences are involved in the binding event of the nucleic acid to the target molecule comparatively unlikely. Even if this occurs, it is not very annoying due to the short length of the defined sequences.
  • the design of the nucleic acid to be amplified and the amplification method according to the invention can have a considerable influence on the basic length of the nucleic acids which are obtained in the context of the SELEX method and bind to the target molecule, since the length of the randomized region of the nucleic acid (s) depends on the length.
  • the length of the nucleic acid binding to the target molecule can be significantly influenced.
  • the method according to the invention reduces the need to characterize the nucleic acids biophysically and then to shorten them in rational and semi-rational approaches before they can be synthesized.
  • the chemical-synthetic representability of ribonucleic acids and deoxyribonucleic acids is particularly necessary for RNA or RNA hybrids for several reasons: On the one hand, aptamers require additional modifications to increase their stability in biological media.
  • RNA polymerases available in the state of the art tolerate 2'-modified pyrimidine nucleotide triphosphates, but not 2'-modified guanosine triphosphate, since there are problems with initiation in in vitro transcription. Furthermore, 2'-fluoro-substituted purine nucleotides and nucleosides are more difficult to synthesize synthetically than 2'-F-pyrimidine nucleotides and nucleosides. As a result, only RNA hybrids are obtained from in vitro selections with modified libraries that are more nuclease-resistant than canonical RNA libraries.
  • Spiegelmers must also be produced synthetically before their binding to the natural target can be demonstrated, for example, in biophysical assays, cell assays or animal experiments.
  • Spiegelmers consist of L-RNA or L-DNA, for the synthesis of which no enzymes have yet been identified.
  • the chemical-synthetic representation of ribonucleic acids and deoxyribonucleic acids is possible immediately after the identification of (mirror) target-binding sequences.
  • the methods according to the invention thus significantly reduce the time until the identification of biologically active aptamers / Spiegelmers. About that furthermore, when using this method there is no risk that a shortening that has failed may never show the biological activity of an aptamer / Spiegelmer.
  • aptamers / Spiegelmers available with good target binding as soon as larger amounts are needed (e.g. for animal experiments, clinical studies, therapeutic agents on the market and as adsorbent materials).
  • Each additional nucleotide not only increases the consumption of raw materials, but also reduces the yield in the synthesis and makes cleaning more difficult.
  • the molecules are already 30 nucleotides shorter than conventional aptamers / Spiegelmers.
  • single-stranded DNA oligonucleotides can recognize and bind to target structures (Bock et al., Selection of single stranded DNA molecules that bind and inhibit thrombin, Nature (1992), 355: 564-6; Green et al ., Inhibitory DNA ligands to platelet derived growth factor B-chain, Biochemistry (1196), 35: 14413-24; Leva et al., GnRH binding RNA and DNA Spiegeimers: a novel approach toward GnRH antagonism, Chem Biol (2002), 9: 351-9). They are therefore also called DNA aptamers.
  • DNA aptamers In contrast to those aptamers which contain ribonucleotides, DNA aptamers have the advantage that they are alkali-stable and autoclavable. This is of great advantage if, for example, the aptamer is to be used in chromatography columns as an affinity matrix which is to be regenerated by a “cleaning in place” process. Diluted alkalis are generally used here. For applications in the medical field, such as, for example, extracorporeal Adsorbers for blood washing, sterility is an indispensable condition. Sterilization is therefore necessary here, possibly also for reuse. The most common method is autoclaving.
  • RNA and Spiegelmers also apply to DNA the reaction sequences - even without the attachment and removal of the adapter molecules, in particular the respective first nucleic acid strands thereof, the first nucleic acid strands as primer binding sites in reverse transcription, in second-strand synthesis, in particular in PCR, and as a promoter for the RNA polyme serve lawn and are therefore also referred to as amplification sequences - an advantage over the technologies of the prior art, since in the end no gel cleaning is necessary for strand separation.
  • the method according to the invention for the amplification of nucleic acids is therefore outstandingly suitable for use in the context of a SELEX method, ie a method for the selection of nucleic acids binding to a target molecule.
  • the methods according to the invention can also be carried out in an automated manner. Automated implementation is understood here to mean that the method is carried out using an automatic machine, for example a robot. It can be provided that one or more of the steps of the method according to the invention are carried out by an automatic machine or a robot. The individual steps of the method according to the invention are designed in such a way that they can be carried out easily by a laboratory robot.
  • the nucleic acid to be amplified is a ribonucleic acid or a deoxyribonucleic acid.
  • the nucleic acid to be amplified can be present as single-stranded or as double-stranded nucleic acid.
  • the nucleic acid to be amplified can be a modified or an unmodified nucleic acid.
  • a modified nucleic acid is one which, for example, has a fluorine atom or an amino group at the 2 'position of the ribose or deoxyribose portion of the nucleotide.
  • Another form of modification is modification of the base portions of the nucleotides known to those skilled in the art, e.g. described by Kusser (W.
  • nucleic acids to be amplified can be used as nucleic acids to be amplified in the sense of the present invention.
  • the use of single-stranded nucleic acids is particularly preferred, in particular if the method according to the invention is used for amplification in the context of methods in which nucleic acid binding to target molecules is selected or generated, such as, for example, in the context of the SELEX method or the method for producing it of L-nucleic acids that bind to a target molecule, as described, for example, in international patent application WO 98/08856.
  • the method for amplification according to the invention can comprise a different number and sequence of steps.
  • the possibility that one or more of the steps does not necessarily have to be carried out in order to be able to successfully carry out the methods according to the invention was discussed herein in the description of the method with the designation “optional”. It is within the knowledge of the experts in this field to determine and carry out the specific sequence of steps in the light of the present invention depending on the type of nucleic acid to be amplified.
  • Tables 1 (use of RNA) and 2 (use of DNA) indicate the different embodiments of the amplification method according to the invention, the sequence of steps actually required depending on the type of nucleic acid to be amplified.
  • the method can basically be used to amplify any nucleic acid, e.g. also for the amplification of cDNA or genomic DNA.
  • the genomic DNA can be the total DNA to be amplified or the cutscene.
  • all that is ultimately required is knowledge of the sequences referred to herein as the first and second defined partial sequences in order to construct suitable adapter molecules.
  • the preferred first and second defined partial sequences disclosed herein it is particularly preferred if the said partial sequences are specifically added to the nucleic acid actually to be amplified and this thus represents the intermediate sequence.
  • kits can additionally or alternatively contain one or more of the following components: adapter molecules with one or more different first defined partial sequences and one or more different second defined partial sequences, ligase, reverse transcriptase, DNA polymerase, RNA polymerase, R&D polymerase , Restriction enzyme, standard solutions for lye and acid as well as buffer solution.
  • sticky-end ligation requires an oligonucleotide matrix, which in the present case is provided with the adapters according to the invention.
  • an oligonucleotide matrix which in the present case is provided with the adapters according to the invention.
  • an intermediate sequence which is called a randomized region in the case of carrying out a SELEX method, and of fixed partial sequences at the 5 'end and 3' end, which are also defined here as partial sequences is designated
  • the corresponding adapter molecules a stable complex is formed, so that the DNA ligase - with appropriate 5 'phosphorylation of the nucleic acid to be amplified and the first strand of the second and possibly third adapter molecule - the adapter molecules with the one to be amplified Can link nucleic acid.
  • a particularly preferred ligase is T4 DNA ligase, very particularly preferably a highly concentrated T4 DNA ligase, as disclosed in the examples herein.
  • the defined partial sequences of the nucleic acid to be amplified are located at the 5 'end and at the 3' end of the nucleic acids to be amplified and can be selected independently of one another.
  • the minimum length of the double-stranded region between the ligation site and the randomized region ie the length of the fixed partial sequences
  • the minimum length of the fixed partial sequence at the 5 'end is two nucleotides, especially if the nucleic acid to be amplified is a DNA.
  • the nucleic acid to be amplified is a ribonucleic acid
  • the length of the fixed partial sequence at the 5 'end should preferably be 4 nucleotides long. This finding is in agreement with the selected transcription initiation sequence, which ensures uniformly high transcription yields for all molecules.
  • T7 promoter which is at least partially contained in the adapter molecules and is used for the transcription of the nucleic acid to be amplified, is directly followed by the randomized region, molecules that start on GG purine would have an amplification advantage in the transcription (Milligan , JF and OC Uhlenbeck (1989). "Synthesis of small RNAs using T7 RNA polymerase.” Methods Enzymol 180: 51-62.), which is not in the sense of an in vitro selection.
  • This second, fixed partial sequence arranged at the 3 'end of the nucleic acid to be amplified is preferably selected such that the sequence complementary to this sequence represents the 3' end of the reverse primer in the polymerase chain reaction. This should not be too GC-rich in order to prevent a mispairing of the reverse primer in the randomized range.
  • LNA Locked Nucleic Acid
  • DNA-based adapter molecules are preferably used. Only in the case of RNA amplification is the first strand of the first adapter molecule preferably a ribo-oligonucleotide, since this can be ligated to the 5 'end of the RNA library with greater efficiency and is a suitable substrate for the reverse transcriptase, such as this 07B is also shown.
  • a surprising effect of the amplification method according to the invention is that the reaction sequence can be carried out without purification steps and only the transcription products, if they are used in a SELEX method, before their further use in a next selection round in the context of the SELEX method of purification be subjected.
  • This purification is preferably carried out by gel cleaning.
  • the basic sequence of the amplification reaction when using RNA libraries, consisting of 5 'ligation of adapter molecules, 3' ligation of adapter molecules, reverse transcription, optional second strand synthesis, optional PCR, shortening of the transcription starting product and transcription, is shown in FIG Inclusion in a selection round of the SELEX procedure shown. Sequence of reactions of RNA amplification
  • RNAs or RNA hybrids to be amplified can carry a hydroxyl group, a monophosphate or a triphosphate when they are provided at the 5 'end.
  • the corresponding reaction sequences are shown in Table 1. If other derivatizations are present at the 5 'end of RNAs or RNA hybrids, these are preferably removed before the start of the reaction sequence. A correspondingly modified starter nucleotide must then be used in the transcription.
  • the first adapter molecule which is also referred to herein as a forward adapter
  • the first strand of nucleic acid which is also referred to herein as a forward ligate
  • the use of a ribonucleic acid is particularly advantageous when the molecule to be amplified is a modified or unmodified RNA and a second strand synthesis is provided.
  • the advantage of this embodiment is that it saves one step.
  • only the forward ligate or the forward primer i.e. the first nucleic acid strand of the first adapter molecule, or the first defined partial sequence present at the 3 'end of the nucleic acid to be amplified, which is present at the 5' end of the nucleic acid to be amplified, or which comprises a sequence complementary thereto, hybridizes to the cDNA.
  • forward ligate i.e. the first nucleic acid strand of the first adapter molecule consists of DNA
  • the second strand of the first adapter molecule preferably consists of DNA.
  • the first strand, which is also referred to here as a reverse ligate, of the second adapter molecule, which is also referred to herein as a reverse adapter, preferably consists of DNA molecules, while the second strand, which is also referred to herein as a reverse matrix, consists at least on the Positions which are complementary to the second fixed (defined) part-sequence, it being sufficient if the overhang is made of DNA and the region 5 'excluding itself can be hydrolyzed alkaline.
  • cleavage site which is designed such that at least one first and one second cleavage product is formed after cleavage, the first cleavage product being one which is base-paired or complementary to the second defined partial sequence.
  • One way to bring about a correspondingly specific cleavage is to design as a ribonucleotide the nucleotide which, in the second nucleic acid strand of the second adapter molecule, directly 5 'adjoins the region of the second nucleic acid strand of the second adapter molecule which is base-paired with the second defined partial sequence of the nucleic acid to be amplified or is complementary.
  • the cleavage can be carried out using restriction enzymes.
  • the restriction enzyme recognition site and / or the interface is arranged in such a way that the second strand of nucleic acid is cleaved at the site described above.
  • the forward adapter corresponds to the first adapter molecule
  • forward ligate corresponds to the first nucleic acid strand of the first adapter molecule
  • forward matrix corresponds to the second nucleic acid strand of the first adapter molecule
  • reverse adapter 1 corresponds to the second adapter molecule
  • reverse ligate corresponds to the first nucleic acid strand of the second adapter molecule
  • Rev Primer Ribo U herein sometimes also referred to as reverse matrix because of its function in the ligation
  • the second nucleic acid strand of the second adapter molecule and the reverse adapter 2 the third adapter molecule
  • the 3 'ends of the adapter molecules are preferably blocked. This blocking of the 3' ends of the adapter molecules, which are not to be used later as primers in the reverse transcription reaction or polymerase chain reaction, suppresses undesirable PCR artifacts. Labor and time-consuming cleaning steps can thus be saved. Furthermore, the formation of concatemer is counteracted in the ligation.
  • the basic concept of suitable adapter molecules in terms of determining the respective nucleic acid sequences can be determined by bioinformatics methods, for example using the Oligos 8.2 program (Ruslan Kalendar, Institute of Biotechnology, University of Helsinki, Finland). However, preferred adapters have been determined by the methods and studies disclosed herein.
  • the sequence of the forward ligate and thus also of the forward primer is dominated by the T7 RNA polymerase promoter. Upstream of this there are additional bases which result in an increase in the melting temperature, so that PCRs can be carried out at high annealing temperatures. With highly structured aptamers, this can favor primer annealing over intramolecular secondary structure formation. This is to prevent the less structured, moderately binding nucleic acid species from being favored in the PCR.
  • the sequence of the entire pool with adapters is disclosed in the example section.
  • the reverse matrix which corresponds in sequence to the second strand of nucleic acid of the second adapter molecule, also serves as a primer for the subsequent reverse transcription in the same reaction vessel.
  • a pure DNA oligonucleotide DNA reverse matrix
  • a DNA reverse primer with a ribonucleotide is preferably used in the PCR at the site to be cleaved.
  • a second ribonucleotide is built into the primer, which is then used for further fragmentation it leads.
  • the resulting fragments should be at 37 ° C, i.e. Transcription conditions can no longer be hybridized on the forward strand.
  • the reverse transcription reaction is carried out under conditions known to those skilled in the art and described for example in Example 4 herein for the amplification of RNA.
  • the polymerase chain reaction (PCR) is a form of the second-strand synthesis reaction and takes place after the reverse transcription, preferably according to the invention without prior purification or concentration. PCR artifacts were prevented by using 3 '-blocked adapter molecules. In most cases, the PCR was carried out over 15 cycles as described in Example 5. Taq DNA polymerase from Röche was used as the polymerase.
  • Second strand synthesis reactions are the fill-in with the Klenow fragment or the Stoffel fragment, which are well known and described in the art, for example, in Sambrook, J., Fritsch, EF, Maniatis, T., Molecular Cloning, 2 nd Edition, Cold Spring Harbor Laboratory Press 1989, Cold Spring Harbor.
  • the major part of the reverse primer, which had been incorporated in the reverse strand, is removed by an alkaline cleavage of the phosphodiester bonds in the nucleic acid backbone.
  • the reaction takes place, for example, in 0.3 M NaOH within 10 min at 95 ° C.
  • the DNA is precipitated from the solution, for example neutralized with HC1 and buffered with 0.1 M NaOAc, for example with ethanol.
  • the cleavage site is located 3 'of the riboUs or ribols built into the reverse primer, as shown in FIG. 02D. This leaves a 5'-OH group on the remaining reverse strand.
  • the PCR product and the two products of the alkaline cleavage can be made visible, for example by analytical denaturing polyacrylamide gel electrophoresis (PAGE) with subsequent ethidium bromide staining.
  • PAGE polyacrylamide gel electrophoresis
  • the cleavage takes place quantitatively and does not produce any undesired by-products, as is shown in FIG. 10.
  • the randomly matching restriction enzyme recognition sequences are not cut in the randomized area.
  • Another advantage over digestion with restriction enzymes is the fact that restriction enzymes generally require double-stranded substrates, but that single strands increasingly occur in PCR in later cycles.
  • the reverse strand generated in the PCR or second strand synthesis is transcribed into RNA.
  • the reaction preferably runs for 2-16 hours at 37 ° C with T7 RNA polymerase which is commercially available, for example, from Stratagene. Other sources of such enzymes are known to those skilled in the art.
  • T7 RNA polymerase which is commercially available, for example, from Stratagene. Other sources of such enzymes are known to those skilled in the art.
  • the position of the T7 RNA polymerase promoter in the 5 'primer region which is provided in a preferred embodiment, does not transcribe it.
  • the resulting RNA library is prepared for later ligation in the 27 transcription.
  • RNA molecules carry a 5 'phosphate group, which is a prerequisite for the ligation.
  • GMP 5'-guanosine monophosphate
  • the present inventors have surprisingly found that an 8-fold excess of GMP over GTP (32 mM: 4 mM) does not detract from the transcriptional yield. This trick can save complex enzymatic steps, such as dephosphorylation and kinization, which never run quantitatively and require cleaning steps.
  • the in vitro transcription product obtained in this way can be purified, for example, by a denaturing polyacrylamide gel.
  • the band of the transcript can then be further treated and, if necessary, subjected to further purification steps, such as an ethanol precipitation.
  • the transcription product obtained in this way then represents a nucleic acid to be amplified, which can be further treated according to the invention or used accordingly in selection processes such as the SELEX process.
  • a transcript is generated from a double-stranded deoxyribonucleic acid at least in the promoter area. This can be:
  • a modified or partially modified ribonucleic acid a.) 2'-fluoro-RNA b.) 2 * -amino-RNA c.) Base-modified RNA, 2'-F-RNA, 2'-NH 2 -RNA.
  • RNA or RNA / DNA polymerases are used for the various desired transcription products: 1.) for sugar-modified RNA: "SP6 R & DNA polymerase” and “T7 R & DNA polymerase”
  • RNA polymerase 77 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase.
  • N + 1 transcripts As a result of the in vttr ⁇ transcription, in addition to the RNA molecules that are complementary to the template strand or template strand, 50% also result in transcripts extended by any nucleotide, the so-called N + 1 transcripts (FIG. 08). N + 2 transcripts have not been observed by the present inventors.
  • the method for amplifying double-stranded ribonucleic acids basically follows that for single-stranded RNA, as shown in Figs. 2A to E.
  • the first and the last six nucleobases of the dsRNA to be amplified are known so that the corresponding ligation matrices can be created.
  • the 5'-terminal nucleotide is a G, preferably the 5'-terminal nucleotides of both strands are GG, in order to ensure sufficient transcription yields.
  • RNA polymerases in particular the SP6, T3 and 77 RNA polymerase.
  • the transcription is carried out in each case only with a part, preferably half, of the PCR batch in the presence of, for example, SP6, T3 or 77 RNA polymerase.
  • the separately synthesized strands are combined again after hybridization and hybridized.
  • the reaction sequence of the amplification when using DNA differs from that when using RNA and is shown in detail in FIG. 39.
  • DNA can be provided with or without 5'-monophosphate.
  • the corresponding reaction sequence is shown in Table 2.
  • An advantage of the DNA amplification step in the context of the SELEX method, as shown in FIG. 39, is, for example, that compared to the prior art (Williams KP, Liu XH, Schumacher TN, Lin HY, Ausiello DA, Kim PS , Bartel DP: Bioactive and nuclease-resistant L-DNA ligand of vasopressin. Proc Natl Acad Sei USA (1997), 94: 11285-90.) PCR is not intended as the last amplification step. Therefore, no preparative gel purification of the DNA, ie gel purification and elution from the gel and precipitation, as described in the prior art, is necessary.
  • the two DNA strands that are written in the PCR are usually separated from one another by shortening one strand beforehand by cleaving a ribo-primer. Preparative gel cleaning cannot be automated well.
  • strand separation Another possibility of strand separation already described is based on the use of a biotinylated primer.
  • the PCR product is then immobilized on streptavidin particles and the strand which does not contain the biotinylated primer is eluted using sodium hydroxide solution, heating or the like.
  • the capacity of the particles is often quite limited, as biotinylated but not incorporated primers also occupy biotin binding sites.
  • the particles often suffer from the alkaline conditions or heat, so that streptavidin or other particle components are eluted together with the DNA strand. These then have to be separated again, for example by a phenol / chloroform extraction which cannot be automated easily.
  • Another advantage of the method according to the invention is that the DNA for the following selection round is not produced only by preparative PCR, but by PCR and in vitro transcription. Therefore fewer PCR cycles are required. Since the PCR exerts a strong selection pressure and often PCR artefacts, so-called amplification parasites, occur with a high number of cycles, this is of great benefit.
  • reaction cycle can be started at any point.
  • a preferred embodiment of the method according to the invention for the amplification of single-stranded deoxyribonucleic acids follows the procedure in FIGS. 40 A to E described scheme.
  • Each of the features described below can also be used individually or in combination with other molecules in other embodiments of the method.
  • the single-stranded DNA to be amplified or a mixture of ssDNA is provided, for example by oligonucleotide synthesis.
  • the sequence (s) of the ssDNA (s) may be unknown. Only the preferably 4 first and the preferably 4 last nucleobases must be known so that corresponding ligation matrices or first and second adapter molecules can be provided. It is preferred that the 3'-terminal nucleotide of each strand, also referred to synonymously herein as the nucleic acid strand, is a C in order to be sufficient in the in vitro transcription used for amplification
  • CC More preferred is pyrimidine CC, i.e. so
  • the first nucleotides of the transcription product are accordingly G,
  • ssDNA to be amplified or a mixture of many ssDNAs with the same defined partial sequences does not yet have 5'-terminal monophosphates, these must be added.
  • the reaction is preferably carried out in ligase buffer (Fermentas T4-DNA ligase buffer) with the addition of ATP and T4 polynucleotide kinase (Gibco Life Technologies).
  • two double-stranded DNA adapters which correspond to the adapter molecules described herein are added to the reaction mixture of the 5 'phosphorylation.
  • the adapters are distinguished by the fact that they carry an overhanging ligation matrix which is complementary to the respectively appropriate end of the ssDNA to be amplified.
  • the reverse ligate ie the first strand of the second adapter molecule, is 5'-phosphorylated in order to ligate to the 3 'end of the ssDNA enable (the 5 'phosphate can be added directly to the oligonucleotide synthesis).
  • the reverse matrix which is identical to the reverse primer in second-strand synthesis / PCR, is furthermore distinguished by the fact that it contains a strong promoter sequence for an RNA polymerase.
  • a strong promoter sequence for an RNA polymerase for example the T7, T3 or SP6 RNA polymerase promoter.
  • the promoter is arranged so that the transcription begins with the defined partial sequence at the 3 'end of each DNA strand to be amplified.
  • the forward matrix and reverse ligate i.e. the first nucleic acid strand of the first adapter molecule and the second nucleic acid strand of the second adapter molecule can be blocked at the respective 3 'end in order to prevent undesired ligation products and mispriming in the PCR of these constructs.
  • This is particularly useful when amplifying nucleic acid libraries, since in this case complementary sequences are often somewhere in the randomized range, i.e. for example in the cutscene.
  • the blocking can be such that the 3'-terminal nucleotide is a 3'-deoxynucleotide. In our case it is a 2'-3'-dideoxynucleotide.
  • the ligation is carried out.
  • the ligase e.g. T4 DNA ligase from Fermentas
  • Primer binding sites (forward ligate and reverse ligate) within 2-12 h at 16 ° C.
  • the DNA can then be amplified using PCR. No purification between the ligation and PCR reaction steps is necessary.
  • the missing primers, dNTPs, PCR buffers, and a thermostable DNA polymerase e.g. Taq DNA polymerase
  • the forward primer corresponds to the forward ligate or a part thereof, evör can optionally be extended at its 3 'end by the defined partial sequence at the 5' end of the ssDNA to be amplified and is also referred to herein as a PCR primer molecule.
  • the reverse primer corresponds to the reverse matrix or a part thereof.
  • a second strand synthesis e.g. with a Klenow or Stoffel fragment, a PCR also being referred to here as second-strand synthesis.
  • the PCR reaction or second strand synthesis is optional. Instead, the in vitro transcription can be continued directly.
  • RNA polymerase (Stratagene or Ambion).
  • T3 / T7 / SP6 RNA polymerase (Stratagene or Ambion).
  • One strand provides about 20-100 copies of RNA. Buffers and NTPs are also required. If there is enough PCR product, this step does not require any purification either. Up to 20% (v / v) PCR product / second strand synthesis can be used in the transcription. If the ligation approach is used directly, up to 40% (v / v) can be used.
  • RNAs can then be added with the addition of cDNA primers, which are also referred to as primer molecules in step (g) of the method according to the invention for the amplification of DNA and which contains at least one ribonucleotide directly 5 'from the fixed partial sequence at their 3' ends
  • Buffers, dNTPs and reverse transcriptase e.g. Superscript II (Gibco Life Technologies)
  • cDNA complementary DNA
  • the sequences of the cDNA obtained correspond to the DNA strands to be amplified with additional nucleotides at the respective 5 'end.
  • the regions of the cDNA primers which are 5 'before the fixed partial sequence are then cleaved off by adding lye and, if appropriate, heating (310 mM NaOH, 95 ° C., 10 min as exemplary reaction conditions).
  • the RNA strands are digested. If further ribonucleotides were incorporated into the cDNA primers, they will disintegrate into several parts, which then remain in the supernatant when ethanol is precipitated with linear polyacrylamide as carrier (described by Gaillard C, Strauss F: ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res (1990), 18: 378). Purification via a suitable molecular sieve (e.g. Microcon YM 10 from Amicon / Millipore) is also possible. If gel cleaning of the ssDNA is performed, the primer fragments would not co-migrate with the longer ssDNA strand.
  • the batch of alkaline cleavage is preferably first adjusted to the desired pH (for example pH 5.3) using HC1, NaOAc or NHOAc. Acidification must be prevented, otherwise the DNA tends to depurinate. The amplification cycle is now closed.
  • the part of the cDNA primer which is 5 'from the defined partial sequence of the original DNA and must be cleaved off can be cleaved off with a restriction enzyme.
  • an RNAse H-positive reverse transcriptase e.g.
  • At least the DNA strand in the heteroduplex of RNA and complementary DNA is cut by a restriction enzyme directly 5 'from the first fixed partial sequence.
  • the primer sequences required for the steps are already contained in the defined first and second partial sequences. This would eliminate the ligation step.
  • the reverse primer bears an RNA polymerase promoter at its 5 'end in addition to the primer sequence, preferably the T7 RNA polymerase promoter, so that the primer region would also be transcribed.
  • the cDNA primer would not have to have a ribo cleavage site.
  • AMV Avian Myeloblastosis Virus
  • the method according to the invention for the amplification of DNA offers, inter alia, the advantage that no preparative unification (ie gel purification + elution from the gel + precipitation) is necessary. This is normally indispensable to separate the two strands of DNA that are written in the PCR, one strand being shortened beforehand by cleaving a ribo-primer. Preparative gel cleaning cannot be automated well. Description of dsDNA amplification
  • the method according to the invention for the amplification of a double-stranded DNA is described below with the aid of a preferred embodiment, which results in individual features or feature conditions, which as such can also be implemented in other embodiments.
  • the double-stranded DNA comes e.g. from DNA libraries.
  • double-stranded cDNA can also be amplified as part of the method.
  • oligonucleotide matrix-assisted ligation works in both cases.
  • the method for the amplification of double-stranded deoxyribonucleic acids basically follows the scheme described for single-stranded DNA, as is shown in FIGS. 40 A to E is shown.
  • the dsDNA to be amplified or a mixture of dsDNA is provided, for example by oligonucleotide synthesis or restriction digestion of genomic DNA.
  • Sequence (s) of the dsDNA (s) may be unknown. Only the preferably 4 first and preferably 4 last nucleobases need to be known, so that corresponding ones
  • Ligation matrices can be created. It is recommended that the 3'-terminal nucleotide of each strand is a C in order to ensure sufficient yields in the in vitro transcription used for amplification. CC is more preferred, pyrimidine is more preferred.
  • the first nucleotides of the transcription product are accordingly G, GG, GGG or GGA.
  • ssDNA to be amplified does not yet carry a 5 'terminal monophosphate, this must be added.
  • the reaction takes place in ligase buffer (Fermentas T4-DNA ligase buffer) with the addition of ATP and T4 polynucleotide kinase (Gibco Life Technologies).
  • two double-stranded DNA adapter molecules are added to the reaction mixture of the 5 ′ phosphorylation for each DNA strand.
  • STAR-2 - (+) adapters or as STAR-2 - (-) - adapters, depending on whether they hybridize to the (+) or (-) strand of the dsDNA.
  • STAR-1 also denotes the methods according to the invention insofar as they amplify RNA
  • STAR-2 the methods according to the invention insofar as they amplify DNA.
  • the adapter molecules are distinguished by the fact that they carry an overhanging ligation matrix which is complementary to the respectively appropriate end of the ssDNA to be amplified. Both reverse ligates are 5 'phosphorylated to enable ligation to the 3' ends of the dsDNA (the 5 'phosphate can be added during oligonucleotide synthesis).
  • the reverse matrices which are identical to the reverse primers in second-strand synthesis and / or PCR, are furthermore distinguished by the fact that they contain strong promoter sequences for two different RNA polymerases.
  • the 77 RNA polymerase promoter in the STAR-2 - (+) - reverse matrix and the SP ⁇ 5-RNA polymerase promoter in the STAR-2 - (-) - reverse matrix are arranged so that the transcription begins with the defined partial sequence at the 3 'end of the DNA to be amplified. Both can also contain the same promoter region.
  • the forward matrix and the reverse ligate can be blocked at the respective 3 'end in order to prevent undesired ligation products and mispriming in the PCR of these constructs. This is particularly useful in the amplification of nucleic acid libraries, since in this case complementary sequences can often occur anywhere in the randomized range.
  • the blocking can be such that the 3'-terminal nucleotide is a 3'-deoxynucleotide. In our case it is a 2'-3'-dideoxynucleotide.
  • the primer binding sites are ligated, for example, within 2-12 h at 16 ° C.
  • the DNA can then be amplified using PCR. No purification between ligation and PCR is necessary.
  • the (+) forward primer corresponds to the (+) forward ligate or a part thereof and can be extended at its 3 'end by the defined partial sequence at the 5' end of the ssDNA to be amplified.
  • (-) - Forward primer and (-) - ligate have the same relationship to each other.
  • the (+) reverse primer corresponds to the (+) - reverse matrix or a part thereof. The same applies to the (-) reverse primer and the (-) - reverse matrix.
  • a second strand synthesis e.g. with Klenow fragment (available from Röche) or Stoffel fragment (e.g. from Applied Biosystems).
  • Klenow fragment available from Röche
  • Stoffel fragment e.g. from Applied Biosystems.
  • the PCR reaction or second strand synthesis is optional. Instead, the in vitro transcription can be continued directly.
  • the product of the ligation or the optional PCR or the optional second-strand synthesis is then divided into several, preferably two, parallel batches.
  • One of the strands is transcribed into RNA by in vitro transcription.
  • the approaches have to be separated in order to prevent the resulting complementary RNA strands from hybridizing with one another and thus making cDNA synthesis impossible.
  • Buffers, NTPs and T7-RNA polymerase or SP6-RNA polymerase are required or added, although T3-RNA polymerase can also be used if the promoter sequence is present in the reverse matrix.
  • One strand provides about 20-100 copies of RNA. If there is enough PCR product, this step does not require any purification either. It is possible to use up to 20% (v / v) PCR product / second strand synthesis in the transcription. If the ligation approach is used directly, up to 40% (v / v) can be used.
  • the division into two aliquots, or several, can also be dispensed with. Then transcripts of both strands are created in one reaction tube. These then hybridize with each other. Before the reverse transcription, the RNA must then be denatured in the presence of an excess of cDNA primer (s) as described as (are) the primer molecule in step (g) of the method for amplifying DNA, for example for 5 min at 95 ° C. This is particularly useful if both strands are transcribed by the same enzyme. (7)
  • RNA can now be added with the addition of a cDNA primer which contains at least one ribonucleotide directly 5 'from the fixed partial sequence at its 3' end, as well as buffers, dNTPs and reverse transcriptase, e.g. Superscript (Gibco Life Technologies), converted into complementary DNA (cDNA).
  • cDNA primer which contains at least one ribonucleotide directly 5 'from the fixed partial sequence at its 3' end, as well as buffers, dNTPs and reverse transcriptase, e.g. Superscript (Gibco Life Technologies), converted into complementary DNA (cDNA).
  • the sequences of the cDNA correspond to the DNA strands to be amplified with additional nucleotides at the 5 'ends.
  • the regions of the two cDNA primers which are 5 'from the fixed partial sequence are then cleaved off by adding lye and, if necessary, heating. Typical reaction conditions are 310 mM NaOH, 95 ° C, 10 min. At the same time, the RNA strand is digested. If further ribonucleotides have been incorporated into the cDNA primers, these will disintegrate into several parts, which then remain in the supernatant when ethanol is precipitated with linear polyacrylamide as carrier (described in Gaillard C, Strauss F: ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res (1990), 18: 378). If the DNA was gel purified, the primer fragments would not migrate with the longer DNA strand.
  • the preparation of the alkaline cleavage is first adjusted to the desired pH (e.g. pH 5.3) with HC1, NaOAc or NEUOAc before precipitation or further purification. Acidification must be prevented, otherwise the DNA tends to depurinate. The amplification cycle is now closed.
  • a particular embodiment of the method according to the invention is obtained if highly structured nucleic acids represent the nucleic acid to be amplified or if such highly structured nucleic acids are to be obtained as part of a selection process and, if appropriate, are to be used in further selection rounds. It was found that it is possible to use the ligation strategy disclosed herein to provide such highly structured nucleic acid species at both ends by ligation with corresponding adapter molecules.
  • the procedure described in Example 8 herein using a model substrate can generally be used for nucleic acids to be amplified in the sense of the present invention.
  • the present invention relates to antagonists of CGRP and amylin, antagonists of the CGRP receptor, CGRP-binding and amylin-binding nucleic acids, the use of such nucleic acids as antagonists of CGRP and / or the CGRP receptor system or of amylin, the use of a said nucleic acids for the manufacture of a medicament, a composition, in particular pharmaceutical composition, comprising said nucleic acid (s), a complex comprising CGRP or amylin and one of said nucleic acids, the use of said nucleic acids for the detection of CGRP or amylin and a Method for screening CGRP antagonists or amylin antagonists and a kit for the detection of CGRP or amylin.
  • the present invention is also based on the knowledge that it is possible to generate CGRP-binding nucleic acids, these binding in particular human CGRP and not binding to rat CGRP and likewise not to the structurally related amylin. It is within the scope of the invention that the nucleic acids binding to CGRP can enter into a certain, in general to be described as not very specific, binding with CGRP other species than human and / or amylin. Regardless, the nucleic acids disclosed herein can also be considered to bind to amylin and / or rat CGRP.
  • ⁇ CGRP The 37 amino acid neuropeptide ⁇ CGRP, calcitonin gene-related peptide, was identified in 1982 as an extremely potent vasodilator (Amara et al., 1982, Nature 298, 240-244). CGRP arises from alternative splicing of the CGRP gene. In addition to the ⁇ CGRP, there is a second CGRP, ßCGRP, which has a high sequence homology to the former, but is transcribed by another gene. Both peptides show similar biological effects as vasodilation, increased blood pressure, hypotension and tachycardia.
  • ⁇ CGRP and calcitonin result from alternative splicing of the calcitonin gene (Amara, GS et al., 1982, Nature 298, 240-244).
  • the structure for hCGRP was determined in part by 1H NMR.
  • the peptide consists of a defined N-terminal loop, which is formed from amino acids 2 to 7 by linking two cysteines via a disulfide bridge, to which about three are attached Connect turns of an ⁇ helix.
  • the C-terminal phenylalanine is in amidated form.
  • the amino acid sequence of human ⁇ CGRP and ßCGRP differ in three amino acids, the amino acid sequence of ⁇ CGRP and ßCGRP and the rat differ in one amino acid.
  • the different sequences are described in Hakala and Vihinen (Hakala J.M.L. and Vihinen M., 1994, Protein Engineering 7 (9), 1069-1075. (Accession numbers: human ⁇ CGRP P06881, human ßCGRP P10092, rat ⁇ CGRP P01256, rat ßCGRP P10093.
  • Amylin is a 37 amino acid peptide hormone. It is secreted in both diabetic and healthy individuals together with insulin from the ß cells of the islets of Langerhans. Amylin was only discovered in 1987 and is currently considered the third active hormone in the pancreas, which helps to control blood sugar levels. Amylin prevents the stomach from emptying too quickly and thus slows down glucose intake after meals. It was also shown that amylin inhibits both glucagon and somatostatin secretion. Like other members of the calcitionin-like peptides, amylin binds to a G protein-coupled receptor.
  • the amylin receptor is made up of various subunits that, in addition to the calcitonin receptor gene product (CTR), either the protein RAMP1 (receptor activity modifying protein 1) or the protein RAMP3 (receptor activity modifying protein 3) contains. Both isoforms form the amylin receptor; however, there are also reports that RAMP 2 can also be involved in the construction of the receptor (G. Christopoulos et al. Mol Pharmacol. 1999 Jul; 56 (l): 235-42 and N. Tilakaratne J Pharmacol Exp Ther 2000 Jul; 294 (l): 61-72)
  • CTR calcitonin receptor gene product
  • CGRP is seen as a key link in connection with migraines.
  • migraines with and migraines without aura The previous division into simple, classic and complicated migraines was abandoned.
  • Migraines are now understood as a neurovascular dysfunction that affects about 10% of the adult population. This disease is characterized by attacks of intense recurring headaches (Doods, H., 2001, Current opinion in investigational Drugs 2 (9), 1261-1268), nausea and exaggerated sensitivity to external stimuli such as light and noise.
  • the often half-sided headache (hemicrania) is pulsating and throbbing and usually occurs on one side. Often the side of the headache changes from one migraine attack to another or even during an attack. The attack is often accompanied by other symptoms. Those affected complain of anorexia, nausea and vomiting. They are particularly sensitive to light and noise and are extremely sensitive to odors and show nervous and visual disorders. The headache disappears between the migraine attacks. Before and after the attacks, mood and appetite, fluid balance and bowel function can change.
  • CGRP plasma concentrations in human subjects showed that the concentration of CGRP in the plasma is an important parameter in migraine diagnosis. Increased plasma concentrations were found in patients with acute migraine, in patients with cluster headache and in people after trigeminal stimulation (Edvinsson, L. et al., 1994, Cephalalgia 14, 320-327). In accordance with the animal studies just described, the elevated CGRP levels described in migraine sufferers can be reduced by sumatriptan or dihydroergotamine (Goadsby, P. et al, .1993, Ann. Neurol 33, 48-56).
  • the present invention was therefore also based on the object of providing an agent which is suitable for the treatment of migraine and the associated range of other diseases.
  • Another object underlying the present invention is to provide an antagonist for CGRP and the CGRP receptor system.
  • this latter object is achieved in a first aspect by an antagonist of CGRP, the antagonist being a nucleic acid and preferably binding the nucleic acid to CGRP.
  • the CGRP is ⁇ -CGRP.
  • the CGRP is ⁇ -CGRP.
  • this latter object is achieved according to the invention by an antagonist of amylin, the antagonist being a nucleic acid and preferably binding the nucleic acid to amylin.
  • this latter object is achieved according to the invention by an antagonist of the CGRP receptor, the antagonist being a nucleic acid and wherein the nucleic acid preferably binds to a ligand of the receptor and wherein the ligand is preferably CGRP.
  • the ligand is ⁇ -CGRP. In an alternative embodiment it is provided that the ligand is ⁇ -CGRP.
  • this latter object is achieved according to the invention by an antagonist of the amylin receptor, the antagonist being a nucleic acid and preferably the nucleic acid binding to a ligand of the receptor, and preferably the ligand being amylin.
  • the nucleic acid comprises at least one L nucleotide.
  • the antagonist is an L-nucleic acid.
  • this latter object is achieved according to the invention by a nucleic acid that binds to CGRP.
  • the CGRP is ⁇ -CGRP.
  • the CGRP is ⁇ -CGRP.
  • this latter object is achieved according to the invention by a nucleic acid which binds to amylin or an amyloid polypeptide.
  • nucleic acid with a sequence, the sequence being selected from the group comprising the sequences according to SEQ J_D No. 142 to SEQ ID No. 147.
  • nucleic acids referred to herein as nucleic acids according to the invention are in particular the nucleic acids of the various aspects of the invention, in particular aspects one to seven and preferably the nucleic acids according to SEQ ID No. 142 to 147.
  • the nucleic acid comprises at least one L nucleotide. In one embodiment of the various aspects of the invention it is provided that the nucleic acid is an L-nucleic acid.
  • the nucleic acid is selected from the group comprising DNA, RNA and combinations thereof.
  • the K u value of the nucleic acid is less than 0.5 ⁇ M, preferably less than 0.1 ⁇ M, preferably less than 0.05 ⁇ M and most preferably less than 0.01 ⁇ M is.
  • the Ko value of the nucleic acid is more than 100 nM, preferably more than 10 nM, preferably more than 1 nM and most preferably more than 0.01 nM.
  • the nucleic acids according to the invention regardless of possible further features and properties, have an IC50 value, the IC50 value preferably 0.5-30 nM, preferably 0.5-10 nM, more preferably 0.5-3 nM, and most preferably 1-2 nM.
  • the nucleic acid comprises a minimal binding motif.
  • the nucleic acid has a length, the length being selected from the group consisting of 15 to 150 nucleotides, 20 to 100 nucleotides, 20 to 80 nucleotides, 20 to 60 nucleotides, 20 to 50 nucleotides and 30 to 50 nucleotides, and the length is most preferably 25 to 45 nucleotides.
  • the nucleic acid has a two, three or more part structure.
  • this latter object is achieved according to the invention by using one of the nucleic acids according to the invention as an antagonist of CGRP and / or the CGRP receptor system.
  • this latter object is achieved according to the invention by using a nucleic acid according to the present invention as an antagonist of amylin and / or the amylin receptor system.
  • this latter object is achieved according to the invention by using one of the nucleic acids according to the invention for the manufacture of a medicament.
  • this latter object is achieved according to the invention by using an antagonist according to the invention for the manufacture of a medicament.
  • the medicament is for the treatment and / or prevention of a disease which is selected from the group consisting of migraines, cluster headaches, anorexia, nausea, vomiting, neurogenic inflammation, in particular neurogenic inflammation which is mediated with other neuropeptides, vasodilation, increased blood pressure, hypotension, tachicadia, diseases which are due to activation of trigeminal afferent sensory neurons and central “nociceptive” neurons, in particular higher pain centers, and include chronic inflammatory pain / or to treat pain, particularly chronic pain, acute pain, inflammatory pain, visceral pain and neuropathic pain.
  • a disease which is selected from the group consisting of migraines, cluster headaches, anorexia, nausea, vomiting, neurogenic inflammation, in particular neurogenic inflammation which is mediated with other neuropeptides, vasodilation, increased blood pressure, hypotension, tachicadia, diseases which are due to activation of trigeminal afferent sensory neurons and central “nociceptive” neurons, in particular higher pain centers, and
  • the nucleic acid or the antagonist binds to CGRP.
  • the medicament is for the treatment and / or prevention of a disease which is selected from the group consisting of high blood pressure, diabetes, gastric emptying disorders, diabetic gastroparesis, polydipsia, and degeneration and / or destruction and / or functional loss of Langerhans islet cells, in particular diabetes mellitus.
  • diabetes is one in which the amylin release is upregulated, as is the case in an early form of diabetes.
  • the diabetes is one in which arnylin plaques occur, as is the case in a late form of diabetes.
  • nucleic acid or the antagonist binds to amylin or an amyloid polypeptide.
  • composition comprising a nucleic acid according to the invention and preferably a pharmaceutically acceptable carrier.
  • composition comprising an antagonist according to the invention and preferably a pharmaceutically acceptable carrier.
  • this latter object is achieved according to the invention by a complex comprising CGRP and at least one nucleic acid according to the invention.
  • this latter object is achieved according to the invention by a complex comprising amylin and at least one nucleic acid according to the invention.
  • this latter object is achieved according to the invention by using a nucleic acid according to the invention for the detection of CGRP, preferably ⁇ -CGRP or ⁇ -CGRP and most preferably human ⁇ -CGRP or ⁇ -CGRP.
  • this latter object is achieved according to the invention by methods for screening CGRP antagonists comprising the following steps:
  • the CGRP is ⁇ -CGRP and / or ⁇ -CGRP, preferably human ⁇ -CGRP and / or ⁇ -CGRP.
  • this latter object is achieved according to the invention by methods for screening CGRP agonists comprising the following steps:
  • CGRP preferably immobilized CGRP
  • nucleic acid according to the invention preferably a labeled nucleic acid according to the invention
  • the determination is carried out by determining whether the nucleic acid is displaced by the candidate CGRP agonist.
  • this latter object is achieved according to the invention by a kit for the detection of CGRP, preferably ⁇ -CGRP or ⁇ -CGRP, comprising at least one nucleic acid according to the invention.
  • this latter object is achieved according to the invention by using a nucleic acid according to the present invention for the detection of amylin and / or amyloid polypeptides and / or amyloid plaques.
  • this latter object is achieved according to the invention by a method for screening amylin antagonists comprising the following steps:
  • this latter object is achieved according to the invention by a method for screening amylin agonists comprising the following steps:
  • nucleic acid according to the present invention preferably a labeled nucleic acid according to the present invention
  • the determination is carried out by determining whether the nucleic acid is displaced by the candidate amylin agonist.
  • this latter object is achieved according to the invention by a kit for the detection of amylin, comprising a nucleic acid according to the present invention.
  • ⁇ -CGRP antagonists such as the CGRP antagonists disclosed herein or the nucleic acids according to the invention can also be used effectively for the treatment of chronic inflammatory and visceral pain.
  • CGRP receptor basically refers to any CGRP receptor.
  • Preferred CGRP receptors are the so-called CGRP 1 receptors and CGRP2 receptors, to which the nucleic acids of the invention bind or for which the nucleic acids disclosed herein are antagonists.
  • the receptor CGRP1 is one for alpha-CGRP and CGRP2 one for beta-CGRP.
  • the nucleic acids or the antagonists of amylin according to the present invention are also those of the amylin receptor, which is also referred to herein as the amylin receptor system.
  • the amylin and / or the amylin receptor is preferably amylin or amylin receptor from humans or rats.
  • Amylin receptors are described as such in Christopoulos G, Perry KJ, Morfis M, Tilakaratne N, Gao Y, Fraser NJ, Main MJ, Foord SM, Sexton PM. Multiple amylin receptors arise from receptor activity-modifying protein interaction with the calcitonin receptor gene product.
  • nucleic acids or antagonists according to the invention can thus be used to inhibit the physiological effect of amylin, in particular the inhibition of the amyloid-dependent stimulation of the renin-angiotensin-aldosterone system or the effect on the central nervous system.
  • Such nucleic acids or antagonists can also be used to avoid amyloid plaques, for example in the pancreas. It is also within the scope of the present invention that the amyloid plaques are dissolved using the antagonists or nucleic acids according to the invention.
  • the group of nucleic acids according to the invention which bind to amlyin or which represent an amylin receptor antagonist includes, in particular, those nucleic acids with the SEQ ID NO. 142 to SEQ ID No. 147th
  • nucleic acids according to the present invention are also intended to include those nucleic acids which are essentially homologous to the sequences specifically disclosed herein.
  • the term essentially homologous is to be understood here so that the homology is at least 75%, preferably 85%, preferably 90% and most preferably more than 95, 96, 97, 98 or 99%.
  • nucleic acid according to the invention or nucleic acid according to the present invention is also intended to include those nucleic acids which comprise part of the nucleic acid sequences or nucleic acids disclosed herein, preferably to the extent that the said parts are involved in the binding of CGRP.
  • This type of nucleic acids can be derived from, for example, those disclosed herein Shortening or truncation.
  • the shortening is intended to refer to either one or both ends of the nucleic acids disclosed herein.
  • the shortening can also refer to a nucleotide sequence within the respective nucleic acid or nucleic acid sequence, ie it can refer to one or more nucleotides between the 5 'and the 3' terminal nucleotide.
  • truncation is intended to include the deletion of as little as a single nucleotide from the sequence of the nucleic acids disclosed herein. Shortening can also affect more than one area of the nucleic acid (s) according to the invention. Examples of nucleic acid truncation are taught in the example portion of the present description.
  • the nucleic acids according to the present invention can be either D-nucleic acids or L-nucleic acids.
  • the nucleic acids are preferably L-nucleic acids.
  • one or more parts of the nucleic acid are designed as D-nucleic acid (s), or at least one or more parts of the nucleic acid are designed as L-nucleic acid.
  • the term “part” of the nucleic acid is intended to denote as little as a nucleotide.
  • Such nucleic acids are generally referred to herein as D- or L-nucleic acid.
  • nucleic acids according to the invention are part of a longer nucleic acid, this longer nucleic acid comprising several parts, at least one part being a nucleic acid according to the present invention or part thereof.
  • the other part or parts of these longer nucleic acids can be either a D-nucleic acid or an L-nucleic acid. Any combination can be used in connection with the present invention.
  • This other part or these other parts of the longer nucleic acid can have a function that is different from binding and in particular binding to CGRP. One possible function is to interact with other molecules, e.g. B. for the purposes of immobilization, cross-linking, detection or amplification.
  • the L-nucleic acids are thus nucleic acids which consist of L-nucleotides, preferably consist entirely of L-nucleotides.
  • D-nucleic acids are thus those nucleic acids which consist of D-nucleotides, preferably consist entirely of D-nucleotides. Regardless of whether the nucleic acid according to the invention consists of D-nucleotides, L-nucleotides or a combination of both, the combination z. B. is a random combination or a defined sequence of sequences of nucleotides, which consists of at least one L-nucleotide or a D-nucleotide, the nucleic acid can consist of one or more deoxyribonucleotide (s), ribonucleotide (s) or combinations thereof ,
  • L-nucleic acid is associated with advantages for a number of reasons.
  • the L-nucleic acids are enantiomers of the naturally occurring nucleic acids.
  • the D-nucleic acids are not very stable in aqueous solutions and in particular in biological systems or biological samples due to the widespread use of nucleases.
  • Naturally occurring nucleases in particular nucleases from animal cells or tissues or cell or body fluids, are not able to degrade L-nucleic acids.
  • the biological half-life of L-nucleic acid in such systems, including the human and animal body is significantly prolonged.
  • L-nucleic acid Due to the lack of degradability of L-nucleic acids, no nuclease degradation products are produced and no side effects due to this are observed. This aspect distinguishes the L-nucleic acid from all those other compounds which are used in the therapy of diseases which are based on the presence of CGRP.
  • nucleic acids according to the invention regardless of whether they are present as D-nucleic acids, L-nucleic acids or D, L-nucleic acids, or whether they are present as DNA or RNA, are present as single-stranded or double-stranded nucleic acids could be.
  • the nucleic acids according to the invention are typically single-stranded nucleic acids which have a defined secondary structure as a result of the primary sequence and can also form tertiary structures.
  • the nucleic acids according to the invention can also be double-stranded in the sense that two mutually complementary strands are paired with one another as a result of hybridization. This gives the nucleic acid a stability which is advantageous if the nucleic acid is in the naturally occurring D-form instead of the L-form.
  • nucleic acids according to the invention can also be modified.
  • a particularly advantageous modification in connection with the present invention is the configuration of the nucleic acid (s) according to the invention in which at least one, o2 preferably several and most preferably all pyrimidine nucleotides forming the nucleic acid have a 2'-fluoro group at the 2 'position of the ribose part of the respective nucleotides.
  • Other modifications are, for example, those with PEG.
  • modification of the nucleic acids according to the invention can be those modifications which relate to a single nucleotide of the nucleic acid and are very well known in the prior art.
  • Corresponding examples are, among others, described in Kusser, W. (2000) J Biotechnol 74, 27-38; Aurup, H. et al, 1994, Nucleic Acids Res 22, 20-4; Cummins, L.L. et al, 1995, Nucleic Acids Res 23, 2019-24; Eaton, B.E. et ⁇ /., 1995, Chem Biol 2, 633-8; Green, L.S. et al, 1995, Chem Biol 2, 683-95; Kawasaki, A.M.
  • the nucleic acids according to the present invention can be in the form of one part, two, three or more parts. Of the multi-part form, the two-part or bipartite form is particularly preferred.
  • a multi-part form of a nucleic acid according to the invention is in particular one which consists of at least two nucleic acid strands. These two strands of nucleic acid form a functional unit, the functional unit being a ligand or binding molecule for a target molecule.
  • the at least two strands can be derived from one of the nucleic acids according to the invention or are obtained by either cleaving a nucleic acid according to the invention which binds to the target molecule in order to form two or more strands, or by synthesis of a nucleic acid which corresponds to a first part of the complete nucleic acid according to the invention and one further nucleic acid, which corresponds to a second part of the complete nucleic acid according to the invention. It is recognized that both cleavage and synthesis can be used to produce a multi-part nucleic acid consisting of more than the two strands exemplified above.
  • nucleic acid strands are typically different from two strands that are complementary to one another and hybridize with one another, although a certain degree of complementarity between the different nucleic acid (parts) can exist.
  • the nucleic acids according to the present invention typically have a high affinity for the target molecule.
  • One way of expressing the affinity, expressed as the binding constant, is the To determine nucleic acids according to the invention is the use of the so-called Biacore device, which is known to those skilled in the art and is described, for example, in Jonsson, U. et al, 1991, Biotechniques, 11 (5), 620.
  • the affinities were also measured by isothermal titration calorimetry (ITC), as in the examples and in Haq, I. & Ladburg, J., 2000, J. Mol. Recognit. 13 (4): 188.
  • the affinity values described here are to be understood as being measured by isothermal titration calorimetry, the temperature for the individual measurement being 25 ° C., unless otherwise stated.
  • Another method used is applied manually; it is a so-called bead assay.
  • constant concentrations of radioactively labeled nucleic acid are combined with different concentrations of biotinylated target molecules such as a target peptide.
  • the complexes formed are removed from the solution by adding streptavidin-loaded beads and the respective amount of radioactivity is determined.
  • the binding constants can be determined from the values obtained by corresponding plotting in graphs.
  • This bead assay is described in more detail, inter alia, in Example 3. If reference is made here to a nucleic acid according to the invention, all nucleic acids according to the invention or all nucleic acids disclosed herein are to be understood in principle, unless otherwise stated.
  • sequences according to the invention either come wholly or partly from the randomized part of the members of a nucleic acid library which are used as starting material for the selection process.
  • sequences according to the invention originate either completely or partially from the non-randomized part of the members of the nucleic acid library which serves as the starting material for the selection process.
  • a non-randomized part is, for example, the part that is used as the binding site for the amplification primer.
  • the nucleic acids according to the invention can be used for the production or manufacture of a medicament.
  • a medicament of this type contains at least one of the nucleic acids according to the invention, optionally together with further pharmaceutically active compounds, the nucleic acid (s) according to the invention preferably itself functioning as a pharmaceutically active compound.
  • one of the nucleic acids according to the invention could be combined with a further active ingredient which has the concentration of CGRP or amylin influenced.
  • the combination of the nucleic acids according to the invention with the further active ingredient appears to be particularly advantageous when both components attack CGRP or amylin and its release via different mechanisms of action.
  • a combination which is advantageous in this sense could consist, for example, of one of the nucleic acids according to the invention and a triptan, such as, for example, sumatriptan, since both substances address different mechanisms of action.
  • a combination preparation and its effect as follows: CGRP occurring in the plasma is captured by a nucleic acid according to the invention and, at the same time, the CGRP release is reduced / prevented by triptans.
  • medicaments comprise at least one pharmaceutically acceptable carrier.
  • Such carriers can e.g. B. water, buffer, starch, sugar, gelatin and the like. Such carriers are known to those skilled in the art.
  • the diseases for which the nucleic acids according to the invention and the CGRP antagonists identified using them can be used are essentially those which belong to the type of migraine, in particular migraines with and without aura, simple migraines, classic migraines and complicated migraines. These include headaches, in particular recurrent headaches, nausea, vomiting, excessive sensitivity to external stimuli such as light and noise, loss of appetite and disturbances in the fluid balance. The headache is particularly one that occurs in patients as pulsating and throbbing. Typically, this headache occurs unilaterally.
  • Further diseases which can be identified using the nucleic acids according to the invention and the candidate CGRP antagonists identified on the basis of their use are vasodilation, increased blood pressure, hypotension and tachycardia, in particular those forms of the aforementioned diseases which are associated with migraine, especially a migraine attack.
  • Other diseases that can be provided using the nucleic acid according to the invention or the candidate CGRP antagonists identified using the method according to the invention are those diseases that involve activation of trigeminal afferent sensory neurons, activation of central nociceptive neurons, and combinations of activation both neuron classes go hand in hand or are connected. In particular, the neurons are those that are assigned to higher pain centers.
  • Other diseases in the sense mentioned above In addition to acute pain these are diseases that are associated with chronic inflammatory pain. Such chronic inflammatory pain includes, in particular, that caused by CGRP along with other neuropeptides, such as substance P.
  • the nucleic acids according to the invention can furthermore be used as starting material for the design of pharmaceutical active substances (English drug design). There are basically two possible approaches to this. One approach is to screen libraries of compounds, such libraries of compounds preferably being libraries of low molecular weight compounds (low or small molecules). Such libraries are known to those skilled in the art. Alternatively, according to the present invention, the nucleic acids can be used for the rational design of active substances.
  • the rational design of active substances can take its starting point from any of the nucleic acids according to the present invention and comprises a structure, in particular a three-dimensional structure, which is similar to the structure of the nucleic acid (s) according to the invention or is identical to the part of the structure of the nucleic acid according to the invention (n), which mediates the binding to CGRP or amylin. In any case, such a structure still exhibits the same or at least similar binding behavior as the nucleic acid (s) according to the invention.
  • the preferably three-dimensional structure of those parts of the nucleic acids binding to CGRP or amylin are mimicked by chemical groups which are preferably different from nucleotides and nucleic acids.
  • This imitation also referred to as mimicry, enables a compound to be constructed which is different from the nucleic acid or nucleic acids used as starting materials for the rational design of the active ingredient.
  • Such a compound or active ingredient is preferably a small molecule or a peptide.
  • Such competitive assays can be structured as follows.
  • the nucleic acid according to the invention preferably a mirror bucket, which is designed as an L-nucleic acid binding target molecule, is coupled to a solid phase.
  • labeled neuropeptides are added to the test system.
  • a potential analogue would compete with the CGRP molecules that bind to the mirror bucket, which would be accompanied by a decrease in the signal obtained from the corresponding label.
  • Screening for agonists or antagonists can involve the use of a cell culture test system known to those of skill in the art. In principle the same approaches can be used using amylin.
  • the nucleic acids according to the invention can be used for target validation due to their characteristic binding behavior to CGRP or amylin.
  • the nucleic acids according to the invention can be used in an ex vivo organ model to study the function of CGRP or amylin. There are basically two ex vivo models in which CGRP agonists / antagonists can be tested. Antagonists for the CGRP2 receptor can be tested in the guinea pig atrium, and antagonists can be checked for their specificity for the CGRP receptor in the rat vas deferens model.
  • this relates to a complex comprising CGRP or amylin and at least one of the nucleic acids according to the invention.
  • the nucleic acids according to the invention are relatively rigid and adopt a precisely defined structure and, in so far, also the target molecule, i. H. the CGRP or amylin itself, a comparatively rigid structure, whereby both the CGRP and the amylin are generally understood to be comparatively flexible due to their length.
  • the kit according to the present invention can comprise at least one or more of the nucleic acids according to the invention.
  • the kit can include at least one or more positive or negative controls.
  • positive controls z. B. CGRP or amylin, against which the nucleic acid according to the invention has been selected, or to which it binds, preferably in liquid form.
  • negative control a peptide can be used, among other things, which behaves similarly to CGRP or amylin in terms of its biophysical properties, but which is not characterized by the nucleic acids according to the invention is recognized or a peptide with the same amino acid composition but of a different sequence from CGRP or amylin.
  • the kit can comprise one or more buffers.
  • the various components can be in the kit in dry or lyophilized form, or dissolved in a liquid.
  • the kit can comprise one or more containers, which in turn can contain one or more of the components of the kit.
  • the tubes contain reaction batches as required for a single experiment using one or more components of the kit.
  • the nucleic acids according to the invention can be used for the detection of the target molecule such as CGRP or amylin or the structures produced therefrom such as for example amyloid plaques or fibrils.
  • the nucleic acids can be labeled directly or indirectly.
  • the label is preferably selected from the group comprising radioactive labels, fluorescent labels or labels suitable for magnetic resonance spectroscopy, such as, for example, europium.
  • nucleic acids, antagonists according to the invention or the medicaments containing them can be administered both systemically and locally. If amylin antagonists or corresponding nucleic acids are used, local administration in the sense of an injection into the pancreas, for example, is basically conceivable. It is also within the scope of the present invention to include the antagonists or nucleic acids in biocompatible gels as depots, which are then released in the abdominal cavity or in the pancreas.
  • FIG. 1 shows a schematic representation of the embodiment of the method according to the invention using RNA, the reaction being integrated in a selection process and the RNA amplification method also being referred to herein as Star-1;
  • 2A to E show the flow diagram of the amplification according to the invention, for example after a first selection round of a SELEX method, starting with the RNA obtained from the previous selection round;
  • 5 shows the result of an experiment to determine the length of the fixed partial sequences at the 5 'end of the nucleic acid to be amplified, shown in the form of denaturing polyacrylamide gels;
  • 7A and 7B show the result of an experiment to optimize the ligation of synthetic RNA on the 5 'and 3' fixed partial sequences, shown as a denaturing polyacrylamide gel;
  • 10 shows the result of an alkaline digestion of the PCR product, as is produced in the context of the method according to the invention, represented as a denaturing polyacrylamide gel
  • 11 shows the result of testing various reverse primers for the alkaline digestion of a PCR product, shown as a denaturing polyacrylamide gel
  • 12A to 12C show a schematic representation of the ligation yield of various terminally structured nucleic acids to be amplified (templates);
  • 12 D shows a schematic representation of the ligation with ligation yields in the case of terminally structured nucleic acids to be amplified when structured at the 5 'end and successive ligation;
  • 12E shows a schematic representation of the ligation with ligation yields in the case of terminally structured nucleic acids to be amplified when structured at the 3 ′ end and successive ligation;
  • Figure 29 shows the dose response curve for rat CGRP
  • Fig. 39 is a schematic representation of the embodiment of the invention
  • Figure 42 shows the binding of grt2-087-Gl 1 to human alpha-CGRP
  • Figure 43 shows the ITC of Spiegelmer NOX-hCGRP-087-Gl 1 and human CGRP
  • FIGS. 2A to E show the flow diagram after a first selection round, for example as part of a SELEX process, starting with the RNA obtained from the previous selection round;
  • the purified RNA library consists of a randomized range of 30-40 nt, which is framed 5 'and 3' by fixed sequences, ie by the first defined partial sequence and the second defined partial sequence.
  • Compare step (1) Double-stranded adapters are added to the RNA in 20-fold excess. They each consist of a ligate that is to be linked to the RNA library and a matrix that can base pair with the overhanging end with the fixed sequences of the RNA library. In the following ligation, the RNA library is linked to the forward ligate and the reverse ligate. Forward matrix and reverse ligate are blocked at the 3 'end (3' deoxy nucleotides) in order not to interfere later in the PCR. Compare step (2) + (3).
  • the reverse matrix serves as a primer for cDNA synthesis.
  • the forward matrix does not interfere with the cDNA synthesis because it was designed so short that it drops at 37 ° C. Compare step (4).
  • the RT can be included in the PCR without purification. Additional primers (forward primer and reverse primer Ribo U) are added. In the PCR, many copies are made from the cDNA. Ideally, these consist of double-stranded DNA. Compare step (5).
  • the reverse primer which is no longer required, and the reverse strand of the PCR product are cleaved by alkaline hydrolysis.
  • the selective alkaline cleavage at this point is possible because the reverse primer (and also the identical reverse matrix that is still present from the ligation) carries a ribonucleotide at this point. Compare step (6).
  • the DNA is transcribed into RNA using an RNA polymerase.
  • the forward ligate and, accordingly, also the forward primer carry the RNA polymerase promoter sequence. Since the transcription only begins downstream of the promoter sequence, the ligate that has now become superfluous is not found in the transcript (RNA).
  • the reverse strand of the PCR product is read in the transcription. Therefore, the transcription terminates where the reverse primer has been cleaved.
  • the transcription is carried out in the presence of an excess of 5'-guanosine monophosphate. This can only be incorporated as a starter nucleotide. Therefore, the vast majority of transcripts begin with a guanosine monophosphate instead of a guanosine triphosphate.
  • the T7 RNA polymerase has the property of attaching one nucleotide more to a proportion of 10-50% of the transcripts than is encoded by the DNA template. This is referred to in technical jargon as 3 'micro-heterogeneities.
  • the RNAs provided are referred to below as n-tran script (correct length) and n + 1-tran script (one nucleotide attached). Compare step (7).
  • FIG. 9 shows the ligation of transcripts with 3 ′ microheterogeneities, such as can be observed in the case of a transcription, including a transcription in connection with the method according to the invention, as a result of the properties of the RNA polymerase.
  • RNAs with the correct length RNAs with an attached nucleotide (n + 1-transcripts).
  • ds reverse adapter 1 is identical to the reverse adapter described in FIG.
  • ds Reverse adapter 2 consists of a reverse ligate (n + 1 ligate) shortened by 1 nt at the 5 'end and carries an inosine ribonucleotide (n + in the reverse matrix at the position opposite the attached nucleotide 1 reverse matrix ribol).
  • n + 1 ligate reverse ligate
  • inosine ribonucleotide n + in the reverse matrix at the position opposite the attached nucleotide 1 reverse matrix ribol.
  • Both reverse matrices can serve as cDNA primers in RT. In PCR they can prime without damage.
  • the alkaline cleavage also runs as before, since the n + 1 Reverse Matrix Ribo I can also be cleaved alkaline. It is of no interest which nucleotide was inserted in the forward strand compared to the inosine in the PCR, since the forward strand is not read by the RNA polymerase.
  • 40 A to E show the flow diagram after a first selection round, for example in the context of a SELEX method, starting with the DNA obtained from the previous selection round.
  • the single-stranded DNA to be amplified or a mixture of ssDNA is provided.
  • the sequence (s) of the ssDNA (s) may be unknown. Only the first 4 and the last 4 nucleobases must be known so that appropriate ligation matrices can be created. Compare step (1). If the ssDNA to be amplified or a mixture of many ssDNAs with the same defined partial sequences does not yet carry 5 'terminal monophosphates, these must be added. Compare step (2).
  • two double-stranded DNA adapters are added to the approach. These are also known as STAR-2 - (+) - adapters or as (+) - forward adapters and ds (+) - reverse adapters.
  • the adapters are distinguished by the fact that they carry an overhanging ligation matrix which is complementary to the respectively appropriate end of the ssDNA to be amplified.
  • the reverse ligate is 5'-phosphorylated to enable ligation to the 3 'end of the dsDNA, and the 5' phosphate can be added directly to the oligonucleotide synthesis.
  • Forward matrix and reverse ligate can be blocked at the respective 3 'end in order to prevent undesired ligation products and mispriming in the PCR of these constructs. Compare step (3).
  • the primer binding sites are ligated. Compare step (4).
  • the DNA can now be amplified using PCR. No purification between ligation and PCR is necessary. The PCR reaction or second strand synthesis is optional. Instead, the in vitro transcription can be continued directly. Compare step (5).
  • RNAs can now be added to complementary DNA (cDNA) be transferred.
  • cDNA complementary DNA
  • the sequences of the cDNA correspond to the DNA strands to be amplified with additional nucleotides at the respective 5 'end. Compare step (7).
  • the area of the cDNA primer that lies 5 'before the fixed partial sequence is now split off by adding lye and, if necessary, heating. At the same time, the RNA strands are digested. The amplification cycle is now closed. Compare step (8).
  • Example 1 Selection of suitable ligases for ligation of DNA or RNA
  • thermostable DNA ligases from New England Biolabs, Röche and Fermentas as well as T4 RNA ligase from Fermentas were tested. Furthermore, the ligation efficiency was tested with thermostable DNA ligases with and without temperature cycling. The following thermostable ligases were included in the experiments: Tsc DNA ligase (Röche), Ampligase (Epicenter), Taq DNA ligase (New England Biolabs).
  • thermostable DNA ligases also showed no activity in the chosen system.
  • Ligation yields of 80% for RNA and 90% for DNA were achieved.
  • 2'F-RNA (2'F-cytidine and 2'F-uridine, 2'OH-adenosine and 2'OH-guanosine) it was also possible to carry out two exemplary selection rounds with the STAR-1 pool.
  • Example 2 Determination of suitable ligation conditions for the ligation of deoxy or ribonucleic acids
  • the excess adapter has the greatest influence on the ligation efficiency.
  • Another decisive factor is the concentration of the ligase, both in relation to the amount of template (see lane 1 and in comparison to 2 and 4) and in relation to volume (data not shown).
  • the columns "5 '-fixed region” and “3' -fixed region” indicate the sequence of the first defined partial sequence and the second defined partial sequence at the 5 'and 3' end of the randomized area, respectively in “5 '-> 3 'direction ".
  • the overhangs of the tested matrices are complementary to these sequence sequences, also referred to herein as reverse-complementary
  • the columns "5 '-fixed region” and “3' -fixed region” indicate the sequence of the "fixed region” at the 5 'and 3' end of the randomized area, in each case in "5 '-> 3' Direction".
  • the overhangs of the tested matrices are reverse complementary to these sequence sequences.
  • RT Reverse transcription
  • Superscript II Invitrogen
  • the reverse transcription takes place immediately after the ligation. With larger quantities of substance, several parallel approaches must be made. The size of the rT approach is determined by the differently concentrated ligation approaches (ligation up to 5 pmol / ligation from 5 pmol).
  • the temperature conditions are:
  • Example 5 Implementation of the polymerase chain reaction and alkaline cleavage when using RNA
  • the (-) strand of the PCR product is digested alkaline at the ribo interface.
  • the DNA is precipitated from the solution or the DNA is separated in another way, or the salts originating from the PCR or from the alkaline hydrolysis are taken into account in the preparation of the buffer for the subsequent transcription reaction.
  • the sample was precipitated after the alkaline hydrolysis.
  • Example 6 Generation of 5'-phosphorylated RNA and removal of the 5'-primer region by in vt ⁇ r ⁇ transcription when using RNA
  • the reverse strand generated in the PCR is transcribed into RNA.
  • the reaction runs 2-16 h at 37 ° C with 77 RNA polymerase from Stratagene. Due to the position of the 77 RNA polymerase promoter in the 5 'primer region (see sequences), this is not transcribed.
  • the resulting RNA library is prepared for later ligation in 77 transcription.
  • GMP guanosine 5'-monophosphate
  • the remaining DNA template can be digested within 20 min after the in vitro transcretion with 20 units DNAse I (Sigma). After adding loading buffer (7M urea, xylene cyanol, bromophenol blue), the mixture is denatured and a 10% solution is added denaturing polyacrylamide gel purified. The band of the transcript is cut out of the gel by UV shadowing and eluted using crush and soak. The resulting eluate is ethanol-precipitated and, after washing once with ice-cold 70% ethanol, the pellet is resuspended in water. The 5'-phosphorylated RNA library obtained in this way is used in the following selection round (FIGS. 01 + 02 E).
  • the transcription was carried out using the following reaction conditions:
  • pyrimidine nucleotides of which have a fluoro atom at the 2'-position of the ribose can also be used within the scope of the present invention
  • Example 7 Preparation of the initial RNA library used
  • a DNA library was created on a standard oligonucleotide synthesizer. This consists of the reverse strand, which contains the regions complementary to the forward primer binding region, which also contains the 77 promoter, to the randomized region and to the six defined nucleotides attached to the 3 'end of the pool. Reverse strand synthesis was chosen for two reasons:
  • RNA polymerase promoter region and the first 4 nucleotides to be transcribed were made double-stranded by simply adding the forward primer. Surprisingly, this was sufficient for transcription with high yields.
  • the overhanging part of the forward matrix cannot therefore hybridize to the corresponding nucleotides of the nucleic acids to be amplified, so that Such an effect still seems to play a role even with longer 3 'overhangs, but it decreases with increasing number of unpaired nucleotides (FIG. 12 C).
  • the concept for the construction of adapter molecules can be used with both DNA and RNA.
  • Example 11 Methoxy modifications of the second and third adapter molecules which are used when using RNA
  • the following adapter molecules can be used.
  • the ligation at the 3 'end of the transcripts can be carried out efficiently without the n + 1 adapter (STAR-1 reverse adapter 2, corresponding to adapter molecule 3).
  • Example 12 Use of nucleic acids without primer binding sites for the selection of D-CGRP binding nucleic acids for the validation of the RNA strategy
  • Rats ⁇ -D-CGRP was synthesized by Bachern, 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 streptavidin or biotin-neutravidin binding.
  • Neutravidin-agarose and Ultralink Plus nmobilized streptavidin gel were used as the matrix.
  • RNA and DNA nucleotides such as primers, start pools, ligation constructs and the like were all synthesized at NOXXON Pharma AG using standard phosphoramidite chemistry. The sequences can be found in the following overview.
  • STAR-1 oligonucleotides used in this application example in italics nucleotides of the ligation matrices that hybridize with the library o / underlined: 77 RNA polymerase promoter N OH : ribo-nucleotide CoM e : 2'-OMethyl-modified cytidine p: 5 'phosphate
  • RNA STAR-1 Forward Ligat
  • the RNA was first incubated at 37 ° C. (or room temperature) for 30 minutes without peptide with the selection matrix (either neutravidin agarose or with Ultralink Plus hnmobilized streptavidin gel). This so-called pre-selection served to separate potential matrix binders. After this incubation step, the unbound RNA was separated from the matrix by means of filtration through MobiTec columns, mixed with various concentrations of biotinylated CGRP and left at 37 ° C (or room temperature) for at least 2 hours - at low peptide concentrations, if necessary also overnight. The biotin-binding matrix was then added to the binding mixture.
  • the selection matrix either neutravidin agarose or with Ultralink Plus hnmobilized streptavidin gel
  • the matrix with the peptide-RNA complexes bound to it was separated from the solution by centrifugation after 30 min and washed with selection buffer.
  • the wash volume used in the first rounds was 5-10 times the amount of the matrix, in later selection rounds it was 25 times the solid phase volume.
  • RNA remaining on the matrix after washing was eluted twice from the matrix material with 400 ⁇ l of 8 M urea with 10 mM EDTA (both from Ambion) for 15 minutes at 65 ° C.
  • the eluted RNA was mixed with 400 ⁇ l phenol / (chloroform / isoamyl alcohol) mixture (l: (l: l / 24)) (Appliches), 5 min Centrifuged at 13,000 rpm at room temperature, the aqueous phase (supernatant) removed, the phenolic phase re-extracted once with 100 ⁇ l water, the aqueous phases combined and shaken with 500 ⁇ l chloroform-isoamyly alcohol mixture (24: 1) (Appischem), 5 min Centrifuged at room temperature at 13,000 rpm and the upper aqueous phase removed.
  • aqueous phase was then precipitated with 2.5 times the volume of 100% ethanol (Fluka), 0.3 M sodium acetate, pH 5.5 (Ambion) and 1 ⁇ l glycogen (Röche) for 30 min at -80 ° C and for 30 centrifuged at 14,000 rpm (4 ° C) for min. The pellet was washed once with ice cold 70% ethanol (Fluka).
  • the reverse transcription took place - directly after the ligation - with up to 15 pmol ligated RNA in a volume of up to 100 ⁇ l.
  • Several parallel approaches were made for larger quantities of substance. The size of the rT approach is determined by the differently concentrated ligation approaches (simultaneous ligation / parallel 2-step ligation up to or from 5 pmol).
  • the temperature conditions were:
  • the PCR was carried out with a maximum of 1 pmol template per 100 ⁇ l PCR. If a larger amount of RNA was eluted, several PCRs were set up in parallel.If you consider that before the start of a visible pool enrichment, only 1 pmol is often eluted and thus the complexity cannot be greater than the number of molecules in a pmol Without a doubt, only amplify a part (> 1 pmol) of the cDNA in later rounds (you do not have to introduce the entire cDNA into the PCR). This was how it started from round 5.
  • the reason for using a maximum of 1 pmol ligation template in the PCR is the ligation matrix for the n + 1 transcripts (3 'micro-heterogeneities in the transcription).
  • the ligation matrix used with the "universal" base (2'-deoxyinosine) is carried over into the PCR and can inevitably also prime there as a reverse primer. This creates a non-cleavable PCR product.
  • the more template that is brought into the PCR the more the more ligation adapters are carried over and the more non-cleavable PCR product is formed, therefore more than 1 pmol of template should never be used in the PCR.
  • inosine is used as the “universal base” for the ligation matrix
  • up to 5 pmol template can be used in the PCR.
  • the PCR products primed by the ligation matrix are also alkaline digestible.
  • ligation and reverse transcription there are two different PCR protocols for simultaneous ligation / parallel 2-step ligation up to and from 5 pmol.
  • the pH was then checked using indicator paper.
  • the pH " should be around pH 5-6.
  • the digested PCR product was quantified using an ssDNA standard (length 78 nt, 3.55 ⁇ mol NaCl (Ambion) was added to the sample to compensate for the salt effect) on a 10% denaturing PAGE to determine how much alkaline digested PCR product must be used in the following in vz ' tro transcription (target amount: 50 pmol PCR product per 100 ⁇ l batch). As a rule, 100 pmol PCR product was digested alkaline (for two 100 ⁇ l in vttro transcriptions).
  • Radioactive alpha- 32 P-GTP was incorporated during T7 transcription. 50 pmol of template (per 100 ⁇ l batch) were introduced into the in vitro transcription. As a special feature compared to other transcriptions, an 8-fold excess of guanosine 5'-monophosphate (8 x GMP) was added via GTP, so that most transcripts should have carried a 5'-monophosphate.
  • the lOx Txn buffer contains 800 mM Hepes / KOH (biochrom), 220 mM MgCl 2 (Ambion), 10 mM spermidine (Fluka), pH 7.5.
  • the in vitro transcription was carried out at 37 ° G for 8-16 hours.
  • the remaining DNA was then optionally digested with about 10 units of DNAse I (Sigma) (20 min at 37 ° C.).
  • the digestion was stopped by adding EDTA (Ambion) (final concentration 25 mM EDTA).
  • RNA (50mer) and DNA (78mer) size standard were additionally applied as size standards. The bands were made visible using "UV shadowing" (at 256 im).
  • the cut-out gel band was eluted using "Crush &Soak".
  • the gel pieces were taken up in 360 ⁇ l H 2 0 and 140 ⁇ l 5 M ammonium acetate (final concentration approx. 2 M).
  • the crush-and-soak elution was carried out twice 1.5 hours at 68 ° C.
  • Selection round 6 was repeated, with several parallel approaches being selected. These differed only in a falling peptide concentration. In the following rounds, the peptide concentration was reduced further and further. The peptide concentrations were selected as follows: the highest peptide concentration corresponded to the concentration at which selection could still be successfully carried out in the previous round (signal> 1%, at least 3-fold via empty selection). In addition, peptide concentrations reduced by a factor of 3.16 and a factor of 10 were used. An empty selection without any peptide served as a control. Rounds 11-14 were selected with a reduced RNA: peptide ratio of 5: 1, round 15 as a double round (2x selection with no amplification). The result is shown graphically in FIG. 15 and in a table in FIG. 16.
  • RNA 02MW matrix change selection
  • Rounds 13xx, 14xx, 15xx were selected as double rounds.
  • a double round is a test procedure which is characterized in that not only one but two successive selection steps take place between two amplification steps. The result is shown graphically in FIG. 19 and in a table in FIG. 20.
  • Cloning and sequencing provided 40 evaluable clones with 17 different sequences (3 of which carry an N, where N stands for a defined nucleotide but which could not be elucidated during the sequencing). 6 sequences (5 without a seq. With N) carry the typical motif 1 (CATACGGTGAAAGAAACGAT). 4 (3 without N) sequences have a partial motif (GAAAG) of motif 1 above and are almost identical. A clone shows the characteristic motif 1/1. 7 clones (6 of which are identical) carry the motif 1/1 with four successive variable positions (ggacGACATGTTC NN GAACATACGGTGAAAGAAACGATTGTCGgacagg).
  • the clones R02-15xx-All and R02-15xx-F12 emerged from this selection strand and were characterized in cell culture in example 13 and in micro-calorimeter in example 15.
  • Modified molecules have already been synthesized from two clones (shortened and terminal star stabilized). These are: STAR-R03-11-F10-45-001 and
  • Example 13 Inhibition of cAMP production by rat CGRP-binding Spiegelmers
  • Cells of the human neuroblastoma line SK-N-MC with the accession number ACC 203 are sown in a number of 4 x 10 4 per well of a 96 well microplate and at 37 ° C and 5% CO 2 in DMEM (1,000 mg / L glucose), which additionally contains 10% heat-inactivated fetal calf serum (FCS), 4 mM L-alanyl-L-glutamine (GLUTAMAX), 50 units / ml penicillin, 50 Contains ⁇ g / ml streptomycin, cultivated. 48 hours after sowing, the cells are 80-90% confluent and are used for the experiments.
  • DMEM 1,000 mg / L glucose
  • FCS heat-inactivated fetal calf serum
  • GLUTAMAX 4 mM L-alanyl-L-glutamine
  • 50 units / ml penicillin 50 Contains ⁇ g / ml streptomycin, cultivated. 48 hours after sowing, the cells are 80
  • the Spiegelmers are mixed with 1 nM human or rat CGRP (Bachern) in Hank's balanced salt solution (HBSS) + 1 mg / ml BSA for 15-60 min at 37 ° C in a 0.2 ml "low profile 96-tube "Plate incubated. Shortly before addition to the cells, 2 ⁇ l of a 50 mM IBMX solution are added. The cells are pretreated with 1 mM IBMX 20 min before the addition of the CGRP / Spiegelmer batches.
  • HBSS Hank's balanced salt solution
  • the medium is aspirated from the cells and the pre-incubated batches are added. After incubation for 30 min at 37 ° C., the cell supernatants are suctioned off and the cells are lysed with 50 ⁇ l / well lysis buffer for 30 min 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 is carried out as described by the manufacturer:
  • 10 ⁇ l / well of the lysate are 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 are added. This is followed by an incubation period of 1 h with shaking at room temperature. The solutions are then removed from the wells and washed six times with the washing buffer provided.
  • Example 14 Cell culture experiments - dose-response curve for CGRP
  • SK-N-MC cells were cultivated in 96-well plates as described in Example 13. 20 min before stimulation with rats alpha-CGRP, the medium was replaced by 100 ⁇ l Hank's balanced salt solution (HBSS) + 1 mg / ml BSA and 1 mM 3-isobutyl-1-methylxanthine (IBMX) was added. The stimulation with CGRP was carried out by adding 100 or 30 times concentrated stock solutions of L-Rat- ⁇ -CGRP (Bachern) dissolved in PBS. After 30 min at 37 ° C., the supernatant was suctioned off and the cells were lysed with 50 ⁇ l lysis buffer. The cAMP content of the extracts was determined as described in Example 13. The plotting of the amounts of cAMP formed against the CGRP concentration used for stimulation, as shown in FIG. 29, resulted in a half-maximal activation of approximately 1 nM.
  • Example 15 Calorimetric determination of the binding constants and the activity
  • the mirror bucket STAR-R03-F10 was measured at 25 ° C. The result is shown in FIG. 33.
  • the binding constant or dissociation constant K D is the reciprocal of the association constant K A (K in the enthalpy diagram) and was found to be 143 nM for the mirror bucket STAR-R02-15xx-All.
  • the activity was 44% (Fig. 30). This is worse than cell culture data, but of the same order of magnitude.
  • the mirror bucket R02-15xx-F12 showed a K D of 44 nM with an activity of 47% (FIG. 31).
  • a dissociation constant of 171 nM with an activity of 64% was measured with this mirror bucket against human ⁇ -CGRP (FIG. 32).
  • the DNA to be amplified does not carry a 5'-monophosphate, it must first be phosphorylated in order to be ligated at the 5 'end.
  • the now 5'-phosphorylated DNA can be transferred directly to the ligation batch.
  • (+) strand to be amplified In the case of ssDNA, only the (+) strand to be amplified is present; the corresponding (+) - forward primer and (+) - reverse primer are used.
  • (+) - forward primers and (+) - reverse primers as well as (-) - forward primers and (-) - reverse primers are used.
  • PCR is optional if enough ligated DNA is available as a template for the transcription reaction. Carrying out in vitro transcription
  • the PCR approach is divided into two transcription approaches.
  • One approach is chosen for the synthesis of the (+) strand (addition of the corresponding RNA polymerase) and the other for the synthesis of the (-) strand (addition of the second RNA polymerase).
  • RNAs can now be converted into complementary DNA (cDNA) by adding the respective cDNA primers which contain at least one ribonucleotide directly 5 'from the fixed partial sequence at their 3' ends.
  • the sequences of the cDNA correspond to the sequences of the DNA strands to be amplified with additional nucleotides at the respective 5 'end.
  • the temperature conditions are:
  • the areas of the cDNA primers which are 5 'before the fixed partial sequence are then cleaved off by adding lye and, if appropriate, heating (310 mM NaOH, 95 ° C., 10 min). At the same time, the RNA template strands are hydrolyzed.
  • restriction of the 5 'end is only necessary in the case of DNA selection without a primer and is an alternative to alkaline digestion. This does not apply to DNA selection with primers.
  • the restriction with Mnl I is equally suitable for ssDNA and dsDNA.
  • the two approaches of reverse transcription are first combined if the approaches for the (+) strand and the (-) strand were separated before the in vitro transcription. If a restriction digest is carried out, the forward primers / cDNA primers must be adapted accordingly.
  • Mnl I also cuts the following sequence on single-stranded DNA using the rT approach. 5 '... CCTC (N) 7'-3'
  • the temperature conditions are:
  • the restriction enzyme can be separated using an enzyme filter (eg Micropure-EZ, Millipore) if necessary.
  • an enzyme filter eg Micropure-EZ, Millipore
  • RNA STAR-1 Forward Ligat
  • Library and deoxyoligonucleotides for the amplification of dsRNA in italics nucleotides of the libraries and of the ligation matrices which hybridize with one another underlined: 77 RNA polymerase promoter or SP ⁇ 5 RNA polymerase promoter
  • NQH ribo-nucleotide pN: 5 'phosphate of a nucleotide 3'dN: 2'-3'-dideoxy nucleotide
  • nucleotides of the libraries and the ligation matrices that hybridize with one another underlined 77 RNA polymerase promoter or SPo RNA polymerase promoter
  • Ribo-nucleotide pN 5 'phosphate of a nucleotide
  • Library and deoxyoligonucleotides for the amplification of dsDNA in italics nucleotides of the libraries and of the ligation matrices which hybridize with one another underlined: 77 RNA polymerase promoter or ⁇ P5 RNA polymerase promoter
  • Ribo-nucleotide pN 5 'phosphate of a nucleotide
  • T7 Forward Matrix short 2 (2 nucleotide overhang, shortened at the 3 'end)
  • Sequence is self-complementary, but: 5 'end is free to 1 nt, at the 3' end there are 6 nt single-stranded pGGA CAA CGG TAG CCA CAG TCC TC TTC TGT GGC TAC CGT TGTC GAC AGG
  • Structure 2/6 sequence is self-complementary, but: 5 'end is 2 nt free, at the 3' end 6 nt are single-stranded pGGA CAA CGG TAG CCA CAG TCC TTC TTC TGT GGC TAC CGT TGT GAC AGG
  • Structure 4/8 sequence is self-complementary, but: 5 'end is free to 4 nt, at the 3' end 8 nt are single-stranded pGGA CAA CGG TAG CCA CAG TCC TTC TTC TGT GGC TAC CGT TAC GAC AGG
  • Structure 4/0 sequence is self-complementary, but: 5 'end is free to 4 nt, at the 3' end there are 0 nt single-stranded pGGA CCC TGT CGT AAC GGT ACA GTC CTT CTT CTG TAC CGT TAC GAC AGG
  • Structure 6/0 sequence is self-complementary, but: 5 'end is 6 nt free, at the 3' end there are 0 nt single-stranded pGGA CTA CCT GTC GTA ACG GAC AGT CCT TCT TCT GTC CGT TAC GAC AGG
  • Structure 8/0 sequence is self-complementary, but: 5 'end is free to 8 nt, at the 3' end there are 0 nt single-stranded: pGGA CTA GG CCT GTC GTA ACG AC AGT CCT TCT TCT GT CGT TAC GAC AGG 48 nt
  • Structure 12/0 sequence is self-complementary, but: 5 'end is free to 12 nt, at the 3' end there are 0 nt single-stranded: pGGA CTA GGCA AT CCT GTC GTA C AC AGT CCT TCT TCT GT GT AC GAC AGG 48 nt Structure 8/8 RNA: sequence is self-complementary, but: 5 'end is free to 8 nt, at the 3' end 8 nt are single-stranded pGGA CAC ACA ACG GUC CAC AGU CCU UCU UCU GUG GAC CGU UAC GAC AGG
  • Example 18 Use of nucleic acids without primer binding sites for the selection of human D-CGRP binding nucleic acids
  • Example 12 For the investigations described herein, the same materials as described in Example 12 were used, with the exception of the subsequent changes relating to the use of human CGRP and the amount of substance in the starting pool.
  • Human ⁇ -CGRP was synthesized by Bachern (Heidelberg).
  • the peptide used for the selection carries a biotin group at the carboxyl terminus in order to separate unbound nucleic acids by means of the biotin-streptavidin or biotin-neutravidin linkage enable.
  • Neutravidin-Agarose (NAAg) and Ultralink Plus Immobilized Streptavidin Gel (SAul, both from Pierce) were used as the matrix.
  • the amount of substance in the start pool “STAR-1 initial pool” was 10 nmol.
  • the amount of substance in the start pool corresponds to a complexity of 6 ⁇ 10 15 molecules.

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Abstract

L'invention concerne un procédé d'amplification d'un acide nucléique à brin simple ou double contenant une séquence partielle définie centrale, laquelle séquence est flanquée, à l'extrémité 3' et à l'extrémité 5', de deux séquences partielles définies. L'amplification de l'acide nucléique peut s'effectuer sans étape de nettoyage intermédiaire grâce à l'utilisation de deux molécules d'adaptation présentant des excédents qui sont au moins partiellement complémentaires aux séquences partielles définies de l'acide nucléique. La molécule d'adaptation est séparée après l'amplification de l'acide nucléique amplifié.
PCT/EP2003/004747 2002-05-06 2003-05-06 Procede d'amplification d'acides nucleiques Ceased WO2003093504A1 (fr)

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WO2009091719A1 (fr) * 2008-01-14 2009-07-23 Applera Corporation Compositions, methodes et kits de detection d'acide ribonucleique
EP2456888B1 (fr) * 2009-07-23 2016-04-20 Agilent Technologies, Inc. Sondes pour l analyse spécifique des acides nucléiques
EP1747285B1 (fr) * 2004-05-18 2016-10-12 Olink AB Procede d'amplification d'acides nucleiques specifiques en parallele
WO2020176362A1 (fr) * 2019-02-25 2020-09-03 Twist Bioscience Corporation Compositions et procédés de séquençage de nouvelle génération
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