DE19916417A1 - New amyloid-specific aptamer, useful for diagnosis and/or treatment of e.g. Alzheimer's disease, is stabilized against nucleases - Google Patents

Abstract

Amyloid-specific aptamer (I) that is stabilized against nucleic acid-degrading enzymes, and its alleles and/or derivatives, is new. An Independent claim is also included for a method of preparing and/or isolating (I).

Description

The invention relates to amyloid-specific substances.

Amylolde referred to insoluble in the invention che peptides, which in particular in extrazellulä deposit the area of a tissue and thus form plaques you can. Go with the amyloid plaque formation ver various diseases, for example in particular Alzheimer's, spongiform encephalopathies and type II diabetes mellitus. In the case of Alzheimer's Amyloid βA4 is of particular importance. The βA4 is a peptide of 39-43 amino acids with one molecule large weight of about 4 kDa. βA4 is a fragment of the like called amyloid precursor protein APP (amyloid Precursor protein), which is a transmembrane glycopro tein is. APP is powered by 3 different proteases, cleaved the so-called α-, β- and γ-secretases. in the Case of α-secretases, a soluble APP- Fragment with neuroprotective properties. In the event of the β- and γ-secretases, however, the βA4, wel Ches outside the cells to aggre soluble plaques yaws. Similar plaque deposits were also found in the Parkinson's disease, Huntington's or the prion proven diseases.

In any case, in the case of Alzheimer's disease is based Plaque formation is ultimately thought to be due to genetic defects, in which the APP metabolism is altered so that the βA4 (1-42) and the βA4 (1-40) amyloid are formed. The βA4 (1-40) preferably attaches to the already formed nuclei of the extremely insoluble βA4 (1-42)  and thus forms the actual plaques. The amyloidab Storage leads to a permanent Mikrogliaakti with the consequence of chronic inflammation, where be harmed by even healthy neurons. But also apoptosis is probably triggered by βA4. Apoptosis inevitably leads to shrinkage of the Ge brain, because neurons unlike other cells not regrow.

From a pharmaceutical and medical point of view, it is of particular interest to develop substances by means of which β-amyloids are detected in the body you can. For so far, Alzheimer's disease only diagnosed by talking and learning tests who why therapy could only begin when Dementia has already made itself felt and the irreversible brain degradation has progressed. By means of substances which bind to β-amyloids, könn He diagnosed the disease very early on be stigmatized and treated so that the degradation of the Brain prevented or at least significantly delayed becomes. Also for a therapy it is important to use Substan zen, which bind to β-amyloids.

Substances of the type mentioned are known from the document DE 197 25 619 A1. This is it relatively short peptides, which are the amyloid bil inhibit. A disadvantage of the extent known Substances that is such peptides, especially short Peptides, usually a relatively low affinity for Have target substances. In the case of short peptides the apparent dissociation constants are typi usually in the μmol range. Also known  monoclonal antibodies to βA4, which is a Quantifi allow amination of amyloid. Such monoclonal However, antibodies are only with great effort, including in-vivo process steps, produced and therefore relatively expensive.

In contrast, the invention is the technical Pro They are based on finding amyloid-specific substances those who are both highly affine and simple are adjustable.

To solve this technical problem, he teaches finding a against nucleic acid-cleaving enzymes stabi aptamer which is amyloid-specific, or Alleles and / or derivatives of such an aptamer. When Aptamers are called nucleic acids which bind to a Bind protein as a target molecule. It can be an aptamer have a DNA or an RNA structure. aptamers become stable against nucleic acid-cleaving enzymes siert by modification. The stabilization touched the affinity of the modified aptamers practically not, but prevents the rapid decomposition of Aptamers in an organism by RNases or DNases. An aptamer is considered stable in the context of the invention referred to when the half-life in biologi sera greater than one minute, preferably above one hour, most preferably greater than one day is. In contrast, for example, unmo modified RNA aptamers degraded in seconds. Alleles are conditional forms of nucleic acids which can be converted into each other by mutations. For this purpose are: Translolcation of affine Partial sequences, point mutation inside or outside  an affine partial sequence, deletion within or outside of an affine subsequence. Derivatives net chemical derivatives by separation, a leadership or exchange of atoms or groups of atoms one or more nucleotides from a nucleic acid to be obtained. It is essential that the derivatives and / or alleles against nucleic acid-cleaving enzymes stabilized and amyloid-specific. The own Each of the allele or derivative can be used separately or simultaneously.

An aptamer according to the invention can be produced by the following method steps: a) a randomized DNA pool is created, b) an RNA pool is optionally generated from the randomized DNA pool and placed in a selection cycle with the following steps: c) the amyloid or a partial sequence of the Amy loids binding RNA of the RNA pool or DNA of the DNA pool is selected, d) selected RNA or DNA is amplified, e) the RNA or DNA from step d) is the RNA pool or DNA pool in step c), wherein steps c) to e) are repeated until the proportion in step c) of selected RNA or DNA increases by less than 50%, preferably less than 10%. This method is known from other contexts known in vitro method for the selection of aptamers. For the process stages is to be carried out in detail as follows. For step a), a nucleic acid library of great complexity can be obtained with chemically-synthesized DNAs. For this purpose, using the automatic solid-plate synthesis, a randomized sequence is synthesized, which is flanked by 2 constant primer regions with, for example, 20 ± 5 nucleotides. The selections are performed, for example, with randomized areas between 30 and 80 nucleotides. The two flanking constant regions serve to complete and amplify single-stranded DNA to double-stranded DNA using the polymerase chain reaction (PCR). By using primers that extend beyond the hybridization region, it is possible to further extend the DNAs. As a result, the recognition region for the T7 RNA polymerase, the T7 promoter, can be introduced, which is required for the possible subsequent transcription of DNA into RNA (step b). The selection (step c) can be carried out by means of affinity chromatography. Here, the target molecule is immobilized on the column material, whereby non-binding aptamers are separated. It may be advisable to prepare and preconnect a precolumn with column material but immobilized target molecule. As a result, an enrichment of column material-affine aptamers is prevented ver. It is understood that the aptamers before Aufga be in the selection cycle if necessary renatu ration, so that they are present in a reproducible conformation. For the removal of selected aptamers from the affinity chromatography column, it is advisable to use the affinity elution in detail. It is eluted with dissolved in binding buffers target molecule. Another procedure is the reversible immobilization of the target molecule. Thus, the target molecule z. B. be coupled via a free sulfide group to form disulfide bridges to the Säulenma material. After the aptamers have bound, the disulfide bridges are reductively cleaved so that the target molecule is eluted together with the aptamer. Finally, elution with a de naturierungs buffer is possible. By denaturing the aptamers change their confirmation and are released from the target molecule. This is relatively unproblematic, since aptamers, unlike proteins, are easily renaturable. The amplification (step d) can be carried out as follows. The selected aptamer is optionally transcribed into DNA by reverse transcriptions. In any case, the amplification is carried out by means of the well-known PCR method, which will not be explained in more detail here. After the amplification, T7 transcription is again optionally carried out to obtain an RNA aptamer, which is then returned to the RNA pool. This process is repeated in cycles, whereby the affine aptamers are accumulated exponentially. The complexity of a startup pool is typically reduced by a factor of 10 ¹⁰ -10 ¹⁵ after 5 cycles. For example, libraries can be of 10 ¹⁵ and more different Apta mers want with little effort and great efficiency. With a complexity of the start pool of 10 ¹⁵ usually 6-15 selection rounds are needed to enrich the desired aptamers. For the identification of aptamers: in step d) DNAs of the last cycle are ligated into plasmids, cloned and sequenced. The determined sequences are compared with each other to identify commonalities in the primary and secondary structure and thus the binding motifs. It is also possible to attach an in vitro evolution to the process described above. In the context of the above method, the RNA aptamers are more important, as well as of course also DNA Apta mers can be produced in a corresponding manner. It then eliminates the T7 transcriptions and reverse transcription.

In the stabilization of aptamers against nucleic acid Re-cleaving enzymes can basically be between 2 An be distinguished, namely the subsequent Modification of aptamers and selection with be already modified RNA or DNA. The advantage of Subsequent modification is the implementation of the Selection under normal conditions. It should however, not all nucleotides, but usually modified only selected sites of RNA or DNA so that it does not cause any unwanted changes the binding properties comes. Can be bypassed this problem by the modifications already in the Introduces output pool. It is understood that the modes fied blocks from the during the selection ver used enzymes (DNA, RNA polymerases) tolerated who have to. Specifically, it is preferable if the 2'-hydroxyl group of the sugar group of one or more Pyrimidine nucleotides by a fluoride, amino or Methoxy group is substituted and / or if in termi nal phosphate groups through an oxygen atom Sulfur atom is substituted and / or if the apta Mer is a spiegelmer. In the latter case the selection of the aptamers takes place against an enantio meres target molecule. The mirror image Nu Kleine acid is then called spiegelmer and binds the actual target molecule, but can, as in the Natural non-occurring molecule, from the enzymes not recognized and therefore not dismantled. These However, this method can only be applied to target molecules  apply that are not chiral and where the en is available.

An inventive aptamer is opposite Peptides characterized by a very high affinity. typi Apparently, apparent dissociation constants are Range below 1 μmol, in particular in the range 1 nmol-100 nmol, while short peptides (<30 Amino acids) typically have values in the μmol range Gen. Favorable to monoclonal antibodies is that the aptamers according to the invention only in require vitro selection methods while the Gewin monoclonal antibodies, the use of Ver Searching animals or cell lines with the risk of uner Wanted variations. Unlike mono clonal antibodies can also aptamers chemically be synthesized, thereby producing a high level of reproducibility guaranteed. Also, the installation of Reporter molecules at precisely defined positions in the Framework of chemical synthesis easier. Also let Aptamers are much easier than monoclonal ones Antibodies to reversibly denature. After all is the development due to the in vitro mode of operation of aptamers against toxins or little immonogenic sub punching possible.

In a preferred embodiment of the invention the aptamer against Alzheimer's β-amyloid 4 (1-42) or Partial sequences thereof, in particular (1-40) specific. Preferably, it is an RNA aptamer. in the Case of a specific against Alzheimer β-amloid 4 Aptamers according to the invention it is preferred that the Aptamer has a loop structure. The loop structure  may in particular be 4 bases, preferably 4 uridines or 3 uridines and a cytidine exist or the have mentioned bases. As part of the loop structure it is preferred if the selected sequence is a Partial sequence GUUU or GUCU has. The loop structure can then be formed by this sequence opposite to a primer sequence AUUC, wherein the respective middle bases unpaired and respectively outer bases are paired. In detail, he can inventive aptamer have a sequence from the Group consisting of the sequences according to patent claim 6.

The invention also relates to the use of a he inventive aptamer or a mixture thereof Aptamers for the preparation of a diagnostic agent and / or treatment of Alzheimer's, spongiform ence Phalopathies, Type II Diabetes Mellitus, Parkinson's and Chorea huntington. In case of the diagnostic agent emp Is it appropriate to mark it in a manner known per se? Atoms and / or reporter molecules enter the aptamer to build. It is understood that these atoms or these Molecules selected in terms of type and position and arranged that the affinity of the aptamer to the target molecule not or only slightly influenced becomes. Use as a treatment agent may be included For example, a modification of aptamers to such note that β-amyloids are complexed and so Plaque formation is prevented or already formed Plaques are degraded. Then the aptamers cause even a complexing or are so modified that a complexation is effected, or serve as Mediators for complexing agents.  

In the following the invention is based on only Embodiments performing experiments closer explained.

1 methods 1.1 Chemical Nucleic Acid Synthesis 1.1.1 Synthesis of the Randomized DNA Pool

The RNA pool for selection was transcribed of a synthesized DNA pool. The DNA pool was chemically attached to the solid phase using an Applied Biosystems synthesis machines (PCR-Mate EP 391 and 394) manufactured. The synthesis is based on the phosphoramidite Method (Beaucage & Caruthers, 1981) and has been incorporated Use of the commercially available phosphoramidites with dC-coupled column material (CPG) of pore size 100 mm performed in 0.2 .mu.mol scale. The phosphoramidi te were dissolved in anhydrous acetonitrile (0.15 M). The Coupling time of the amidites was the standard synthesis tokoll extended from 15 s to 35 s and the splitting off of the Trityl protecting group at the beginning of each cycle by 20% shortened.

The DNA template strand consists of a 110mer, which consisting of two flanking primer regions of 20 bases each and known sequence as well as a randomized region composed of 70 bases in the middle. For the synthesis The randomized region used an amidite mixture det, in which the different Reaktovotät the amidites  already considered. The ratio of the 4 amidites DA: dG: dT was 3: 2, 5: 2, 5: 2.

The cleavage of the protecting groups after completion of the synthesis and the cleavage of the column material was carried out in 1.5 ml of conc. Ammonia (33%) for 24 h at 55 ° C. The ammonia was then removed in a vacuum centrifuge and the DNA was purified on a denaturing 8% polyacrylamide gel, identified by UV shadowing, eluted from the gel and desalted by ethanol precipitation (see 1.2).
Sequence: 5'-d [CCA AGC TTG CAT GCC TGC AG- (N) ₇₀ -GGT ACC GAG CTC GAA TTC CC] -3 '.

Determination of complexity

The complexity was determined by determining the molar amount of DNA by UV absorption (see 1.2.6). Since the probability that two identical DNA sequences were synthesized, is extremely low (4 ⁷⁰ = 1,4.10 ⁴² ver different possible sequences), it is assumed that each sequence is different. It follows that the amount of DNA is equal to the pool complexity.

Since not every sequence of the polymerase during the PCR is duplicated because one unfavorable for the enzyme Sequence is present or not all protecting groups separated were a PCR cycle with radioactively labeled dATP performed. After removal of the excess dATPs by ethanol precipitation or NAP column can be replaced by Ce renkovzählung be determined how much radioactive markier te DNA strands were synthesized (see 1.2.7). Because the DNA output concentration was known, and only maximum  One copy per molecule could be obtained the ratio of initial concentration of DNA to radioactive mar labeled DNA the amplified portion of the produced Pools. 60% of the synthesis pool could be from the Po lymerase be copied.

1.1.2 Synthesis of primers

The two primers for the amplification of the DNA pool by PCR were carried out analogously to the synthesis of the DNA library. The synthesis was carried out on a 1 μmol scale according to the standard protocol. The purification was carried out as described above. Column material with a pore size of 50 nm was used.
Primer A: 5'-d [TAA TAC GAC TCA CIA TAG GGA ATT CGA GCT CGG TAC C] -3 '
Primer B: 5'-d [CCA AGC TTG CAT GGC TGC AG] -3 '

1.2 Purification of Nucleic Acids 1.2.1 Gel electrophoresis

The most common method for the purification of nucleic acids is the gel electrophoresis. It is based on the hike the charged molecules in an electric field. you works here at a pH of about 8.5 at the Nucleic acids are present as polyanion and the positive ge load anode hiking. This one makes oneself of the Length-dependent mobility of the nucleic acid in a gelma trix. As a gel matrix are most commonly agarose  and polyacrylamide used. While with agarose gels separates the nucleic acids under native conditions, can be treated with polyacrylamide gels both under native as well by adding urea under denaturing condition be separated. Agarose gels are usually used at molecular sizes of 100-20000 base pairs, Polyacrylamide gels in molecular sizes up to 500 bases.

Polyacrylamide gel electrophoresis (PAGE)

In polyacrylamide gel electrophoresis (PAGE), the gel matrix by radical polymerization of acrylamide with N, N'-methylenebis (acrylamide) as cross-linker. The polymerization initiator is ammonium loxodisulfate (APS) and N, N, N ', N-tetramethylethylenediamine (TEMED) as Radical stabilizer. The pore sizes of the gel can be through the concentration of the monomers and the concentration of the Crosslinker can be adjusted.

Denaturing polyacrylamide gel electrophoresis

In the separation of single-stranded nucleic acids work If you are under denaturing conditions, the Ausbil secondary tertiary structures that are too different run behavior in the gel can be prevented. To this is added 7 M urea to the polymerization.

The separation is done on a 21.5 x 19 cm gel with a thickness of 1 mm at 70 mA in 1 x TBE buffer. Approaches:

The batch is made up to 50 ml with water and the Polymerization by adding 300 μl of APS (10%) and 30 μl TEMED started. After 45 minutes of polymerization, the gel becomes for at least 15 min at 600 V pre-run, so that can set a constant temperature.

The samples are ver with the same volume of formamide puts. The course of the electrophoresis can be described by the Be using a solution of the two dyes bromophenol blue and xylene cyanol can be observed in formamide.

Native polyacrylamide gel electrophoresis

The separation of double-stranded DNA is reported under native Be conditions. You go as described above ben, but eliminates the addition of urea. The Samples are taken with the same volume of native samples buffer offset. In contrast to the denaturing PAGE The gel should not get too warm on the native PAGE (Denaturation of the DNA). That's why at a tension separated from 300-400V.

sequencing gels

For the separation of the products of the radioactive Sequen 6% denaturing gels were used. These Gels measuring 42 × 33 cm are only 0.4 mm thick. So that after completion of the electrophoresis the gel on one Glass plate sticks, and you remove the other plate  Can, is a special treatment of the two glass plates necessary.

This is on the large, adhesive glass plate a solution from 200 μl of silane A-174 (methacryloxypropyltrimethoxysilane, Pharmacia) and 1 ml of glacial acetic acid in 20 ml of ethanol with a lint-free paper towel evenly distributed. This process dur will be repeated 20 minutes later. After 60 minutes, the Plate thoroughly rubbed with ethanol and for at least Dried for 60 min. The small, non-adhesive glass plate is treated with a 2% solution of dichlorodimetylsilane in 1,1,1-trichloroethane (Repel silane, Pharmacia) as long as one rubbed until the solution in the form of minute droplets the glass plate rolls off. This procedure was after 30 min repeated, and after another 60 min was the plate rubbed thoroughly with water and for at least 60 min let it dry.

Gel solution: 42 g urea, 12.5 ml acrylamide / bisacrylamide (40% / 2%), 20 ml 5x TBE, add 100 ml with H ₂ O.

Before the addition of APS and TEMED, the polymerization batch is again filtered through a pleated filter. The gel is allowed to polymerize for at least 45 minutes and preceded by closing at 55 W for 30 min. Using a so-called "shark comb" 48 ³⁵ S-labeled samples (= 12 sequencing) can be applied to the gel.

After completion of electrophoresis, the small glass plate carefully lifted off. The gel on the big glass plate is used to elute the urea for 6 min with dist. What rinsed and then in a drying oven at 60 ° C.  dried for 30 min. Subsequently, for the autora diography an x-ray film. One exposes the film for at least 12 h on the gel.

When using an ABI sequencer the Vorbe act of the glass plates. Here are the glass plates before used and checked for use in the sequencer, that no basic absorption due to impurities is available. It is allowed to run for 1 h at 55 W. Because the Samples are marked with fluorescent dyes, is omitted the after-treatment of the gel. After completed electric phensically, the gangs are assigned to the samples on the computer nete and the analysis software started.

Gels for cleavage analysis

For the secondary structure analysis of aptamers by enzyma Table RNA cleavage was 25% denaturing butt lyacrylamide gels used (42 × 33 cm, 0.4 mm thickness) Here were the two monomers acrylamide and bisacrylamide used in the ratio 50: 2. A pretreatment of Glass plates is due to the higher stability of the gel unnecessary. It is allowed to run for 60 min at 1500 V and then separates the samples at 55W. After be Ended electrophoresis, a glass plate is lifted and transferred the gel to an old x-ray film and into Cling wrap wrapped. Subsequently, for the Autoradiography an X-ray film to be launched.

Gel solution: 42 g urea, 50 ml acrylamide / bisacrylamide (50% / 2%), 20 ml 5x TBE.  

agarose gel electrophoresis

Agarose gels are used to separate large DNA molecules used under native conditions. In this work wur they are used to analyze the products after the PCR and the Analysis of plasmid DNA from minipreparations used. The pore size can be altered by altering the agarose concentrations varies and depends on the use ten agarose (chain length).

2% (w / v) agarose is poured into an Erlenmeyer flask in 1 × TBE buffer dissolved in the heat (microwave). One lets the Cool the mixture down to about 50 ° C and then apply for the detek DNA 5 μl ethidium bromide (10 mg / ml) to 100 ml Approach to and pour the gel. After cooling the gel Can the samples with the same volume native Sample buffer were added, applied and at 100-110 V electrophoresed. There were flat gel chambers used by BioRad (Mini and Wide Mini Sub DNA Cells).

Gel elution of nucleic acids

The detected by UV shadowing or autoradiography cut Gelbande is in a Eppeldorf vessel over with 400 μl of water and 40 μl of sodium acetate (3 M, pH 5.5). Shake at 4 ° C for at least 12 h or at 70 ° C for 1 h and gives the eluate in a new Eppeldorfgefäß. By ethanol precipitation or gel filtration The solution can be freed from urea and salts.

1.3.2 Detection methods for nucleic acids in gels ethidium bromide staining

The use of Ethidiumbromid is the fastest and most common method for the analytical detection of Nucleic acids, ethidium bromide is a heterocyclic, cationic fluorescent dye incorporated into the nucleic acid is intercalated. The work with the dye should be done carefully as ethidium bromide is strongly mutagenic. Polyacrylamide gels are in for 20 min of an ethidium bromide solution (0.5 μg / ml) in 1 × TBE colored. In agarose gels, ethidium bromide has already become added to the gel solution. On a transilluminator can be at 306 nm transmitted light, the nucleic acids as recognize glowing bands.

UV shadowing

In the preparative purification of nucleic acids, one would like to avoid interactions with dyes. For the detection one makes use of the absorption of the nucleic acids at 260 nm. For this purpose, the gel is placed on a holding foil wrapped in a thin-layer chromatography plate, which was treated with a fluorescent dye (60 F ₂₅₄ ). By irradiating UV light (254 nm), the nucleic acids can be recognized as dark shadows and cut out. In order to avoid damage to the nucleic acids by the UV light, this step should be carried out as quickly as possible. The disadvantage of this method is its low sensitivity. (0.5-1 A ₂₆₀ ), which is why it is mainly used in preparative purification.

autoradiography

Radiolabeled nucleic acids ( ³² P or ³⁵ S) can be detected in gels, depending on their specific activity, in the smallest amounts by blackening of an applied X-ray film (<fmol).

Gels with ³² P-labeled oligonucleotides are wrapped in a cling film after a glass plate has been removed. If one wants to cut out the nucleic acids from the gel and elute, the gel is marked with three phosphorescent points. In an X-ray film cassette with amplifier film (Kodak) to put an X-ray film (Fuji) on the gel. Depending on the amount and specific activity of the nucleic acid exposed to the film for 5 min to 24 h. For longer exposure times, the cassette is frozen at -80 ° C to prevent diffusion of the oligonucleotides

Sequence gels containing ³⁵ S-labeled nucleic acids are dried on the large glass plate after electrophoresis. Subsequently, an X-ray film is exposed on the dry gel at room temperature for 1 to 2 days.

The exposed films are developed (1-2 min Genentwickler G150, Agfa-Gefaert N.V.), fixed (3 min, X-ray fixator G350, Agfa-Gefaert N.V.), watered (5 min) and dried.

If you want to elute the nucleic acids, then cut the black bands on the x-ray film, puts the film like that on the gel that the 3 markers with the black dots on the film, the bands cut out and eluted as described above.

1.2.3 Gel filtration

With the help of gel filtration can nucleic acid solutions of salts and other low molecular weight compounds be separated. You make the difference Permeation behavior of the molecules in the pores of the gel uses beds (gel permeation chromatography). The big ones In contrast to the small ones, molecules can not be found in the Pores of the column material penetrate and therefore eluted immediately. Prefabricated columns were used Company Pharmacia with Sephadex G-25 (NAP-5, NAP-10, NAP-25) or G-50 (NICK). The implementation was carried out according to information of the manufacturer.

1.2.4 ethanol staining

Nucleic acids can be prepared from aqueous solution by ethanol Presence of sodium acetate at pH 5.5 precipitated who while salts and other impurities in solution stay. For this purpose, the nucleic acid solution with 1/10 of the Volume of a 3 M sodium acetate solution (pH 5.5) and the 2.5 times volume of ethanol and for 10 min at -20 ° C. posed. Then it is centrifuged for 30 min 10000-14000 rpm / 4 ° C, discards the supernatant Ethanollö solution, the pellet washes with 400 μl of 70% ethanol and zen centrifuged for a further 5 min. One takes off the supernatant and the pellet dries at room temperature. At very small Nucleic acid levels may be precipitated by the addition be improved by glycogen. The after the selection Round eluted RNA was always at -20 ° C for 1 h precipitated in the presence of 40 μg glycogen.  

1.2.5 Phenol and chloroform extraction

For the removal of proteins from aqueous solutions z. B. after enzyme reactions, for the separation of the Peptide-RNA complex after selection or at Isolation of plasmids from cells, are added to the Solution with the same volume of Tris-saturated Phenols (pH 8.0) and vortex vigorously. One separates the both phases by centrifugation at 3000 rpm and take Carefully remove the aqueous upper phase. The phenolic Phase can be repeated with half the volume of water be added, shaken and centrifuged. The combined aqueous phases are washed twice with the double volume of chloroform extracted to residual Remove phenol from the aqueous solution. Already small Traces of phenol can subsequent enzyme reactions considerably restrict or even completely inhibit.

1.2.6 Measurement of radioactivity

The radioactive labeling of nucleic acids is a practical method to detect even the smallest amounts. Quantification is at ³² P-labeling by Cerenkov counting. For this purpose, the Eppendorf tube with the sample is measured in a scintillation vial in the scintillation counter (LS 6000 SC, Beckman) with program 1 ( ³² P, Cerenkov count) for one minute. The results are obtained in cpm (counts per minute).

1.2.7 Concentration determination of nucleic acid solutions

Due to the absorption maximum of the bases of the nucleic acids in the UV range at 260 nm, the concentration of DNA and RNA solutions can be determined with a photometer. Approximately the following equations apply:

ssDNA: number of μmole = total OD ₂₆₀ / (10 number of bases)
dsDNA: 1 OD = 50 μg
pmol = μg.1000000 / (2.number of bases.325)
RNA: 1 OD = 40 μg
pmol = μg.1000000 / (number of bases.337)

1.3 Molecular biological methods 1.3.1 Polymerase Chain Reaction (PCR)

The polymerase chain reaction (Polymerase Chain Reaction, PCR) is a method for exponential amplification of DNA. There are two in addition to the DNA to be amplified 15-30 base oligonucleotides (primers) with defined Sequence needed to reach the 3 'ends of the DNA template or its complementary strand are complementary. After one Denaturation step hybridize these primers in one Primer annealing step with the to be amplified DNA template. The annealing temperature becomes so high chosen that no nonspecific hybridization occurs. With a thermostable DNA polymerase takes place in Extension step, the extension of the primer along the Templates in 5'-3 'direction. Ideally, you get that way from each DNA duplex, two new DNA strands. By repeating these three steps several times (up to 30 Cycles) gives an exponential increase in DNA.

As a DNA template can be both double and single stranded DNA can be used. By using Overhanging primers may be an extension of the DNA  be achieved. This was for the introduction of the T7 promoter into the chemically synthesized DNA exploited. All reactions were with the Pfu polymerase carried out by the Taq polymerase through their Proof reading activity and its exonuclease activity which ensures a lower error rate. Following the PCR, a phenol-chloroform Ex traction and an ethanol precipitation carried out to the Pfu Polymerase (with exonuclease activity) to remove.

Standardized approach temperature conditions 10 μl of 10 × Pfu buffer 94 ° C / 4 min Init.denat. AL = L <4.8 μl MgCl ₂ (100mM) 0.5 μl BSA (2 μg / μl) 94 ° C / 2 min denaturation 4 μl of primer A (100 μM) 55 ° C / 2 min annealing 4 μl of primer B (100 μM) 75 ° C / 6 min extension 4 μl dNTP mix (10 mM per dNTP) 12-20 cycles AL = L <1 μg DNA template AL = L CB = 3 <1 μl Pfu polymerase (2.5 U / μl) @ AL = L CB = 3 <ad 100 μl with H 2 O fill up

Preparative PCR

For the amplification of the synthesized DNA library The reaction was not in PCR tubes, but in 10 ml glass jars (6 × 5 ml), which in three Water baths were swiveled corresponding temperature. Due to the large volume, the reaction times were extended and the temperatures increased by 2 ° C and the Number of cycles limited to 10. Furthermore, the duration of the doubled last extension phase.  

preparative approach temperature conditions 300 μl of 10 × Pfu buffer 96 ° C / 4 min Init.denat. AL = L <1440 μl MgCl ₂ (100mM) 15 μl BSA (20 μg / μl) 96 ° C / 5 min denaturation 1200 μl primer A (120 nmol) 56 ° C / 5 min annealing 1200 μl of primer B (120 nmol) 77 ° C / 12 min extension 480 μl dNTP mix (100 mM per dNTP) 10 cycles 30 μl of Pfu polymerase 75 ° C / 12 min additional text. AL = L <ad 30 ml with H ₂ O fill up.

Following the PCR, a phenol-chloroform Extraction performed. To determine the DNA concentration a sample (30 μl) was applied over an 8% denaturing polyacrylamide gel purified, eluted and desalted. By comparing the absorption at 260 nm before and after purification, the concentration of the pool be calculated.

PCR after reverse transcription

The reverse transcription approach was extended to four 100 Distributed μl lances. The times of the three steps were something extended, too, the duplication bad to ensure amplifiable DNA sequences. The Number of cycles was after the amount of eluted RNA after adapted to the selection. After the phenol-chloroform Ex traktion and ethanol precipitation, the DNA was in 200 ul Water dissolved.

reaction temperature conditions 20 μl from rev. transcription 94 ° C / 4 min Init.denat. AL = L <40 μl 10 × Pfu buffer 19.2 μl MgCl ₂ (100 mM) 94 ° C / 2 min denaturation 16 μl primer A (100 μM) 55 ° C / 2 min annealing 16 μl of primer B (100 μM) 75 ° C / 10 min extension 2 μl BSA (2 μg / μl) 6-16 cycles AL = L <16 μl of dNTP mix (10 nM per dNTP) AL = L CB = 3 <4 μl Pfu Polymerase @ AL = L CB = 3 <ad 400 μl with H 2 O fill up

PCR for the amplification of cloned sequences

After cloning and isolation of the plasmids, the Insert sequence by PCR from the complete vector out amplified. Purification is as above described.

Standardized approach temperature conditions 40 μl 10x Pfi buffer 94 ° C / 4 min ID AL = L <19.2 μl MgCl ₂ (100mM) 2 μl BSA (2 μg / μl) 94 ° C / 2 min denaturation 16 μl primer A (50 μM) 94 ° C / 2 min annealing 16 μl of primer B (50 μM) 75 ° C / 6 min extension 16 μl dNTP mix (10 mM per dNTP) 25 cycles AL = L <2 μl vector (1/10 volume of miniprep) AL = L CB = 3 <4 μl Pfu polymerase (2.5 U / μl) @ AL = L CB = 3 <ad 400 μl with H 2 O fill up

1.3.2 T7 Transcription

With the help of in vitro transcription, an RNA complementary to a DNA sequence can be produced. For this, the DNA template must contain a 3'-terminal recognition region of 17 bases, the T7 promoter. Immediately behind this promoter, transcription is started by T7 RNA polymerase from bacteriophage T7. One can increase the efficiency of the polymerase by starting the transcript with two guanosine nucleotides.

Standard approach:
10 μl 10 × transcription buffer
10 μl DTT (100 mM)
6 μl BSA (2 μg / μl)
16 μl NTP mix (100 mM per NTP)
50 μl DNA template (about 1/4 of a PCR assay)
2 μl T7 polymerase (50 U / μl)
Add 100 μl with H ₂ O.

By adding α- ³² P-CTP, the RNA can be radiolabeled. After 2-3 h incubation at 37 ° C, the RNA is purified on a 6% denaturing polyacrylamide gel, eluted and desalted by ethanol precipitation. After ethanol precipitation, only siliconized Eppendorf tubes were used to minimize loss of RNA by adsorption to the vessel wall.

Preparative transcription

To prepare the RNA start pool for selection, a preparative transcription of 3.8 ml volume was used (19 × 200 μl). In addition, the DNA concentration was increased to 40 μmol / 100 μl. 1.6 nmol DNA was used, corresponding to a pool complexity of 1 × 10 ¹⁵ . The reaction time was increased to 5 h.

Preparative transcription approach:
320 μl DNA pool (1.6 nmol)
380 μl 10 × transcription buffer
380 μl DTT (100 mM)
22.5 μl BSA 120 μg / μl)
600 μl NTP mix (100 mM per NTP)
10 μl of α- ³² P-CTP
75 μl T7 polymerase
Add 3.8 ml to H ₂ O ad.

If radioactively labeled RNA is to be prepared by addition of α- ³² P-CTP, a small aliquot (1/50 of the mixture) of the reaction mixture is taken as reference value in order to be able to determine the specific radioactivity of the RNA. The radioactivity in this sample corresponds to 1/50 of the added amount of CTP. The amount of substance added radioactive CTPs is negligible due to the high activity. From the activity of the control cpm _comparison and the actual sample cpm _sample , one can calculate the amount of the incorporated cytidine n _C in the sample. By dividing by the number of cytidine nucleotides in the RNA, the amount of RNA n _{RNA is obtained} . To calculate the number of Cs in the randomized area, assume that every fourth base is a cytidine. In addition to the cyticlin bases in the two primer regions, statistically 31.5 Cs are contained in the RNA.

1.3.3 Reverse Transcription

For a later amplification by PCR was the RNA after each round of selection with the reverse transcriptase transcribed into cDNA. A transcriptase was used with an RNase H deletion, d. H. the RNA is at the Transcription is not digested, and it is possible to have several  Make DNA copies of an RNA strand. Similar to in the PCR, too, a complementary to the 3 'end Oligonucleotide needed to form a DNA-RNA hybrid, which subsequently from the reverse transcriptase is extended.

hybridization Reverse transcription 10 μl of RNA (selected) 12 μl annealing mixture 2 μl of primer B (100 μM) 4 μl 5x RT buffer Incubation at 70 ° C / 10 min 2 μl DTT (100 mM) 1 μl dNTP mix (10 mM / dNTP) AL = L <incubation at 46 ° C / 2 min 1 μl Rev. Trans. (200 U / μl)

Incubate for one hour at 46-48 ° C and then drive immediately with the PCR (1.3.1).

1.3.4 Sequencing

The sequencing takes place by addition of a Sequencing primer to the single-stranded DNA, then extended by a DNA polymerase along the plasmid becomes. By adding dideoxyribonucleotides to the no further dNTPs can be coupled, it comes statistically to a demolition of the chain extension behind every single base.

Radioactive sequencing with ³⁵ S

In radioactive ³⁵ S sequencing, in addition to the dNTPs, ³⁵ S-dATP (or ³⁵ S-dCTP) is also incorporated to allow detection by autoradiography.

The sequencing was carried out with the sequencing kit from Pharmacia. By using the universal and the reverse primer, both the strand and the complementary strand are sequenced, whereby a faulty sequencing z. B. can better recognize by forming a stable secondary structure. In order for the DNA to be single-stranded, it is denatured in the first step. After hybridization of the primer, the sample is distributed for the sequencing reaction on four Eppendorf tubes corresponding to the four bases and the respective dideoxynucleotide added. It is allowed to react for exactly 5 min at 37 ° C and breaks the polymerization by adding the stop solution on ice. Half of each sample is applied to a 6% sequencing gel for separation (see 3.2.1). From the gel an autoradiogram is created on which the respective sequence can be read.

1.) Denaturation 2.) hybridization 10 μl of plasmid DNA 10 μl of plasmid, denat. 2.5 μl NaOH (2 M, fresh) 2 μl annealing buffer Incubation for 10 min / RT 2 μl primer (5 pmol / μl) Inkub. 20 min / 37 ° C and 10 min / kT

3.75 μl sodium acetate (3 M, pH 5.5)
8.75 μl H ₂

O
75 μl ethanol
Ethanol precipitation, take up in 10 μl H ₂

O

3.) marking 4.) Abort reaction 14 μl annealing approach 2.5 μl per A, C, G or T mix 3 μl labeling mix (A-Mix) 4.8 μl labeling approach 1 μl [α- ³⁵ S] -dATP (10 μCi / μl) 37 ° C / 5 min 2 μl T7-DNA polym. (1.5 U / μl) 6 μl stop solution AL = L <4 min / RT

Sequencing with the ABI sequencer

Detection of the DNA fragments occurs in the ABI sequencer by fluorescence measurement. You work with it Dideoxynucleotides with four different Fluorescent dyes were modified and therefore at different wavelengths fluoresce. This must be the Reaction and separation on the gel for the individual Bases are not carried out separately, causing the Number of samples reduced by 75%. The sequencing was using the kit of Perkin-Elmer after the method of Cycle sequencing performed in thermocycler. As in In a normal PCR, the polymerase forms the opposite strand to the template DNA, except that in cycle sequencing only a primer is present, so only one strand multiplied and that by incorporating the ddNTPs the Chain extension statistically at each point of the Templates is aborted. This reduces the required amount of DNA to 1/10 of a plasmid isolation.

sequencing approach temperature conditions 5 μl of plasmid 96 ° C / 2 min Init.denat. AL = L <5 μl primer (5 μmol / μl) universal / reverse 10 μl sequencing mix 96 ° C / 15 sec denat. 50 ° C / 15 sec annealing 60 ° C / 4 min extension 25 cycles

Since the excess of fluorescently labeled ddNTPs in the interfering with fluorometric analysis, these are going through  Gel filtration with spin columns previously separated. there were CentriSep (Princeton Separation) and Centriflex, respectively (Advanced Genetic Technologies Corp.) Spin Columns used according to the instructions. The Centriflex Columns have the advantage that they are already pre-swollen are and therefore directly operational. A separation the ddNTP by ethanol precipitation is also possible. That's just for a large number of samples faster and cheaper, but succeeds Separation of ddNTPs is not quantitative, which is why the Separation of the first 20 bases on the Sequenzgel not clean succeed. But because the sequencing primer sequence in is a greater distance from the insert, this is not big disadvantage. The purified samples are collected in the Vacuum centrifuge to dryness and concentrated in 2 ul Sample buffer dissolved. 1-1.5 μl of this sample then the 6% denaturing sequencing gel applied. After completion of the electrophoresis, the Assignment of the samples and the evaluation of the evaluation of the computer.

1.3.5 3 'Labeling of RNA

The radioactive 3'-labeling is carried out by esterification of the free 3'-hydroxyl group of the RNA with the 5'-monophosphate group of a cytidine-3 '- [5'- ³² P] -di phosphate (pCp). This ligation is catalyzed by T4 RNA ligase under ATP hydrolysis.

After an 18-hour incubation at 4 ° C, the reaction mixture is purified over a 12% denaturing polyacrylamide gel, eluted and precipitated with ethanol.

Reaction:
12 μl RNA (2 μmol / μl)
12 μl of 3 × ligase buffer
3 μl ATP (150 μM, freshly diluted)
10 μl of [5'- ³² P] pCp
3 μl T4 RNA ligase (10 U / μl)

1.3.6 5 'Labeling of RNA

The 5'-end of the RNA is tagged by kinasing a radioactive phosphate group to the 5'-hydroxyl group. For this purpose, the triphosphate group of RNA present after transcription must first be cleaved off with the aid of alkaline phosphatase. The alkaline phosphatase from shrimp was used because it can be inactivated by heating to 80 ° C for 10 min for subsequent kinase. The T4 polynucleotide kinase can now transfer the radioactive phosphate residue from the [γ- ³² P] ATP to the RNA. After completion of the reaction is purified on a 12% denaturing polyacrylamide gel and the RNA precipitates with ethanol.

dephosphorylation:
7 μl RNA (2 μmol / μl)
1 μl of 10x phosphatase buffer
2 μl of alkaline phosphatase, shrimp (1 U / μl)
Incubation at 37 ° C / 1 h
3 μl EDTA (0.1 M)
80 ° C / 10 min. then 0 ° C / 5 min

Kinasing:
23 μl Dephosphorylierungsansatz
3 μl H ₂ O
5 μl 10x kinase buffer
1.6 μl DTT (100 mM)
3 μl MgCl ₂ (100 mM)
10 μl [γ- ³² P] -CTP
4 μl T4 polynucleotide kinase (10 U / μl)

1.3.7 Nucleolytic cleavage of RNA

There are many ribonucleases that are RNA dependent cut their secondary structure. From the splitting pattern a limited cleavage with these RNases should therefore a statement about the secondary structure of the be possible. The enzyme concentration and the incubation period must be chosen so that no complete digestion of the RNA occurs. About 50% The RNA used should remain undigested, so that statistically only one cleavage occurs per molecule.

Before the enzymatic cleavage, the RNA was as well during selection for 5 min at 90 ° C in Binding buffer denatured and then in 20 min on RT cooled. The cleavage reactions were in one Volume of 6 μl in siliconized Eppendorf tubes carried out. The reaction is stopped by adding 6 μl Sample buffer stopped and the samples placed on ice. 54 of the reaction mixture are on a 25%  denaturing polyacrylamide gel separated and subsequently visualized by autoradiography.

Cleavage with ribonuclease T ₁

Ribonuclease T1 (from Aspergillus oryzae) cleaves specifically single-stranded RNA on the 3 'side of guanosine nucleotides (Gp ↓ Np). This nuclease is capable of taking native as well as under denaturing conditions (7 m Urea). The split under denaturing Conditions allows a later assignment of the bases on the autoradiogram, while looking from the native Cleavage obtained via single-stranded regions can.

denaturing division native split 2 μl RNA (about 150000 cpm) 2 μl RNA (about 150000 cpm) 1 μl t-RNA (6 μg / μl) 3 μl t-RNA (6 μg / μl) 2 μl of T ₁ cleavage buffer 0.7 μl H ₂ O 0.4 μl H ₂ O 0.3 μl RNase T ₁ 0.6 μl RNase T ₁ 20 min / room temperature AL = L <20 min / 55 ° C

Cleavage with ribonuclease T ₂

Ribonuclease T ₂ (from Aspergillus oryzae) cleaves single-stranded RNA with sequence-independent release of oligonucleotides with 3'-phosphate groups. RNase T ₂ can therefore be used in combination with RNase 51 for the identification of non-base-paired regions.

native cleavage:
2 μl RNA (about 150000 cpm)
3 μl t-RNA (6 μg / μl)
0.7 μl H ₂ O
0.3 μl RNase T ₂ (5.75 U / μl)
20 min / room temperature

Cleavage with ribonuclease S ₁

The nuclease S ₁ (from Aspergillus oryzae) cleaves like the RNase T ₂ sequence-independent single-stranded regions of the RNA. In contrast to RNase T ₂ , however, oligonucleotide 5'-phosphates are released here.

native cleavage:
2 μl RNA (about 150000 cpm)
3 μl t-RNA (6 μg / μl)
0.7 μl H ₂ O
0.3 μl RNase S ₁ (0.4 U / μl)
20 min / room temperature

Cleavage with ribonuclease V ₁

With the ribonuclease V ₁ (Cobra venom), preferably double-stranded regions of the RNA can be cleaved independently of the sequence. As in the case of cleavage with RNase 5 ', oligonucleotide 5'-phosphates are also released here.

native cleavage:
2 μl RNA (about 150000 cpm)
3 μl t-RNA (6 μg / μl)
0.6 μl H ₂ O
0.4 μl RNase V ₁ (0.72 U / μl)
20 min / room temperature

1.3.8 Partial alkaline hydrolysis of RNA

Under alkaline conditions RNA is unstable in contrast to DNA. Cleavage products with a 5'-hydroxyl group are formed via a cyclic 2 ', 3'-phosphate transition state. By limiting the time of alkaline hydrolysis, the RNA can be cleaved to produce oligoribonucleotides of any length. When this reaction mixture is applied to a denaturing polyacrylamide gel, an alkali metal hydroxide is obtained after autoradiography, which can be used for the assignment of the cleavage products of the enzymatic RNA cleavage. The reaction was stopped by adding 6 μl sample buffer and placed on ice until applied to the gel.

Reaction:
3 μl of RNA (about 150000 cpm)
1.5 μl t-RNA (6 μg / μl)
4.5 μl of alkaline buffer (50 mM NaOH, 1 mM EDTA, freshly prepared)
45-60 sec / 120 ° C

1.4 Preparation of Affinity Chromatography 1.4.1 Immobilization of Peptides on Sepharose

For affinity chromatographic selection, the Peptides are immobilized on a carrier material. The  Immobilization should be reversible to elution the specific binding RNA as an RNA-peptide complex too enable. Therefore, peptides on disulfide bridges to Thiopropyl-Sepharose 6B can be coupled. To that allow the peptides to have a free thiol group which is why the βA4 (1-40) and βA4 (1-16) with a Cysteine were synthesized at the N-terminal end and consequently, or contain 17 amino acids.

To swell, 1.4 g of thiopropyl-Sepharose 6B are suspended in 30 ml of water and swirl for 15 min. All used Solutions should be run for 10 min before use. Ultrasonic treatment degas to oxidation to avoid sulfide groups by atmospheric oxygen. you filtered off and washed with 3 × 50 ml of water. The swollen gel is dissolved in 25 ml of a solution of 88 μM βA4 (1-16), 12 μM Tris, pH 7.5, 500 mM NaCl, 1 mM EDTA and suspended for 1.5 h at room temperature Swirling incubated. Immobilization of βA4 (1-40) due to the great tendency for aggregation and the associated insolubility immobilization in a 60% hexafluoroisopropanol solution (LTFIP) be performed. This is suspended in 25 ml a solution of 45 μM βA4 (1-40), 10 mM Tris-HCl, pH 7.7 and rock for 1.5 h at RT.

The gel is filtered off and washed with 2 × 50 ml of a 0.1 M sodium acetate solution, pH 6.0. To be unresponsive Thiol groups of the gel to split off the protecting groups and to block reactive thiol groups, the gel became 45 while swirling in 20 ml of 2-mercaptoethanol (5 mM, 0.1 M NaOAc, pH 6). The column material will be twice washed with 50 ml of binding buffer and at 4 ° C under  Binding buffer stored.

For the precolumn, the column material became as above described, but without the addition of the peptide. Here all thiopyridyl groups were through Substituted 2-mercaptoethanol.

1.4.2 Determination of the peptide concentration on the column material

To determine the loading of the column material, the Peptide of 100 μl column material by reductive cleavage split off the disulfide bridges. This is eluted Peptide from the column by adding 500 μl twice Allow elution buffer to flow slowly through the column (30 ul / min). The concentration of the peptide in the eluate was on determined two types.

Protein Assay

The concentration of the peptide in the eluate was at βA4 (1-16) determined by the Coomassie protein assay become. To this is added 500 .mu.l of the eluate with 500 .mu.l Coomassie solution (Pierce) together, shakes and measures the Absorption in a 1 ml disposable cuvette at 595 nm. The Concentration is obtained by comparison with one Calibration curve obtained at various known concentrations has been recorded. Because the absorption is not timed is constant, it is important that the measurement is always after the same time after the addition of the Coomassie solution is carried out. Due to the aggregation in aqueous  Solutions, the peptide concentration of βA4 (1-40) could could not be determined by the protein assay and was therefore determined by amino acid analysis.

amino acid analysis

200 μl of the eluate (1 ml) were concentrated to dryness in a Dansyl glass and acid hydrolysed by gas phase hydrolysis. For this purpose, put the Dansylgläschen in a 40 ml vessel with screw cap and tap to about 3 ml of 5.7 M hydrochloric acid. By evacuating the vessel and heating to 150 ° C for 1 h, the sample is hydrolyzed. The sample is concentrated in vacuo and taken up in 100 ul of water. 20 μl of this solution were derivatized with phenylisothiocyanate for the amino acid analysis after the coupling steps of Edman-Ahhaus. The amino acids were separated by HPLC on an ABT PTH RP C18 column (5 μm, 220 × 2.1 mm) with a binary gradient system (buffer A: H ₂ O, buffer B: 5% w / v tetrahydrofuran, 6 mM sodium acetate , pH 4.2). The amounts of the amino acids of the peptide were calculated by comparing the peak height of the amino acids with a reference substance of known concentration. After dividing the amount of substance by the number of residues in the peptide and averaging over 10 amino acids to obtain the concentration of the peptide in the eluate and thus the loading of the column material. The amino acid analysis was carried out by Dr. med. Schröder performed.

1.4.3 Siliconization of the columns

To adsorb the RNA on the surface of the columns  or to keep the frits as low as possible, these were silanized before use. This is done by rinsing the columns with toluene and then filled with a 5% Solution of dichlorodimethylsilane in toluene. After a to two hours, this solution is removed, the columns with Toluene and then with water several times thoroughly washed.

1.4.4 Optimization of the binding buffer

To suppress non-specific interactions of the RNA, the salt concentrations in the binding buffer were matched to the column material. For this, the NaCl concentration was varied between 150-500 mM and the MgCl ₂ concentration in the range of 5-50 mM. The conditions under which least RNA was bound on the column should be used for selection.

1.4.5 Performance of in vitro selection

The selection of high-affinity RNA molecules was affinity chromatography on small columns (Mobicols, Mo Bi Tec) with 100 μl column volume. In each Selection round was a precolumn (without peptide) and a Main column (affinity column) used. The radioactive labeled RNA was transformed into binding buffer on a Concentration of 2.5 μM set. So that the RNA in their stable conformation exists, one denatures for 5 min at 90 ° C and renatured by 20 minutes Cool to room temperature. 200 μl of this RNA is carried on the guard column. Only in the first Selection round was due to the large amount of RNA of  deviated from this protocol and twice as large concentration about 2.5 nmol RNA used. The Column volume of the main column was increased to 250 μl. The fore and main column was in the first round with 3 ml Binding buffer washed while remaining in all following Rounds were washed only with 2 ml of buffer. in the Following the peptide with the binding RNA after Removal of the guard column with 1 ml of elution buffer from the Column eluted into a siliconized Eppendorf tube.

To ensure a constant flow through the column received, a peristaltic pump was used. The Binding buffer was added to the column in 500 μl portions given and passes by applying a light Overpressure with the help of the pump through the precolumn on the Main column. Now the pump is first to the main column connected before the next portion of buffer on the Pre-column can be applied. The river through the Columns should be about 33-35 μl / min.

After completion of the elution, the RNA concentrates the Washing and elution solution and the columns through Cerenkov count determined. The Fluat is in one Vacuum centrifuge to about 400 ul and concentrated to Removal of the peptide with phenol and chloroform extracted. With ethanol with the addition of glycogen (40 μg) the RNA is precipitated and then reversed transcribed. The DNA is amplified by PCR and used for the next round of selection by transcription into RNA umgeschriehen. If no further increase in the specific eluted RNA is more observed, the selection be canceled. To the mixture of the different  To analyze sequences, you separate the Sequences by cloning in bacterial cells.

1.4.6 Determination of dissociation constants

The affinity chromatographic determination of Dissociation constants were determined using Econo columns (Bio-Rad), as these have a larger volume own than those used during the selection Mobicols. A sufficiently large column bed volume is crucial so that the applied amount of RNA (10 μl, 100 nM) can pass through the column as a sharp band. It was worked with 800 ul column material, whereby a column volume of 810 μl gives (volume of the tap Etc.). After application of the radioactively labeled RNA washes Keep the column with binding buffer until no Radioactivity can be detected more on the column. At first you catch the eluate drop by drop To be able to determine the dead volume as accurately as possible. Later suffice 500 ul fractions. By using a pump and a fraction collector can automate the determination become.

1.5 Microbiological methods

For the transformation of Epicurian Coli cells, the DNA is ligated into a vector. Was used here the pPCR script Amp SK (+) cloning vector (Stratagene), a Derived from pBluescript II SK (+) phagemid, which is a gene for ampicillin resistance as well as a lac promoter and a T7 RNA polymerase promoter contains. Within the SK multiple cloning siles is a rare srf I interface (5'-GCCC / GGGC-3 '), which is a ligation of the  blunt-ended PCR products of selection with the sliced vector allows. As for the PCR by default the Pfu DNA polymerase was used a workup of the DNA is not necessary. Otherwise that would be the case Use of Taq polymerase, as these are often a another base hangs on the end of the chain. The Restriction splitting of the vector, as well as the ligation of the DNA inserts can be placed in a vessel at the same time be performed. Due to the simultaneous activity of the Restriction enzyme and ligase is in equilibrium between cut and uncut vector, until by ligation of the insert the Srf I restriction interface is lost, and the vector stops can be split.

These plasmids are now in super competent Epicurian Coli cells XLI-Blue MRF 'Kan (Stratagene) introduced and spread on agar plates, the X-gal (5-bromo-4-chloro-3-indolyl-β, D-galactopyranoside), IPTG (Isopropylthio-β, D-galactoside) and the antibiotic Contain ampicillin. Due to the antibiotic can only the cells grow on the medium by recording a plasmid have an ampicillin resistance. The Cells that received a non-recombinant plasmid have an intact gene for β-galactosidase, the cleavage of the X-Gals a blue dye releases. These cells turn blue. The white ones Colonies represent the transformed bacteria that contain exactly one recombinant plasmid, any can be increased.

1.5.1 Ligation

Before ligation, the DNA is added with the addition of the same Volume of a 4 M ammonium acetate solution (instead of the Sodium acetate) and 2.5 times the volume of ethanol like. The pellet is taken up in TE buffer.

The ratio of insert. Plasmid should be between 40: 1 and 100: 1. A ratio of 70: 1 was chosen, which corresponds to an amount of 364 fmol insert per standard batch.

Ligation reaction:
1 μl pPCR-Script Amp SK (+) cloning vector (10 ng / μl)
1 μl 10x PCR Script buffer
0.5 μl ATP (10 mM)
3 μl PCR product (364 fmol)
1 μl SrfI restriction enzyme (5 U / μl)
1 μl of T4 DNA ligase (5 U / μl)
Add 10 μl with H ₂ O

It is allowed to react for one hour at RT, heated for 10 min to 65 ° C and set up the sample until transformation Ice.

1.5.2 Transformation

Through a special salt treatment it is possible To make cell membranes DNA-permeable (competent) and to to bring a recording of a plasmid. For the Transformation, however, were commercially available used competent cells. These are already opposite small temperature fluctuations very sensitive and must therefore always be stored at -80 ° C or while aliquoting permanently on ice.  

Furthermore, it is important to strictly adhere to the duration of the 45 s temperature interval. About half an hour prior to cell streaking, ampicillin-containing agar plates (100 μg / ml) should be overlaid with 20 μl of IPTG (0.2 M) and 20 μl of X-gal (10% (w / v) in DMF). Prepare three different agar plates for each transformation, apply 100, 150 or 200 μl of the mixture and dilute it with a curved glass rod on the agar surface. The agar plates are allowed to incubate for about 18 h in the incubator at 37 ° C.

Transformation approach:
40 μl Epicurian Coli XLI-Blue MRF 'Kan supercompetent cells
0.7 μl of b-mercaptoethanol (1.5 M)
Incubate for 10 min on ice, shake every 2 min
2 μl ligation batch
Incubate on ice for 30 min
45 sec / 42 ° C, immediately afterwards on ice
450 μl SOC medium (preheated to 42 ° C)
Shake 37 ° C / 1 h
Streak on agar plates with X-gal, IPTG and ampicillin, incubate at 37 ° C / 18 h.

1.5.3 Isolation of Plasmids

The obtained after the transformation on the agar plate White clones are made with the help of a sterile wooden stick (Toothpick) and in 3 ml overnight culture at 37 ° C multiplied in a shaking water bath. From every culture becomes a glycerin culture for eventual later use kept. Homogenize 0.5 ml of a culture the same volume of glycerol, the sample freezes in  liquid nitrogen and stored at -80 ° C. The remaining 2.5 ml are in two portions in one Transferred Eppendorf vessel and the cells at 13000 rpm, 2 Centrifuged for min. The supernatant is discarded and shake the cells in 500 μl of STE buffer suspended. Pellet again and suspend then for 10 min in 100 ul lysate I. Man gives 200 μl lysate solution 11 is added and the samples are swirled gently during the 20 minute incubation on ice. In This step is followed by the lysis of the cells. By adding of 150 μl lysate solution ITT is added to the chromosomal DNA 0 ° C / 15 min like. The samples may only Gently mix (do not vortex) to one To avoid tearing the DNA by the shear forces. The Cell debris and the chromosomal DNA are at 13000 rpm / 15 min centrifuged and the supernatant in a new Transferred Eppendorf vessel. It is extracted with phenol (pH 4.5-5) and chloroform, precipitate with ethanol and dissolve the Pellet in 50 μl TE buffer. Because in these samples the mRNA the cell is still included, this is the result of However, sequencing on an ABI sequencer interferes with it another RNA digestion by adding 250 ul ST buffer the plasmid isolation kit (see below) become. It is again precipitated with ethanol and dissolves the sample 50 μl of water.

An alternative to this isolation was also a kit (Nucleobond AX20, Macherey-Nagel). This was proceeded in accordance with the regulation.

The purity and size of the plasmids were gel electrophoretically on a 1% agarose gel checked (see 1.2.1).  

1.6 Characterization of aptamers

The primary structures of aptamers were determined using a Computer program examined for homologous areas. To a program was written in Pascal that the Read sequences as a text file and on motives of variable Length compares. There will be all the motifs several times occur with the number of sequences in which they are were found in a text file. These File can be edited with a word processor or printed out.

2 results

The great importance of amyloid in the course of Alzheimer's disease is undisputed, although the Causes of the disease are still unknown. It However, it could be shown that the deposits of this Peptides in the brains of patients neurotoxic. By using the PNA technologies, with the help of in In vitro selection of high-affinity RNA molecules against amyloid and an amyloid fragment. These aptamers can for example as a tool for further research can serve the disease, but can also aid the Diagnosing Alzheimer's. There are studies that state that the amyloid content in the blood of Alzheimer's patients are different from the healthy person differs (Li et al., 1995, Rosenberg et al., 1997, Di Luca et al., 1998). This shows that for this widespread disease with the invention a diagnosis and effective medicines are opened.  

Aptamers have similar properties to antibodies, but have some advantages over the antibodies. you are smaller and can be synthesized by high chemical synthesis Purity are produced. The chemical synthesis allows also a specific modification or the installation of Reporter molecules, what a research assignment and diagnosis is of great interest.

In the amyloid plaques in the brains of Alzheimer's patients, the amyloid is found in the insoluble β-sheet structure. The transition from the soluble α-helix or coil form in the β-sheet structure proceeds relatively quickly in aqueous solution and is dependent on some parameters, such. As the salt concentration and the pH. It could be shown that the conformational change starts from the non-polar amino acids at the C-terminal end of the amyloid. By shortening the amyloid one can therefore produce peptides, which convert much slower or hardly at all into the insoluble β-sheet structure. Since these shortened peptides are much easier to handle and also more cost-effective, aptamers against a fragment of the amyloid are also presented in the exemplary embodiments. Since it is not certain that the aptamer recognizes the amyloid against the fragment, it has also been selected against the complete βA4. It would be conceivable that in this way aptamers could also be obtained against the soluble and insoluble conformation. The two peprides were chemically synthesized and provided by Schering AG.
Cys-βA4 (1-16):
NH ₂ -Cys-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His Gln-Lys-COOH
Cys-βA4 (1-40):
NH ₂ -Cys-Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr-Glu-Val-His-His-Gln-Lys-Leu-Val-Phe-Phe-Ala-Glu-Asp -Val-Gly-Ser-Asn-Lys-Gly-Ala-Ile-Ile-Gly-Leu-Met-Val-Gly-Gly-Val-Val-COOH

2.1 Immobilization of the peptides

For the selection by affinity chromatography were the two peptides reversible on a matrix immobilized. These were the peptides with a cysteine synthesized at the N-terminal end to form a Immobilization on thiopropyl-Sepharose 6B (Pharmacia) to enable disulfide bridges. The free thiol group of the peptide substitutes the pyridyl group which is replaced by Conversion to the stable 2-thiopyridone a very good Leaving group represents. By washing with dithiothreitol (DTT), the disulfide bridge can be cleaved reductively and the peptide is eluted again from the matrix. Since the released during immobilization 2-thiopyridone absorbed at 343 nm, a determination of the Concentration of the peptide on the column material photometric concentration determination of the released Thiopyridone be possible. This method resulted but too high, not reproducible values. Therefore The peptide was eluted from 100 μl of column material with DTT and the concentration in the eluate with the help of Coomassie Protein assays determined. Since the assay is not linear, had previously a standard curve with different Be included peptide concentrations. By Measurement of absorption at 595 nm and comparison with the  Standard curve was a loading of 46 μM for βA4 (T-16) determined.

Due to the high tendency for aggregation this could Assay for βA4 (1-40) should not be used. That's why The concentration of the peptide in the eluate was through Amino acid analysis determined. This was the eluate hydrolyzed and applied to a protein sequencer. By averaging the peak areas of the individual amino acids and comparison with a sample of known concentration the concentration could also be determined at βA4 (1-40) become. The loading of the column material with βA4 (1-16) was at this determination at 40 μM, while at βA4 (1-40) corresponded to 4 μM.

2.2 In vitro selection Creation of the RNA library

The RNA library is generated by in vitro transcription from a DNA library that has been chemically synthesized on a solid phase. It was prepared using a synthesis machine as a single-stranded DNA, consisting of two primers of 20 nucleotides each and a 70 nucleotide randomized region. For the synthesis of 3 'in 5' direction the usual Phosporamiditchemie was used. The random incorporation of the four nucleotides can be achieved by using a solution of all four DNA phosphoramidites, taking into account the different reactivity in the mixing ratio (dA: dG: dC: dT = 3: 2, 5: 2, 5: 2). In a randomized range of 70 nucleotides theoretically 4 ⁷⁰ or 1.4.10 ⁴² different molecules are conceivable. However, this corresponds to 2.3.10 ¹⁸ mol or 8.2.10 ¹⁹ kg. In contrast, the actually synthesized amount of about 10 ¹⁵ molecules is so small that it is statistically very unlikely that two identical molecules were synthesized. The yield of each individual coupling of the synthesis was over 99% and could be followed by the cleavage of the trityl protecting group. However, due to the many couplings and the large losses in the purification over a polyacrylamide gel, the overall yield was only just under 2% or 3.8 nmol. From this DNA, the molecules must be removed, which are not amplified by PCR due to errors in the synthesis (eg, no complete cleavage of the protective group, cleavage of the bases, etc.). For this purpose, a sample of this DNA was reacted in a PCR cycle with radiolabeled deoxynucleotide and quantified the amount of counterstrand produced. The proportion of PCR amplifiable DNA was 42% or 1.6 nmol. This results in a complexity of approximately 1.10 ¹⁵ molecules.

The single-stranded DNA library was transformed by PCR Double strand completed and duplicated. It was additionally by using an extended primer the 17 nucleotide T7 promoter sequence inserted for a transcription in RNA was needed. At this PCR reaction was the goal in a small multiplication a huge amount of DNA. The DNA pool was around the Factor 13 reproduced.

Carrying out the selection

The selection for the βA4 (1-16) and the βA4 (1-40) were carried out under the same conditions. The binding buffer used (10 mM Tris-HCl, pH 7.5, T50 mM NaCl, 5 mM MgCl ₂ , 1 mM EDTA) was adapted to the selection conditions so that the unspecific interactions on the selection column were minimized in a test selection. The selection was carried out in plastic columns with a small frit at the bottom and a large frit over the column material. The column and the frits were siliconized prior to selection to minimize adsorption of the RNA. Furthermore, following ethanol precipitation of the RNA following T7 transcription, only siliconized Eppendorf tubes were used. In each round of selection, a precolumn with column material without peptide was used to separate RNA molecules that bind specifically to the column material. Since the amount of RNA applied to the column in the first round was many times greater than in subsequent rounds, the column volume of the main column was increased to 250 μl, while the volume of the precolumn was constant at 100 in all subsequent rounds μl was kept. In the first round, 2.3 nmol of the hybridized RNA pool, corresponding to an average of 1.4 copies per sequence, was applied to the column at a concentration of 5 μM and washed with 3 ml of binding buffer. Subsequent washing with 1 ml of elution buffer, the disulfide bridges were cleaved and the peptide eluted with the specific binding RNA. The peptide was separated by phenol / chloroform extraction, the RNA precipitated with ethanol and reverse transcribed into cDNA, amplified by PCR and transcribed into RNA for the next round. In the first round, the complexity of the pool could already be reduced so much that in all other rounds only 500 .mu.mol renaturierte RNA with a concentration of 2.5 pH were applied to the column. In addition, the column bed volume of the main column was reduced to 100 μl and washed with only 2 ml of binding buffer. In both selections, an increase in specific eluted RNA was observed in the 4th round. After the 8th round, the selection could be stopped because no further increase of the specifically eluted RNA could be observed (Table 1). It is noticeable that the proportion of RNA binding on the guard column increases from round to round, although a precolumn was used in each round (Table 1). Also increasing is the proportion of RNA that can not be eluted from the pre- and main column.

2.3 Characterization of aptamers

The eluates after the eighth round of selection were reverse transcribed and amplified by PCR. This gives a mixture of aptamer sequences, which must be separated for analysis. By cloning into competent bacterial cells capable of accurately accepting a DNA plasmid and then streaking on an agar plate, single colonies containing an aptamer sequence are obtained. For the transformation, the DNA was ligated into the pPCR script Amp SK (+) cloning vector (Stratagene). This vector contains a gene for ampicillin resistance as well as a lac promoter. After transformation of the vector into Epicurian Coli XLT-Blue MRF'Kan cells, the cells were plated on agar plates with X-gal, IPTG and the antibiotic ampicillin. Due to the ampicillin only transformed cells can grow, with those with a vector without an insert staining blue due to the X-Gals and the intact β-galactosidase gene. For each selection, about 100 white clones were selected and the plasmids were isolated. The inserts were sequenced according to the dideoxymethod of Sanger et al. (Sanger et al., 1977), with a portion being radioactively sequenced with ³⁵ S and a portion using an ABI sequencer. By using the universal primer and the reverse primer, both strands of the insert were sequenced. A total of 75 samples were sequenced for the βA4 (T-40) and the βA4 (1-16).

2.3.1 Sequence analysis primary structure

The primary structures of the randomized region of aptamers, consisting of about 70 nucleotides, were examined for homologous regions. The existence of conserved regions can reveal which nucleotides are involved in binding to the target molecule. A computer program has been written, which has carried out the search for the same motifs of different lengths. The 75 sequences determined for selection against the βA4 (1-40) can be divided into 5 groups that are highly conserved per se ( Figure 1). 13 of the 75 sequences can not be divided into any of the groups and also have no conserved regions among each other. In all other sequences motif b1 is represented by up to 11 bases (Table 2). Three more motifs that occur in most of the sequences are listed in Table 2.

Table 2

As with the sequences for the βA4 (T-40), the sequences of the aptamers against the βA4 (1-16) can also be grouped into groups that are highly conserved ( Figure 2). Unlike the sequences of βA4 (1-40) selection, there is only one motif that occurs in more than two groups. However, the 9 base a1 motif appears in more than half of the sequences (Table 3). Five more motifs from 6 to 11 bases were found.

Table 3

secondary structure

The aptamers against βA4 (1-40) were also analyzed for their secondary structure, and the secondary structure models were calculated using the computer program RNADraw (Matzura & Wennborg, 1996). As expected, the structures of aptamers within a family are similar. Figures 3-6 show the structures of several aptamers from different families. When comparing the structures, it is noticeable that the motif b2 (Table 2) occurs base-paired in all aptamers with the beginning of the primer A. Since not all bases of the motif are complementary to the primer, a loop also occurs in each structure. This suggests that this stem-loop region is crucial for the interaction with the amyloid. The strain probably has only a stabilizing function, while binding to the peptide could take place via the unpaired bases of the loop. This assumption is supported by the fact that in all sequences always four uridines or three uridines and a cytidine form this loop. For the aptamer β61 in FIG. 3, these are the bases U6, U7, U72 and U73. Although belonging to a different group, the aptamer β124 is very similar to the β61. In addition to the primer-paired motif and the loop, both also have a hairpin with the base sequence AAG ( FIG. 3, A50-G52, FIG. 4, A46-G48).

While the arrangement of motif b2 is similar in all sequences, the similarities in motif b1 are not so clear. Although the unpaired base sequence UAAG occurs in the sequence of β61 as well as in β124 ( Figure 4, C32-G35), in other sequences these bases are paired. Nevertheless, this motif seems to play a role in the binding of the peptide, since an aptamer, β19, was found in which the sequence of motifs b1 and b2 is reversed ( Figure 15). Near the 5'-end lies the motif b2, which again is base-paired with the primer A, while the motif b1 is located near the 3'-end. However, a structural similarity of this region with the other aptamers is not readily apparent.

The aptamer β55, with the lowest dissociation constant, differs somewhat more strongly from the structures of the other aptamers, although again the motifs b1 and b2 are to be found here ( FIG. 6). In contrast to the other structures, fewer bases are paired with the primer A. Also, the loop of the four pyrimidines does not occur in this form. Instead there is a loop from the bases AAU. In motif b1, only two bases UA ( Figure 6, U28, A29) are single-stranded, which could mean that these two bases are involved in the binding of the peptide. It is striking that in all four structural models presented, the last three bases of the primer protrude into a hairpin. However, since these bases are conserved, it does not have to be a significant structural feature. The structural model of this aptamer was investigated in more detail by enzymatic cleavage with RNases. For this purpose, the RNA was radioactively labeled at the 3 or 5 'end and subjected to limited nucleolytic hydrolysis using various specific cleaving nucleases. The single-strand-specific nucleases ribonuclease T ₂ and nuclease S ₁ , the double strand-specific ribonuclease V ₁ and the guanosine-specific ribonuclease T _{1 were used} . The comparison of the structures suggests that the 3 'end of the aptamer to the bases of motif b2 is not essential for the binding of the peptide.

2.3.2 Determination of dissociation constants

The binding of an aptamer to its target and thus the formation of a complex can be considered as an equilibrium reaction. The apparent dissociation constants were determined by means of affinity chromatography. In this method, one makes use of the balance between free and bound RNA by eluting the RNA from the column by washing with binding buffer. The more the RNA binds to the peptide, the later the RNA is eluted from the column. The elution volume is therefore a measure of the binding constant as a function of the column size and the loading of the column with the peptide and can be calculated according to an equation given in the following reference (Famulok & Szostak, 1992, Sassanfar & Szostak, 1993). For this equation to be valid, the volume of the column must be as large as possible relative to the volume of the autografted RNA, so that the RNA runs in a narrow band through the column, as in a column chromatographic purification. In addition, the concentration of the RNA solution should be smaller than the expected binding constant in order to apply the equation within its validity range and thus minimize the error of the dissociation constant (Arnold et al., 1986). It follows that the RNA must be strongly radioactively labeled so that even the smallest amounts in the fmol range can still be detected. It was worked with micro pillars of the Econo system (Bio-Rad), which are characterized by a small cross-section. 10 μl of a 100 nM RNA solution was applied to a column with a column bed volume of 800 μl and eluted continuously by means of a pump. The elution profile of aptamer β55 showed a sharp peak at the beginning of the elution, presumably due to misfolded RNA that can not interact with the peptide. This peak can be used to determine the dead volume V ₀ . In order to determine the dead volume as accurately as possible, a very small fraction volume was chosen at the beginning of the elution, while later significantly larger fractions were collected. In order to be able to compare the activities of different size fractions, the data were normalized to a constant volume of 100 μl.

To show that it is a specific enrichment of binding aptamers was included for comparison Elution profile of the randomized. Added to RNA pools. As expected, no retardation of the RNA could occur here to be observed.

By using a guard column with unloaded Support material in all rounds of selection should be one Enrichment of columnar RNAs already prevented. To still ensure that it in the selected RNAs to peptide-binding aptamers was an elution profile of the RNA on a Guard column added. As in the elution of RNA from the Guard column no retardation could be observed be concluded that it is Aptamere against the Amyloid acts.

The dissociation constants of four aptamers against the βA4 (1-40) could be determined in this way (Table 4). Since the RNA concentration used by a factor of 5 is greater than the best dissociation constant, must be  Errors are factored in, so the real ones Dissociation constants probably slightly larger are.

Although the RNA was completely eluted from the column, became the column material after a maximum of three determinations replaced, since no more retardation of RNA could be determined. This became that Column material to the limiting size, and it could no more samples can be measured.

The determination of the dissociation constant for the aptamers against the βA4 (1-16) was carried out analogously. Here could however, only one binding sequence can be found.

Table 4

sequence Listings

Claims (12)

1. Stabilized against nucleic acid-cleaving enzymes Aptamer, which is amyloid specific, or all le and / or derivatives of such an aptamer. 2. Aptamer according to claim 1, which against the Alzhei β-amyloid 4 (1-42) or partial sequences thereof, in particular (1-40), is specific. 3. Aptamer according to claim 1 or 2 with a Loop structure. 4. Aptamer according to claim 3, wherein the loop structure four bases, preferably four uridines or three Uridine and a cytidine, preferably from the four bases. 5. Aptamer according to any one of claims 1-4 containing a sequence GUUU or GUCU. 6. Aptamer according to any one of claims 1-5, containing a sequence from the group consisting of the Se SEQ ID NO: 1 to SEQ ID NO: 68   7. Aptamer according to claim 6, wherein the 3 'end of a Primer sequence according to SEQ ID NO: 69 and / or the 5 'end has a primer sequence SEQ ID NO: 70. 8. Aptamer according to claim 7, wherein the loop structure is formed from the sequence UUUC or GUCU and the opposite thereto primer sequence AUUC and wherein the respective middle two bases un paired and the respective outer bases paired are. 9. Aptamer according to any one of claims 1-8, wherein the 2'-hydroxyl group of the sugar group of one or more pyrimidine nucleotides by a fluoride, Amino or methoxy group is substituted and / o wherein in terminal phosphate groups an acid atom is substituted by a sulfur atom and / or wherein the aptamer is a spiegelmer. 10. A process for the preparation and / or isolation of an aptamer according to any one of claims 1-9, comprising the following process steps:

• a) a randomized DNA pool is created,
• b) If necessary, an RNA pool is generated from the randomized DNA pool and given up to a selection cycle with the following steps:
• c) RNA of the RNA pool or DNA of the DNA pool binding to the amyloid or a partial sequence of the amyloid is selected,
• d) selected RNA or DNA is amplified,
• e) the RNA or DNA from stage d) is added to the RNA pool or DNA pool in stage c), wherein stages c) -e) are repeated until the proportion in stage c) of selected RNA or DNA increases by less than 50%, preferably less than 10%.

11. Aptamer according to one of claims 1 to 9, receives Lich by a method according to claim 10. 12. Use of an aptamer according to one of the claims 1-9 or a mixture of such aptamers to Her provision of a means for diagnosis and / or loading Treatment of Alzheimer's, Spongiform Encephalopa Tien and type II diabetes mellitus.