DE102004002351A1 - New RNA aptamers specific for lipopolysaccharide-binding protein, useful for diagnosis and treatment of sepsis, inhibit the lipopolysaccharide-induced signaling cascade - Google Patents

Abstract

RNA aptamers (I), or their alleles and/or derivatives, that bind specifically to lipopolysaccharide-binding protein (LBP) are new. An independent claim is also included for a method for preparing and/or isolating (I). ACTIVITY : Antibacterial; Immunosuppressive. No details of tests for these activities are given. MECHANISM OF ACTION : Inhibition of LBP, a key component in the lipopolysaccharide-induced signaling cascade.

Description

invention concerning wound healing

The The present invention relates to the selection of 2'-fluoro-pyrimidine modified RNA aptamers specific to lipopolysaccharide-binding protein (LBP) and have a common lead structure motif.

lipopolysaccharide (LPS) triggers in higher Organize a variety of pathophysiological effects, which are known as a manifestation of bacterial sepsis. Neither the problem of timely diagnosis nor therapy is medically solved. The highly preserved Lipoid A component represents the toxic principle the LPS. Attempts to develop more therapeutically effective, lipoid A-specific antibody have so far been unsuccessful. There is therefore an urgent one Need for new approaches not only for the treatment of sepsis, but also for research their molecular bases.

State of technology

• 1) Auf Grund ihrer Größe von 8–15 kDa (Antikörper: 150 kDa) können Aptamere potenziell an schlecht zugängliche Zielstrukturen besser binden als Antikörper; zudem resultieren aus dem Größenunterschied andere Transporteigenschaften.
• 2) Die Herstellung von Aptameren durch chemische Synthese ist reproduzierbarer, ortsunabhängiger und störunanfälliger als die biotechnologische Herstellung von Antikörpern.
• 3) Aptamere repräsentieren eine andere Klasse polymerer Moleküle als Proteine und besitzen daher andere pharmakologische Eigenschaften, die sich als günstig erweisen können.

So far no successful therapy of sepsis is known. Antibody-based approaches have been unsuccessful. As stated below, aptamers are an unexplored alternative to antibodies in the context of sepsis. Nucleic acid molecules (and their derivatives) that bind specific ligands are called aptamers (Latin aptus, apt). Aptamers fold into defined 3D structures that allow them to specifically bind other molecules, similar to antibody-antigen complexes, with affinities down to the picomolar range. Heretofore, aptamers have been selected for more than a hundred different target molecules or targets, such as amino acids, nucleotides, biological cofactors, sugars, dyes, antibiotics, peptides, proteins, viruses, and whole cells. Aptamers have potential advantages over antibodies:

• 1) Because of their size of 8-15 kDa (antibody: 150 kDa), aptamers can potentially bind better to poorly accessible target structures than antibodies; In addition, the difference in size results in other transport properties.
• 2) The production of aptamers by chemical synthesis is more reproducible, location-independent and störunanfälliger than the biotechnological production of antibodies.
• 3) Aptamers represent a different class of polymeric molecules than proteins and therefore possess other pharmacological properties that may prove beneficial.

Currently There are no effective therapies to combat septic shock.

From the patent DE 198 59 912 C2 a test system for the detection of various markers, its production and use is known, however, no in vitro selection method is used, and it biotin-labeled DNA oligonucleotides and antibodies and an antigen are described. The patent DE 198 46 077 C2 Although "trypanosome-binding nucleic acids and their production and use" show selection in 12 rounds, 20% binding of RNA molecules, the target structure is Trypanosoma, and selection occurs directly at the trypanosomes, and a different selection matrix is used ,

The patent DE 101 16 829 A1 "Neuropeptide-binding nucleic acids" describes neuropeptide Y-binding nucleic acids isolated in 12 rounds of selection using modified RNA Streptavidin agarose is used as the matrix.

The patent DE 696 18 614 T2 "Nucleic Acid Molecules Able to Distinguish PrPc and PrPSc Prion Protein Isoforms and Methods of Making Them" discloses nucleic acids that can distinguish between the isoforms PrP ^c and PrP ^sc , which were randomized from a 111 nucleotide priming sequence with a 74 nucleotide long sequence Area isolated in 11 cycles, using Matix BA85-nitrocellulose for binding assays and immobilizing a fusion protein with glutathione-Sepharose 4B.

The patent US 2003/0175730 A1 "Multivalent RNA Aptamers and their expression in multicellular organisms" used as a target B52 (Drosophila splicing factor), as a library, a 108 nucleotide primary sequence is used with a 40 nucleotide randomized area, the selection is in FIG Cycles. The matrix used is a nitrocellulose membrane. The RNA is unmodified.

The patent DE 199 16 417 A1 "Amyloid-specific aptamer" describes amyloids that accumulate in the extracellular area of a tissue where they form plaques and can cause diseases such as Alzheimer's disease, spongiform encephalopathies, and type II diabetes mellitus.From a 110-nucleotide primary sequence with a 70-nucleotide randomized area, in 6 For the affinity chromatographic matrix, small columns (Mobicols, MoBiTec) are used, whereby the column and column material are used in siliconized form.

Of the The state of the art does not offer any solutions to aptamers specific to lipopolysaccharide-binding protein (LBP). All targets and selection matrices used are very different, and the techniques described therein can not be transferred. nitrocellulose as a selection matrix is not suitable, and also Ni-NTA Magnetic Lead agarose beads alone to no successful selection.

It There is therefore a need for a simple and optimized selection method for providing RNA aptamers specific to lipopolysaccharide binding Protein (LBP) bind and treat and study sepsis be used.

task The present invention is the disadvantages described in State of the art to remedy and a method for the production of RNA aptamers for To provide lipopolysaccharide-binding protein (LBP) - that simple feasible and RNA aptamers for the manufacture of a medicament for treatment to provide sepsis that are specific and highly affine to LBP tie.

Solution of the task

These The object is achieved by the provision of 2'-fluoro-pyrimidine-modified RNA aptamers specific to lipopolysaccharide-binding protein (LBP) and used as a drug for the treatment of sepsis as well as a simple optimized production method of these RNA aptamers, according to claims 9 to 13th

The Manufacturing process has the distinct advantage that it has a optimized combination of the selection matrices Ni-NTA agarose and Ni-NTA magnetic agarose beads. The selection takes place in 8 rounds of selection and is with Ni-NTA agarose in rounds 1-4, 7 and 8 and succeeded with Ni-NTA Magnetic Agarose Beads in rounds 5 & 6.

The selected aptamers are 2'-fluoro-pyrimidine modified and bind specifically to lipopolysaccharide binding protein (LBP). In the core region, they have a conserved single-stranded region bounded by 2 helices, with the 2 helices being part of a three-helix junction. Here, three-helix-junction refers to a region of the nucleic acid molecule in which 3 helices (P1-P3) meet together ( 3 ). The single-stranded region connecting helices P2 and P3 consists of 4 to 5 nucleotides that are part of the consensus sequences ( 1 gray-shaded areas) and which are exclusively 2'-fluoro-pyrimidine residues. The consensus sequence motifs according to which the aptamers in 1 are YG-CUUCY (Y: C or U) for group 1 and RUUUCY (R: A or G, Y: C or U) for group 2 ( 1 ). The helices P3 and P1 are linked by 0-3 single-stranded nucleotides, preferably 2'-fluoro-pyrimidine residues. Helices P1 and P2 are linked by 1-6 single-stranded nucleotides, preferably 2'-fluoro-pyrimidine residues. The described sequence and structure criteria describe the lead structure to be protected.

aptamers are according to the invention nucleic acids or nucleic acid derivatives (DNA and RNA equivalent), due to their structural peculiarities to a particular protein as a target molecule tie. To aptamers against nucleic acid-cleaving enzymes such as DNases and to stabilize RNases, corresponding modifications are made of the sugar phosphate backbone either before or after their selection. Unmodified aptamers be in biological fluids mined in seconds, stabilized by modifications Aptamers have a half-life of> 1 minute, generally> 1 hour, in biological sera.

The selected aptamers bind specifically to the murine lipopolysaccharide binding protein (mLBP), which has a key function in the context of the LPS-induced signaling cascade. The selected aptamers thus fulfill the requirements for testing for diagnostic and therapeutic effects in the treatment of septic shock and can be used as molecular tools in sepsis research.

object The invention also includes alleles or derivatives of the selected aptamers. Alleles are states of nucleic acids caused by mutations be converted into each other can. Mutations are understood to mean: (1) the translocation affine Partial sequences, (2) point mutations inside or outside one affine subsequence, (3) deletions inside or outside an affine subsequence. Derivatives means chemical derivatives, by separation, introduction or exchange of atoms or atomic groups of one or more nucleotides from a nucleic acid to be obtained. Also, the alleles or derivatives are against nucleic acid-cleaving Enzymes stabilize and bind specifically to lipopolysaccharide-binding protein (LBP). Furthermore, orthologous genes (eg for the human LBP) subject of the invention, since corresponding specific aptamers can also be provided according to the method set forth herein.

• a) Bereitstellung eines komplexen Gemisches von DNA-Sequenzvarianten (randomisierter Pool) durch chemische DNA-Synthese.
• b) Herstellung eines komplexen Gemisches von Transkript-Sequenzvarianten aus dem randomisierten DNA-Pool.
• c) Selektion auf 2'-Fluor-Pyrimidin-modifizierte RNA-Moleküle (2'-F-Y-RNA), die spezifisch das Lipopolysaccharid-bindende Protein (LBP) oder Teilsequenzen daraus binden.
• d) Amplifizierung der selektierten 2'-Fluor-Pyrimidin-modifizierte RNA-Moleküle (2'-F-Y-RNAs).

An aptamer according to the invention can be produced in an in vitro selection process by the following method steps:

• a) Provision of a complex mixture of DNA sequence variants (randomized pool) by chemical DNA synthesis.
• b) Preparation of a complex mixture of transcript sequence variants from the randomized DNA pool.
• c) Selection for 2'-fluoro-pyrimidine-modified RNA molecules (2'-FY-RNA) that specifically bind the lipopolysaccharide-binding protein (LBP) or partial sequences thereof.
• d) Amplification of the selected 2'-fluoro-pyrimidine-modified RNA molecules (2'-FY-RNAs).

The Methods of chemical DNA synthesis and reverse transcription, as used in steps a and b are those skilled in the art known and in laboratory standard works such as Sambrook, Fritsch, Maniatis, 1989, Molecular Cloning, CSH Laboratory Press, Cold Spring Harbor, NY. etc in detail described. The selection of 2'-F-Y RNA molecules, the specifically bind the mLBP or partial sequences thereof (step c), takes place for example by means of affinity chromatography. For this purpose is that with an N-terminal, 6 × His tagged mLBP on a columnar material immobilized, incubated with the randomized 2'-F-Y RNA pool and non-binding 2'-F-Y RNA molecules corresponding washing steps separated. Aptamers bound to the mLBP are eluted and collected. Advantageously, the aptamers before the selection of de- and renatured so that they exist in a reproducible conformation. It will be apparent to those skilled in the art that without further inventive benefits alternative methods of protein biochemistry for the selection of nucleic acids and Nucleic acid derivatives, which specifically bind the LBP or partial sequences thereof can be without the scope of the claims to leave.

The amplification of the selected RNA (step d) is carried out for example by means of PCR, wherein the 2'-FY RNA is first transcribed into DNA via reverse transcription. A portion of the DNA is ligated by standard methods in plasmids, cloned and sequenced. Evaluable sequences are first examined by means of an alignment program (eg Clustal X) to identify similarities at the primary structure level. Since such programs optimize the alignment over the entire sequence, the thus pre-grouped sequences are then examined for further similarities, ie on sequences of four or more identical nucleotides and moved in the text editor according to these similarities and sorted. This is an intellectually significant step in the process that allows grouping of aptamers into consensus sequence motifs ( 1 ). In parallel, secondary structural predictions are made for the aptamer sequences (eg with the program Mfold, Zuker, 2003) in order to evaluate the structural context of the consensus sequence motifs. It is necessary that these secondary structure predictions be evaluated by a nucleic acid expert, as alternative structural predictions for a sequence complicate the analysis and predicted structures may even be corrected in individual cases.

Plasmids encoding aptamers are transcribed using T7 RNA polymerase to obtain aptamers for functional studies. The stabilization of the aptamers against nucleic acid-cleaving enzymes such as DNases or RNases is realized by modifications that do not affect the affinity of the aptamers. These modifications are alternatively introduced before or after the selection. Preferably, the modification is that the 2'-hydroxyl group of the sugar group of one or more pyrimidine nucleotides is substituted by a fluoride, amino or methoxy group. The RNA aptamer according to the invention is distinguished from peptides by a very high affinity for lipopolysaccharide binding the protein (LBP).

In a preferred embodiment the aptamer is a modified RNA aptamer and specific for the lipopolysaccharide binding Protein (LBP) or partial sequences thereof.

Preferably, the selected aptamers in the core region have a conserved single-stranded region bounded by 2 helices, said 2 helices being part of a three-helix junction. Three-helix-junction here refers to a region of the nucleic acid molecule in which 3 helices (P1-P3) come together ( 3 ). The single-stranded region connecting helices P2 and P3 consists of 4 to 5 nucleotides that are part of the consensus sequences ( 1 gray-shaded areas) and which are exclusively 2'-fluoro-pyrimidine residues. The consensus sequence motifs according to which the aptamers in 1 are YGCUUCY (Y: C or U) for group 1 and RUUUCY (R: A or G, Y: C or U) for group 2 ( 1 ). The helices P3 and P1 are linked by 0-3 single-stranded nucleotides, preferably 2'-fluoro-pyrimidine residues. Helices P1 and P2 are linked by 1-6 single-stranded nucleotides, preferably 2'-fluoro-pyrimidine residues. The described sequence and structure criteria describe the lead structure to be protected. In detail, an aptamer according to the invention has properties that are described in the 1 - 3 are shown.

The Invention relates to the use of the RNA aptamers according to the invention, or a mixture of such RNA aptamers, for the preparation of a Drug for the treatment of patients with septic shock.

Of the The term patient refers equally to humans and vertebrates. This allows the drug in human and veterinary medicine be used. The therapeutically effective drug of the present Invention is presented to the patient as part of a pharmaceutically acceptable Composition either oral, rectal, parenterally intravenous, intramuscular or subcutaneous, intracisternal, intravaginal, intraperitoneal, intravascular, local (Powder, ointment or drops) or administered in spray form.

pharmaceutical acceptable compositions can which include modifications as salts, esters, amides and prodrugs, provided they are of a reliable medical grade Assess no excessive toxicity, irritation or cause allergic reactions in the patient.

Of the Term "prodrug" refers to Compounds that are transformed to improve uptake, such as by hydrolysis in the blood. Dosage forms for the local Administration of the vaccine of this invention include ointments, Powders, sprays or inhalants. The active component is used under sterile conditions, with a physiologically acceptable excipient and possible Preservatives, buffers or propellants, as appropriate, mixed.

execution

in the The invention will now be described in terms of experimental execution protocols explained in more detail.

Of the The term RNA in the following includes both unmodified RNA and 2'-fluoro pyrimidine-modified RNA. 2'-fluoro pyrimidine-modified RNA is abbreviated with 2'-F-RNA. The embodiment shows RNA aptamers that specifically bind the murine LBP. These results can without further inventive steps also transferred to humans become.

step a) Provision of a complex mixture of DNA sequence variants (randomized pool) by chemical DNA synthesis.

The synthetic pool of randomized DNA molecules contains a randomized region of 60 nucleotides and is flanked by two constant DNA regions of 20 nucleotides each:
5'-CCAAGCTTGCATGCCTGCAG (N) ₆₀ GGTACCGAGCTCGAATTCCC-3 '

It the synthesis method according to Klussmann et al., 1996 is used.

step b) Preparation of a complex mixture of transcript sequence variants from the randomized DNA pool.

The Producing a pool of randomized RNA from the randomized RNA DNA pool is made by a Auffüllreaktion with T7-Sequenase and subsequent T7 transcription. This replenishment reaction is carried out by a genetically modified form of T7 DNA polymerase (T7-Sequenase Version 2.0 DNA polymerase). Unlike the native ones T7 DNA polymerase has no 3'-5 'exonuclease activity.

• – Annealing des Primer A 5 min bei 90°C und Abkühlung der Probe bei Raumtemperatur für 10 min. (Primer A: 5'-TAATACGACTCACTATAGGGAATTCGAGCTCGGTACC-3')
• – Auffüllreaktion (2 h bei 37°C). Die beiden Schritte werden wie folgt wird in zwei separaten Ansätzen durchgeführt.

Annealing: 15,6 μl ssDNA-Pool (80 μM) 12,5 μl Primer A (100 μM) 100 μl Sequenase-Reaktionspuffer (5 x): 200 mM Tris/HCl pH 7,5, 100 mM Magnesiumchlorid, 250 mM Natriumchlorid 325,1 μl H₂O_bidest Auffüllreaktion (500 μl): 453,2 μl Annealing-Ansatz 25,0 μl DTT (100 mM) 18,8 μl dNTP-Mix (je 10 mM) 3 μl T7-Sequenase (13 U/μl) (Amersham) The sequence of the Auftrean reaction with T7-Sequenase is divided into the steps:

• Annealing of primer A for 5 minutes at 90 ° C. and cooling the sample at room temperature for 10 minutes. (Primer A: 5'-TAATACGACTCACTATAGGGAATTCGAGCTCGGTACC-3 ')
• - Replenishment reaction (2 h at 37 ° C). The two steps are performed as follows in two separate approaches.

annealing: 15.6 μl ssDNA pool (80 μM) 12.5 μl Primer A (100 μM) 100 μl Sequenase reaction buffer (5x): 200mM Tris / HCl pH 7.5, 100mM magnesium chloride, 250mM sodium chloride 325.1 μl H ₂ O _bidist Replenishment reaction (500 μl): 453.2 μl Annealing approach 25.0 μl DTT (100 mM) 18.8 μl dNTP mix (10 mM each) 3 μl T7 Sequenase (13 U / μl) (Amersham)

The Purification of the dsDNA is done with Microcon filter units. The purified dsDNA obtained is used in preparative T7 transcription.

T7 transcription allows the transcription of a DNA sequence into the complementary RNA sequence. For transcription by means of the DNA-dependent RNA polymerase isolated from bacteriophage T7, the DNA sequence must contain the T7 promoter. Upon detection, the polymerase begins to polymerize using the counterstrand to the promoter as a template. The transcription efficiency is greatest when the sequence of transcribed RNA begins with two guanosine residues. For the synthesis of modified RNA using 2'-fluoro-UTP and 2'-fluoro-CTP, a mutant (Y639F, Biozym Diagnostik GmbH c / o Epicenter, Hessisch-Oldendorf, Germany) is used instead of the normal T7 RNA polymerase which has a phenylalanine (Y639F) in place of the tyrosine at position 639 (Sousa & Padilla, 1995). While the wild-type T7 RNA polymerase incorporates rNTPs 70-80-fold better than dNTPs, mutant rNTPs are only 4-fold more preferred. 2'-fluoro-pyrimidine nucleotides are thus incorporated much better in the transcription than by the wild-type polymerase (Huang et al., 1997). To prepare the 2'-F-RNA start pools for the corresponding initial selection round, a reaction volume of 750 μl (2 batches of 375 μl) with a total of 2.4 nmol DNA template (from preparative T7 Sequenase reaction) is selected. The use of [α- ³² P] -ATP in a reaction mixture allows the radioluminographic detection of the product in the polyacrylamide gel.

The reaction time is 3-4 h at 37 ° C; transcription is followed by ethanol precipitation followed by purification of the 2'-F RNA via a 10% denaturing polyacrylamide gel. Batch (375 μl): 38.5 μl dsDNA template (32 μM) 37.5 μl 7-transcription buffer (10x): 400 mM Tris / HCl pH 8.0, 200 mM magnesium chloride, 10 mM spermidine, 0.1% ^Triton® X-100] 18.75 μl DTT (100 mM) 46.86 μl 2'-F-YTP mix (10 mM each) [Y = C, U] 46.86 μl 2'-OH-RTP mix (10 mM each) [R = A, G] 6 μl [α- ³² P] -ATP (370 mBg / ml) 9.43 μl T7 RNA polymerase (Y639F) (3.7 μg / μl) 171.06 μl H ₂ O _bidist

The Isolation of the T7 transcripts is carried out by radioluminographic Detection in the gel by excision of the corresponding gel region and "crush & soak" elution followed by ethanol precipitation Amount of activity will over Cerenkov counting and the yield by absorbance measurement of the nucleic acid solution 260 nm determined in the photometer.

step c) Selection for 2'-fluoro-pyrimidine-modified RNA molecules (2'-F-Y RNA) specific the lipopolysaccharide binding protein (LBP) or partial sequences tie from it.

Selection round no. 1: chemically synthesized template DNA (NOXXON Pharma AG, Berlin) is hybridized in a preparative T7 Sequenase reaction with a hanging, the T7 promoter bearing primer A and converted into complete double-stranded DNA.
(Primer A: 5'-TAATACGACTCACTATAGGGAATTCGAGCTCGGTACC-3 ')

The resulting template DNA is transcribed preparatively. About 553 pmol of 2'-fluoropyrimidine-modified RNA (2'-F-RNA), ie 3.3 × 10 ¹⁴ molecules, are used in the first round of selection.

Before the selection round, 80 μl of Ni-NTA agarose suspension for the coupling of mLBP-Hiss (biometec, Greifswald) are washed four times with 2 × 400 μl _bidistilled H ₂ O and 2 × 400 μl binding buffer (PBSM) (140 mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 1.76mM KH ₂ PO ₄ , 2mM MgCl ₂ ). To separate the agarose from the solution is centrifuged for 10 s at 13000 rpm in a table joint and the supernatant carefully removed. After the last washing step, the agarose is resuspended in 360 μl PBSM and mixed with 40 μl mLBP-His ₆ (40 μg). The coupling to the Ni-NTA matrix is carried out for 45 minutes with occasional gentle mixing at room temperature. Subsequently, the agarose is pelleted at 1000 rpm for 5 min in a table joint and washed after removal of the supernatant 2 × with 400 ul PBSM and finally taken up in 160 ul PBSM.

The 2'-F-RNA start pool (553 pmol) is adjusted to a concentration of 2.9 μM in 187 μl PBSM, denatured for 4 min at 90 ° C and cooled to room temperature within 20 min. Before selection, pre-selection is also performed by incubating the 2'-F-RNA start pool with equilibrated Ni-NTA agarose (50 μl) for 20 min at 37 ° C in a 1 ml column (Mobicols, MoBiTec). Subsequently, the solution is centrifuged with the not binding to the Ni-NTA agarose 2'-F-RNA molecules in a reaction vessel, transferred to a second reaction vessel with about 200 ul agarose-coupled mLBP-His ₆ and for 1 h at 37 ° C incubated. After washing the Ni-NTA agarose twice with 200 .mu.l of PBSM and centrifuging for 5 min at 1000 rpm (tabletop centrifuge), the elution of the bound 2'-F-RNA molecules with 200 .mu.l elution buffer (140 mM NaCl, 2.7 mM KCl, 10mM Na ₂ HPO ₄ , 1.76mM KH ₂ PO ₄ , 2mM MgCl ₂ , 300mM imidazole, 7M urea) over 1h at 37 ° C with occasional shaking. From the eluate, the 2'-F-RNA is isolated by phenol-chloroform extraction and ethanol precipitation. The pre-selection agarose, the supernatants of the secondary washing steps and the eluted 2'-F-RNA are quantified by Cerenkov counting. The eluted 2'-F-RNA is reverse transcribed and the ssDNA after previous T7-Sequenase reaction amplified by PCR. This is followed by T7 transcription.

Selection rounds No. 2-8. In all further rounds of selection, murine (m) LBP-His ₆ (0.08, 0.16 or 0.32 nmol) is added to a corresponding amount of equilibrated Ni-NTA agarose (20 μl) (laps 2-4, 7 and 8). or Ni-NTA Magnetic Beads (20 or 40 μl) (rounds 5 and 6). The 2'-F-RNA mixtures (0.048-0.25 nmol) are dissolved in 100 μl of PBSM prior to the respective preselection step and de- and renatured as described above. For rounds 5 and 6, 20 μL or 40 μL Ni-NTA Magnetic Beads Suspension for coupling mLBP-His ₆ (biometec) are equilibrated by washing four times with 2 x 400 μL _bidistilled H ₂ O and 2 x 400 μL PBSM. To separate the beads from the solution, the reaction vessel is ge in a 12-tube magnet and the supernatant removed after 1 min. After the last washing step, the beads are mixed with 5 μg (round 5) or 10 μg (round 6) mLBP-His ₆ in a total volume of 100 μl or 200 μl PBSM. The coupling to the Ni-NTA matrix is carried out for 45 minutes with occasional gentle mixing at room temperature. Subsequently, the beads are washed before selection with 200 ul PBSM.

To a preselection step (for rounds with Ni-NTA Magnetic Agarose Beads according to) the selections are made in a volume of 100-300 μl. Washed and eluted as previously described.

The Table shows the selection parameters of selection with Ni-NTA agarose, the concentration ratios 2'-F-RNA: mLBP each before the preselection step, ie without consideration of 2'-F-RNA molecules, the removed from the pool by binding to the matrix become.

d) Amplification of the selected 2'-fluoro-pyrimidine-modified RNA molecules (2'-F-Y-RNAs)

To is a reverse transcription of the selected RNA with subsequent T7-Sequenase Auffüllreaktion and amplification performed by PCR.

RNA is using an RNA-dependent DNA polymerase (reverse transcriptase) primer-dependent (primer B: 5'-CCAAGCTTGCATGCCTGCAG-3 ') into the complementary DNA counterstrand rewritten.

For this purpose the SuperScript ^™ RNase H is, for example, ^- reverse transcriptase is used, the more DNA copies of producing an RNA template. It is originally from the Moloney murine leukemia virus and is provided with an RNase H deletion.

• – Annealing des Primers (3 min bei 90°C)
• – Abkühlung der Probe bei Raumtemperatur für 10–15 min (dann auf Eis) und
• – Reverse Transkription (60 min bei 42°C).

The process of reverse transcription is divided into the steps:

• Annealing of the primer (3 min at 90 ° C)
• - Cool the sample at room temperature for 10-15 min (then on ice) and
• - reverse transcription (60 min at 42 ° C).

To the reaction, the enzyme by incubation of the mixture for 10 min at 70 ° C inactivated.

(x = 4,4 μl in den Runden 1, 3 & 5; 4,8 μl in Runde 2; 8,8 μl in den Runden 4, 6 & 7; 1,54 μl in Runde 8)The primer is added to RNA at least in 1.1-fold excess.

(x = 4.4 μl in rounds 1, 3 &5; 4.8 μl in round 2; 8.8 μl in rounds 4, 6 &7; 1.54 μl in round 8)

After reverse transcription, the RNA is hydrolyzed by addition of 5.5 μl of a 2 M NaOH solution and allowed to incubate at 37 ° C for 1 h. The cDNAs are isolated by ethanol precipitation and resuspended in 20 _ul H ₂ O _bidest .

The isolated after a reverse transcription ssDNA fragments are coated with an overhanging primer
(Primer A: 5'-TAATACGACTCACTATAGGGAATTCGAGCTCGGTACC-3 ')
hybridized and filled in the T7 Sequenase reaction (see step b) to a complete duplex. The primer is added at least 1.1 times excess to the DNA. annealing: 20.0 μl DNA (from reverse transcription) x μl Primer A (25 μM) 20.0 μl Sequenase reaction buffer (5x): 200mM Tris / HCl pH 7.5, 100mM magnesium chloride, 250mM sodium chloride ad 90.7 μl H ₂ O _bidist (x = 8.8 μl in rounds 1, 3, 9.6 μl in round 2, 17.6 μl in rounds 4, 6, 7, 5.27 μl in round 8) fill reaction (100 μl) : 90.7 μl Annealing approach 5.0 μl DTT (100 mM) 3.75 μl dNTP mix (10 mM each) 0.6 μl T7 Sequenase (13 U / μl)

The Purification of the dsDNA is done with Microcon filter units.

The PCR (Polymerase Chain Reaction) is used for example with DNA polymerase performed from the thermophilic bacterium Thermus aquaficus (Tap) and analyzed using an agarose gel.

When DNA template, the dsDNA from the Sequenase reactions are used. A maximum of 25 cycles are carried out and after every 5, 10, 15, 20 and 25 cycles of samples (10 μl) analyzed this approach on an agarose gel.

After determining the number of cycles with which most copies of the dsDNA template and, if possible, no artifacts are achieved, the remaining dsDNA is preparatively amplified in appropriate PCR approaches. Standard approach: x μl dsDNA template (12.5 nM final conc. per run in rounds 1,2,3 &5; 25 nM in rounds 4, 6, 7 & 8) 10.0 μl Tap buffer (10x): 100 mM Tris / HCl pH 8.8; 500 mM KCl; 0.8% Nonidet P 40 8.0 μl MgCl ₂ (25 mM) 2.0 μl Primer C (25 mM) [5'-TAATACGACTCACTATAG] 2.0 μl Primer B (25 mM) [5'-CCAAGCTTGCATGCCTGCAG] 2.0 μl dNTP mix (10 mM each) 0.5 μl Taq polymerase (5 U / μl) ad 100 μl with H ₂ 0 _redistilled , _top up

Temperature conditions:

• 95 ° C / 0.5 min (denaturation at the beginning)
• 95 ° C / 1.0 min (denaturation)
• 53 ° C / 2.0 min (annealing)
• 72 ° C / 1.0 min (extension)
• 12-17 cycles

General Techniques and sub-procedures

The Purification of nucleic acids is carried out by standard methods of gel electrophoresis. It will be 8-15% Gels with added 8 M urea used. The preparative Purification of T7 products and 5'-labeled RNA is 10% Gelen, the analysis of lead ion-induced and enzymatic RNA cleavage on 10 and 20% gels (each with 8 M urea).

The Examination of PCR products after reverse transcription and after amplification of insert sequences from cloning vectors and the analysis of plasmid DNA from minipreparations done with the Agarose gel electrophoresis.

Nucleic acid bands in polyacrylamide and agarose gels are stained by standard ethidium bromide staining and visualized under UV light. Oligonucleotides are detected due to their high light absorption in gels with UV shadowing. The gel is wrapped in cellophane film and placed on a TLC plate (TLC aluminum foil, silica gel 60 F ₂₅₄ (Merck)) impregnated with fluorescent dye. By irradiating UV light (254 nm), the nucleic acids cast a dark shadow on the silica gel plate. This method is very insensitive and is therefore used exclusively for preparative purposes. The detection of radioactively (eg ³² P) labeled nucleic acids is carried out by means of radioluminography, using a bio-imaging image plate and the bio-imaging analyzer BAS 1000 (Fujifilm). This method is used for the detection of gel-purified, internally ³² P-labeled RNA after T7 transcription or 5'-labeled RNA, as well as for the evaluation of binding assays and cleavage for structure elucidation.

at Polyacrylamide gels become one of the two after electrophoresis Glass plates removed and the gel with the other plate in cellophane wrap taken. For orientation also radioactive markings at the edges applied to the gel. Exposure of the bioimaging image plate takes place for 1-10 minutes or over Night, dependent from the amount of radioactivity applied to the gel.

Nitrocellulose filters for the determination of dissociation constants are fixed on Whatman papers and also wrapped in cellophane film. A bio-imaging plate is exposed overnight and the readout of the data is done as for gels with the Bio-Imaging Analyzer, the digital data analysis and processing followed by appropriate software (PCBAS Version 2.09). The elution of nucleic acids takes place by means of diffusion elution or electroelution. Oligonucleotides separated by denaturing polyacrylamide gel electrophoresis are isolated by means of diffusion elution. For this purpose, the bands of the radiolabelled nucleic acids are first excised from the gel with a 1: 1 print of an imaging image as a template. The nucleic acids in the gel piece are either eluted overnight at 4 ° C. with shaking in 400 μl of 1 M NaOAc pH 4.7 per cm ² piece of gel (or smaller) or by means of the "crush &soak" method 1 ml of ethanol precipitated.

to execution the "crush & soak" method becomes that cut out gel pieces crushed with a sterile pipette tip on the reaction vessel wall. After addition of 400 .mu.l 1 M NaOAc pH 4.7, the reaction vessel is a total of three times in liquid nitrogen frozen and thawed on ice again. Then done the actual elution at 4 ° C for 4-6 h under Shake. The eluate is then prepared by a siliconized glass wool Reaction vessel for separation centrifuged from gel matrix and precipitated with ethanol.

through Phenol and chloroform extraction removes proteins from aqueous nucleic acid solutions become. The removal of proteins takes place z. B. after dephosphorylation of RNA.

After addition of 1 volume of phenol (0.1 M Tris buffered to pH 7.6) to the aqueous solution, shaking vigorously (vortex) for 1 minute, centrifuging and adding 1 volume of chloroform. After vortexing again, centrifuge for 1 to 5 minutes at 13000 rpm (tabletop centrifuge) and room temperature. The upper aqueous phase is removed and the phenolic extracted with 100 ul of water. The combined aqueous phases are extracted with 2 volumes of chloroform. The precipitation of the nucleic acids, in particular the precipitation of PCR products and synthesis products of the reverse transcription from aqueous solutions, takes place by ethanol precipitation. To reduce the coprecipitation of nucleoside triphosphates while ammonium acetate is used. Precipitation with ammonium acetate: x μl nucleic acid solution 1/2 x Vol Ammonium acetate (10 M) 2.5 x vol Ethanol (96%)

For very small amounts of nucleic acid and short-chain nucleic acids, the aqueous solution is adjusted to 20 ug / ml glycogen as a precipitation aid. The RNA eluted after each round of selection is also preferably precipitated in the presence of glycogen. It is precipitated for at least 1 h at -20 ° C and the nucleic acids by a subsequent, at least 30 minutes centrifugation at 13000 rpm / 4 ° C (Heraeus, Biofuge 15R) pelleted; the supernatant is then removed and discarded. The pellets are washed with about 130 μl of ice-cooled 70% ethanol and again centrifuged for 10-15 min at 13000 rpm and 4 ° C. The supernatant is discarded and the pellet dried at room temperature and taken up in H ₂ O _bidistilled or binding buffer (PBSM).

The Concentration and desalting of protein and nucleic acid solutions takes place using Microcon filter units (Millipore). For the purification of DNA after the T7 Sequenase reaction and after the preparative PCR are filter units of type YM-30 used (retention limit for double-stranded nucleic acids: approx. 50 base pairs; For single nucleic Acids: about 60 nucleotides).

A maximum of 500 μl nucleic acid solution is pipetted into the sample reservoir of a filter unit. The subsequent centrifugation at 11000 rpm (Heraeus, Biofuge 13) for 15 min, the solution is forced through the membrane pores, wherein those nucleic acids that exceed the retention limit due to their size, are retained on the membrane. The membrane is then washed twice with 10o .mu.l H ₂ O _bidest and washed by appropriate centrifugation. For elution, 20 μl (after T7-Sequenase reaction) or 25 _.mu.l H ₂ O _bidistilled (after preparative PCR) pipetted onto the membrane of the sample reservoir and allowed to stand for 10 min at room temperature. The sample reservoir containing the purified nucleic acid is then placed upside down in a sterile reaction tube and the nucleic acid solution is collected by centrifugation at 4000 rpm (Heraeus, Biofuge 13) for 5 min. The elution _{step is} repeated during the purification of PCR products with 25 μl H ₂ O _bidest .

The PCR (Polymerase Chain Reaction), for example, with the DNA polymerase from the thermophilic bacterium Thermus aquaticus (Taq) analyzed using an agarose gel.

When DNA template, the dsDNAs from the Sequenase reactions are used. A maximum of 25 cycles are carried out and after every 5, 10, 15, 20 and 25 cycles of samples (10 μl) analyzed this approach on an agarose gel.

After determining the number of cycles with which most copies of the dsDNA template and, if possible, no artifacts are achieved, the remaining dsDNA is preparatively amplified in appropriate PCR approaches. Standard approach: x μl dsDNA template (12.5 nM) 10.0 μl Taq-Pufter (10x) 8.0 μl MgCl ₂ (25 mM) 2.0 μl Primer C (25 mM) 2.0 μl Primer B (25 mM) 2.0 μl dNTP mix (10 mM each) 0.5 μl Taq polymerase (5 U / μl) ad 100 μl fill up with H ₂ O _bidist

Temperature conditions:

• 95 ° C / 0.5 min (denaturation at the beginning)
• 95 ° C / 1.0 min (denaturation)
• 53 ° C / 2.0 min (annealing)
• 72 ° C / 1.0 min (extension)
• 5-25 cycles

to Amplification of insert sequences from a cloning vector for subsequent T7 transcription becomes a preparative PCR (from 5-15 individual approaches consisting) carried out with previously linearized plasmids, thereby For example, a DNA template amount of 1 ng per batch is used, the number of cycles is 35th

The T7 transcriptions as part of the selection rounds for the generation of 2'-F-RNA pool is carried out with reaction mixtures of 150-190 .mu.l and 0.15-0.5 nmol DNA template (from preparative PCR). To prepare the 2'-F-RNA start pools for the corresponding initial round of selection, a reaction volume of 750 μl (2 batches of 375 μl) with a total of 2.4 nmol DNA template (from preparative T7 sequence reaction) is selected. The use of [α- ³² P] -ATP in a reaction mixture allows the radioluminographic detection of the product in the polyacrylamide gel.

The reaction time is 3-4 h at 37 ° C; transcription is followed by ethanol precipitation followed by purification of the 2'-F RNA via a 10% denaturing polyacrylamide gel. Batch (ex. 150 μl): x μl DNA template 15.0 μl T7 transcription buffer (10x): 400 mM Tris / HCl pH 8.0, 200 mM magnesium chloride, 10 mM spermidine, 0.1% ^Triton® X-100] 7.5 μl DTT (100 mM) 18.75 μl 2'-F-YTP mix (10 mM each) [Y = C, U] 18.75 μl 2'-OH-RTP mix (10 mM each) [R = A, G] 3.0 μl [α- ³² P] -ATP (370 mBg / ml) 3.77 μl T7 RNA polymerase (Y639F) (3.7 μg / μl) y μl H ₂ O _bidist

The Isolation of the T7 transcripts is carried out by radioluminographic Detection in the gel by excision of the corresponding gel region and "crush & soak" elution followed by ethanol precipitation Amount of activity will over Cerenkov counting and the yield by absorbance measurement of the nucleic acid solution 260 nm determined in the photometer.

2'-fluoro-pyrimidine RNA aptamers

to Generation of non-radioactively labeled 2'-F-RNA aptamers becomes a reaction volume of 300 μl with 0.37 nmol DNA template was selected. The reaction time is 4 h at 37 ° C; after subsequent ethanol precipitation and gel purification becomes the 2'-F RNA detected by UV shadowing and isolated from the corresponding gel piece by means of "crush & soak" elution. This is followed by an ethanol precipitation.

Unmodified RNA

In order to obtain a further length marker for cleavage analyzes, an unmodified comparison RNA of the same sequence is prepared from a 2'-F-RNA aptamer to be analyzed by means of T7 transcription using the wild-type enzyme. To generate free 5'-hydroxyl groups for a radioactive label at the 5'-end so-called starter nucleotides are used. The prerequisite is that the last nucleotide of the initiator is complementary to the first nucleotide of the template. According to the invention, a dinucleotide serves as a starter, the 5'-ApG. It is used in quadruplicate to GTP to promote the initiation of transcription with 5'-ApG instead of 5'-GTP. The amount of DNA template in the batch is 30 pmol. After a reaction time of 3 h at 37 ° C, the purification and isolation as described for 2'-fluoro-pyrimidine RNA aptamers described. approach x μl DNA template 10.0 μl T7 transcription buffer (5x): 200mM Tris / HCl pH 7.9, 30mM Magne sodium chloride, 50 mM DTT, 50 mM sodium chloride, 10 mM spermidine 4.0 μl NTP mix (25 mM each) 6.3 μl ApG (63.65 mM) 3.77 μl T7 RNA polymerase (200 U / μl) ad 50 μl H ₂ O _bidist

RNA is using an RNA-dependent DNA polymerase (reverse transcriptase) primer-dependent (primer B: 5'-CCAAGCTTGCATGCCTGCAG-3 ') into the complementary DNA counterstrand rewritten.

For this purpose the SuperScript ^™ RNase H is, for example, ^- reverse transcriptase is used, the more DNA copies of producing an RNA template. It is originally from the Moloney murine leukemia virus and is provided with an RNase H deletion.

Of the The process of reverse transcription is divided into the steps Annealing of the primer (3 min at 90 ° C), cooling of the sample at room temperature for 10-15 min (then on ice) and reverse transcription (60 min at 42 ° C). To the reaction, the enzyme by incubation of the mixture for 10 min at 70 ° C inactivated. The primer becomes at least 1.1-fold excess RNA used.

After reverse transcription, the RNA is hydrolyzed by addition of 5.5 μl of a 2 M NaOH solution and allowed to incubate at 37 ° C for 1 h. The cDNAs are isolated by ethanol precipitation and resuspended in 20 _ul H ₂ O _bidest .

The isolated after a reverse transcription ssDNA fragments are coated with an overhanging primer
(Primer A: 5'-TAATACGACTCACTATAGGGAATTCGAGCTCGGTACC-3 ')
hybridized and filled in the T7 Sequenase reaction to a complete duplex. The overhang of the primer contains the promoter necessary for T7 transcription. This replenishment reaction is carried out by a genetically modified form of T7 DNA polymerase (T7-Sequenase Version 2.0 DNA polymerase). Unlike the native T7 DNA polymerase, it has no 3'-5 'exonuclease activity.

The sequence of the replenishment reaction with T7-Sequenase is divided into the steps Annealing of the primer (5 min at 90 ° C), cooling of the sample at room temperature for 10 min and Auffüllreaktion (2 h at 37 ° C). The primer is used at least in 1.1-fold excess to the DNA. The purification of the dsDNA is carried out with Microcon filter units. annealing: 20.0 μl DNA (from reverse transcription) x μl Primer A (25 μM) 20.0 μl Sequenase reaction buffer (5x): 200mM Tris / HCl pH 7.5, 100 mM magnesium chloride, 250 mM sodium chloride ad 90.7 μl H ₂ O _bidist Replenishment reaction (100 μl): 90.7 μl Annealing approach 5.0 μl DTT (100 mM) 3.75 μl dNTP mix (10 mM each) 0.6 μl T7 Sequenase (13 U / μl)

The Reaction becomes additional in the preparative scale (2 × 500 μl) for the DNA template of the start pool of Selection using 1.2 nmol of chemically synthesized ssDNA performed.

All clonings are preferably carried out by means of TOPO TA ^Cloning® kits from Invitrogen in a one-step procedure according to the manufacturer's instructions. PCR products generated by Taq polymerase are directly incorporated into a cloning vector by means of a topoisomerase without prior purification.

topoisomerase is over a phosphotyrosine bond covalently bound to the respective 5 'end of the vector. The preserved Phosphodiester bond energy allows ligation of the vector DNA with PCR product to release the topoisomerase. The tap polymerase has a sequence-independent transferase activity, whereby it attaches a single deoxyadenosine to the 3 'end of PCR products. Of the In contrast, linearized vector has an overhanging deoxythymidine at the 3 'end.

For DNA ligation, the amplified PCR products are directly used in the cloning reaction with the cloning vector (pCR ^® 2.1-TOPO ^® vector). The mixture is incubated for 30 min at room temperature and then placed on ice. Reaction: 4 μl PCR product 1 μl Salt solution 1 μl TOPO ^® vector

The transformation is carried out according to standard methods, for example with chemically competent E. coli cells (One ^Shot® TOP 10). For this purpose, 6 μl of the cloning reaction are pipetted into 50 μl TOP 10 cells and mixed gently. The cells are incubated for 30 min on ice and then for 30 s at 42 ° C (heat shock). After 2 min on ice 250 .mu.l SOC medium are added and shaken for 1 h at 37 ° C and 200 rpm in a hot air shaker. Aliquots of 50, 10 and 150 μl of the transformation approach are per each an agar plate containing antibiotics and X-GAL b and incubated overnight at 37 ° C.

The Analytical plasmid isolation (plasmid minipreparation) is according to manufacturer's instructions performed by Qiagen. The grown after the transformation on the agar plates clones are picked using sterile toothpicks and either on master plates or in each case 3 ml antibiotic-containing LB medium inoculated, wherein the multiplication in liquid medium overnight at 37 ° C in a hot air shaker takes place at 220 rpm. Each 0.5 ml of a culture are with 0.5 ml of autoclaved glycerol (86%) mixed in liquid nitrogen frozen and at -80 ° C as Glycerinkultur stored.

The cells in the remaining 2.5 ml are pelleted at about 13000 rpm for 20 s in a bench centrifuge. The pellet is resuspended in 300 μl of RNase A-containing P1 buffer. After addition of 300 μl of P2 buffer, gentle mixing and incubation for 5 minutes at room temperature, 300 μl of ice-cooled 300 mM potassium acetate (pH 5.5) are precipitated on ice for 5 min and 10 min at 13000 rpm (Heraeus, Biofuge 13) centrifuged. The supernatant is added to a tip-20 column (Qiagen) equilibrated with 1 ml QBT buffer. After washing the column four times with 1 ml of QC buffer, it is eluted with 0.8 ml of QF buffer. The eluted DNA is precipitated with 0.56 ml of isopropanol, centrifuged for 30 min at 13000 rpm and the precipitated plasmid DNA in 50 _{.mu.l of} H ₂ O _redistested resuspended.

Restriction endonucleases cleave DNA to specific sequences. This is used to linearise cloning vectors containing an insert sequence to be examined. For example, in a batch containing 1.5 to 3.0 μg of DNA, Apa I is cut with the restriction enzyme. The cleavage takes place for 2 h at 30 ° C and includes enzyme activation for 20 min at 60 ° C. The linearized DNA is concentrated by ethanol precipitation and resuspended in 20 μl H ₂ O _bidistilled . Restriction mixture: x μl Cloning vector with insert 2 μl Apa I buffer (10x): 100 mM Tris / HCl pH 7.5; 100 mM MgCl 2; 1 mg / ml BSA 1 μl Apa I (10 U / μl) ad 20 μl H ₂ O _bidist

The dephosphorylation reaction is carried out with an alkaline phosphatase (from calf intestine). Alkaline phosphatase catalyzes the hydrolysis of 5'-phosphate residues of DNA, RNA, and ribo- and deoxyribonucleoside triphosphates. Dephosphorylated RNA generated by T7 transcription for the purpose of a subsequent radioactive 5 'label. 20 pmol of RNA are dephosphorylated per batch. The reaction is incubated for 30 min at 37 ° C. The purification of the RNA molecules takes place by means of phenol / chloroform extraction and subsequent ethanol precipitation. Reaction: x μl RNA 5 μl CIAP buffer (10x): 100 mM Tris / HCl pH 7.5; 100 mM MgCl 2 1 μl alkaline phosphatase (1 U / μl) ad 50 μl H ₂ O _bidist

The dephosphorylated and purified RNA is resuspended in 10 μl H ₂ O _bidist .

The polynucleotide kinase of E. coli phage T4 catalyzes the transfer of the γ-phosphate of an ATP molecule to a free 5 'end of DNA or RNA. The γ-phosphate is thereby esterified with the 5'-hydroxyl end of the oligonucleotide. About 10 pmol of RNA are used per batch. The reaction is incubated for 1 h at 37 ° C. After the 5'-phosphorylation, an ethanol precipitation takes place, after which the labeled oligonucleotide is purified via a 10% denaturing polyacrylamide gel. The isolation of the labeled oligonucleotide takes place after the radioluminographic detection in the gel by gel elution with subsequent ethanol precipitation. Subsequently, the amount of activity is determined by Cerenkov counting. Reaction: x μl RNA 2 μl Kinase Buffer (10x): 500mM Tris / HCl pH 7.6; 100 mM MgCl 2; 50 mM DTT; 1 mM spermidine; 1mM EDTA 3 μl [γ- ³² P] -ATP (370 mBq / ml) 1 μl Polynucleotide kinase (10 U / μl) ad 20 μl H ₂ O _bidist

The RNA ligase of E. coli phage T4 catalyzes the ATP-dependent formation of a phosphodiester bond between a 5'-phosphate and a 3'-hydroxyl terminus of single-stranded RNA or DNA. The smallest possible substrate is a nucleoside 3'-5'-bisphosphate. About 10 pmol of RNA are used per batch. After incubation overnight at 4 ° C, the RNA is purified via a 15% denaturing polyacrylamide gel. Reaction: x μl RNA 1.2 μl T4 RNA ligase buffer (10x): 500mM Hepes / NaOH pH 8.0; 100 mM MgCl 2; 100 mM DTT 3.0 μl ATP (50 μM) 3.0 μl [5'-3 ² P] pCp (370 mBq / ml) 1.0 μl T4 RNA ligase (20 U / μl) ad 12 μl H ₂ O _bidist

specific Cleavages by ribonucleases provide information about secondary structures as well as accessible Regions in ribonucleic acids. The endonucleases used produce either 5'-hydroxyl and 2 ', 3'-cyclophosphate termini or 5'-phosphate and 3'-hydroxyl termini. In addition to these enzymatic cleavages are also chemically induced Splits possible, and both give information about that secondary structure the investigated RNA (Ehresmann et al., 1987, Giegé et al. 1999).

For the cleavage of aptamers both native and denaturing conditions are used. In order to obtain native conformations as possible, the 5'-labeled RNA to be characterized is denatured and refolded before the cleavage analyzes in binding buffer (PBSM). For this purpose, the RNA solution is diluted with 2-fold concentrated binding _buffer and H ₂ O _bidistilled to a volume of 20 _ul and then denatured for 3 min at 90 ° C. Subsequently, a cooling takes place at room temperature for 20 min. The renatured RNA molecules are used for both denaturing and native cleavage. Approach: (Σ 20 μl) x μl RNA (~ 1-3 x 10 ⁶ cpm) y μl H ₂ O _bidist 10 μl 2x Binding Buffer (2xPBSM): 280mM NaCl, 5.4mM KCl, 20mM Na ₂ HPO ₄ , 3.52mM KH ₂ PO ₄ , 4mM MgCl ₂ )

For RNA cleavage Ribonuclease (RNase) T1 from Aspergillus orizae is used Endoribonuclease specifically neighboring phosphodiester bonds to the 3'-phosphate of guanosine nucleotides of an RNA cleaves (GP ↓ Np). RNase T1 cleaves both under native as well as denaturing (~ 4.3 M urea) conditions and allows together with the partial alkaline hydrolysis the assignment of fission products. The denaturing cleavage takes place for 30 min at 55 ° C, the native for 20 min at room temperature. Both reactions are mixed with 7 μl RNA sample buffer (2.3 M urea; 1 × TBE; 66% [v / v] formamide; each 0.05% [w / v] bromophenol blue and xylene cyanol) Stopped with EDTA (120mM) stopped.

RNase V1 (Cobra Venom) cleaves double-stranded regions or regions with pronounced base stacking, independent of sequence. Fission products are formed with a 5'-phosphate end. To identify helical regions, cleavage under native conditions is performed with RNase V1. The incubation of the reaction mixture is carried out as in the cleavage by RNase T1 under native conditions for 20 min at room temperature. native cleavage: 1.0 μl RNA (4 - 15 x 10 ⁴ cpm) in PBSM 0.5 μl tRNA (5 μg / μl) 3.5 μl Binding buffer (PBSM) 1.0 μl RNase V1 (0.01, 0.05 U / μl)

Aspergillus orizae nuclease S1 is a single-stranded endonuclease which is stimulated by zinc ions (Np ↓ Np). Fission products are formed with a 5'-phosphate end. The cleavages are also carried out under native conditions. The incubation of the reaction mixture is carried out for 20 min at room temperature. The reaction is stopped with 7 μl RNA sample buffer spiked with EDTA (120 mM). native cleavage: 1.0 μl RNA (4 - 15 x 10 ⁴ cpm) in PBSM 0.5 μl tRNA (5 μg / μl) 2.5 μl Binding buffer (PBSM) 1.0 μl ZnSO ₄ (27 mM) 1.0 μl S1 nuclease (1 U / μl)

nuclease P1 from Penicillium citrinum, like the nuclease S1, is a single strand specific Endonuclease and is also activated by zinc ions (Np ↓ Np).

It Fission products are formed with a 5'-phosphate end. The divisions will be performed under native conditions.

The incubation of the reaction mixture is carried out for 20 min at room temperature. The reaction is stopped with 7 μl RNA sample buffer spiked with EDTA (120 mM). native cleavage: 1.0 μl RNA (4 - 15 x 10 ⁴ cpm) in PBSM 0.5 μl tRNA (5 μg / μl) 2.7 μl Binding buffer (PBSM) 0.8 μl ZnSO ₄ (3 mM) 1.0 μl P1 nuclease (0.01, 0.05 U / μl)

For the detection of single-stranded or flexible regions in RNA molecules, lead-induced cleavages are performed. In contrast to the cleavage with nuclease S1, P1 and V1, fragments with 5'-hydroxyl and 2 ', 3'-cyclophosphate ends are formed. The reaction mixture is incubated for 10 min at 37 ° C and the Re reaction with 7 μl of RNA sample buffer mixed with EDTA (120 mM). Lead fission: 1.0 μl RNA (4 - 15 x 10 ⁴ cpm) in PBSM 0.3 μl tRNA (5 μg / μl) 1.0 μl Pb ²⁺ cleavage buffer (5x): 250 mM Tris / HCl pH 7.0; 0.5 M Mg (OAc) ₂ ; 0.5 M NH ₄ OAc 1.7 μl H ₂ O _bidist 1.0 μl Pb acetate (100 mM)

All splitting approaches after stopping with RNA sample buffer in liquid nitrogen frozen and displayed on denaturing polyacrylamide gels.

Under alkaline conditions it is possible ribonucleic acids to form 2 ', 3'-cyclophosphates to hydrolyze, with the cleavages statistically at each position arise. You get by electrophoresis on a denaturing polyacrylamide gel a so-called alkaline conductor, on the basis of which, together with the RNase T1 conductor under denaturing conditions, the assignment of fission products is possible.

The Hydrolysis of RNA molecules done by two methods. For For general cleavage analyzes, a NaOH-containing solution is used. The reaction is done with 5 μl RNA sample buffer stopped, in liquid nitrogen frozen and applied to denaturing polyacrylamide gels.

For boundary studies, both 5'- and 3'-labeled RNA are hydrolyzed under less strongly alkaline conditions with NaHCO ₃ , and the fragments are then concentrated by ethanol precipitation. The RNA fragments are resuspended in 40 μl of binding buffer

mit [Proteinfrai] = [Proteingesamt] – [Protein-RNA] = [Proteingesamt] – [Proteingebunden] [RNAfrei] = [RNAgesamt] – [RNAgebunden]
und [Protein-RNA] = [RNAgebunden] [RNAgebunden] = [RNAgebunden] (1:1 Stöchiometrie vorausgesetzt)
ergibt sich nach Einsetzen:

und nach Umformungen:

The equilibrium dissociation constant K _d can be determined from binding curves with increasing protein but constant RNA concentration. The RNA is present in negligible concentrations, the following relationship exists (Hardt et al., 1995):
Out

With [Protein frai ] = [Protein total ] - [protein RNA] = [protein total ] - [protein bound ] [RNA free ] = [RNA total ] - [RNA bound ]
and [Protein RNA] = [RNA bound ] [RNA bound ] = [RNA bound ] (Assuming 1: 1 stoichiometry)
results after insertion:

and after transformations:

und daraus: Kd = [Proteingesamt] – 0,5 × [RNAgesamt] At half-maximal saturation, with [RNA bound ] = 0.5 × [RNA total ] follows:

and it: K d = [Protein total ] - 0.5 × [RNA total ]

With negligible RNA concentration compared to the protein concentration results:
K d = [Protein total ] at half-maximal complex formation

Thus, the equilibrium dissociation constant K _d can be read directly from the binding curve.

For the determination of dissociation constants between 2'-F-RNA aptamers and LBP, the same trace amounts of radioactively labeled 2'-F-RNA are used (labeling with ³² P at the 5 'or 3' end according to standard methods). LBP concentrations are selected according to a preliminary experiment with three arbitrarily selected LBP concentrations and a derived estimate of the K _d value so that in each case four concentrations are above or below the estimated K _d value.

For a K _d determination, eight batches are pipetted with either 0.5 or 0.04 pmol 2'-F RNA and made up to 90 μl with binding buffer. To denature the 2'-F-RNA, the batches are heated for 3 min at 90 ° C and cooled within 20 min to room temperature. Then 10 μl of an LBP solution with the following molar amounts are added to the corresponding batches: 1.5, 5, 10, 15, 20, 40, 60 and 100 pmol. This is followed by incubation for 1 h at 37 ° C. An approach with the same amount of renatured 2'-F-RNA but no protein is performed as a negative control or background for the evaluation. After incubation, the batches are each drawn through a nitrocellulose filter, the filters are subsequently washed with 300 μl of PBSM and fixed on a Whatman paper. The determination of the amount of radioactively labeled aptamer which is retained on the filter by the binding to LBP is carried out by radioluminographic detection. A quantitative evaluation of the radioactive spots is carried out by means of the program PCBAS Version 2.09. The further data analyzes are carried out with the program GraFit Version 3.0 (Erithacus Software, UK).

to Determination of the binding minimal sequence will be boundary experiments carried out. So-called boundary experiments are a very simple and informative Method to find out how far an aptamer is in its sequence shortened can be without the binding ability to the target molecule This is a at the 3'- or 5'-terminus radiolabeled aptamer hydrolyzed limited under alkaline conditions and the resulting Fragments examined for binding to the target molecule.

In order to obtain native conformations as possible, the 2'-F-RNA fragments to be characterized are degraded and renatured in binding buffer (PBSM) before the binding assay. Aptamer solution is denatured for 3 min at 90 ° C for this purpose and then cooled at room temperature for 20 min. In approaches of 100 μl then 200 nM or 400 nM mLBP are incubated with small amounts of 2'-F-RNA (2 - 2.5 x 10 ⁵ cpm) for 1 h at 37 ° C and then on PBSM-impregnated nitrocellulose filter (25 mm diameter , 0.45 μm pore size) is filtered. The 2'-F-RNA mLBP complexes are eluted from the filter after a wash with 300 μl of binding buffer. The filter is cut into 8 pieces with a scalpel. The 2'-F-RNA mLBP complexes remaining on the filter are eluted with 200 μl of urea solution (7 M) and then subjected to phenol-chloroform extraction. The 2'-F-RNA fragments are concentrated in an ethanol precipitate and analyzed on a denaturing polyacrylamide gel. Approaches serving as negative controls without protein or with bacterial EF-Tu are worked up in parallel in the boundary experiment.

literature

• - Ehresmann C, Baudin F, Mougel M, Romby P, Ebel JP & Ehresmann B (1987) Probing the structure of RNAs in solution. Nucleic Acids Res. 15: 9109-9128
• - Giegé R, helmet M, Florentz C (1999) Chemical and Enzymatic Probing of RNA Structure. In: Comprehensive Natural Products Chemistry, Vol. 6 edn. pp 63-80
• - Hardt WD, Schlegl J, Erdmann VA & Hartmann RK (1995) Kinetics and thermodynamics of the RNase P RNA cleavage reaction: analysis of tRNA 3'-end variants. J. Mol. Biol. 247: 161-172
• - Huang Y, corner stone F, Padilla R & Sousa R (1997) Mechanism of ribose 2'-group discrimination by an RNA polymerase. Biochemistry 36: 8231-8242
• - Klussmann S, Nolte A, Bald R, Erdmann VA & Prince JP (1996) Mirror-image RNA that binds D-adenosine. Nat. Biotechnol. 14: 1112-1115
• - Sousa R & Padilla R (1995) A mutant T7 RNA polymerase as a DNA polymerase. EMBO J. 14: 4609-4621
• - Zuker, M (2003) Mfold web server for nucleic acid folding and hybridization pre-diction. Nucleic Acids Res. 31: 3406-3415

Figure legends

1 : DNA sequences of isolated 2'-F-RNA molecules from selection with Ni-NTA agarose. The PCR products representative of the eighth round selection 2'-F RNAs were cloned and 100 clones sequenced. Evaluable sequences are divided into three groups (1 to 3) and are shown here from 5 'to 3' in reading direction, with only the originally randomized area is shown. The names of the individual clones are on the left side before the corresponding sequences. The frequency of isolated clones is given on the right. The conserved sequence motifs Groups 1 and 2 are grayed out.

2 : Secondary structure model of aptamer A011. The conserved nucleotides of the consensus motif are circled. The primer sequences flanking the originally randomized region are indicated by lowercase letters. The secondary structure presented here is supported by enzymatic and lead ion-induced cleavage experiments.

3 : Lead structures of the selected aptamers. A 'is subtype of variant A. The nucleotides of the consensus sequences are circled. A is the core region of aptamers A011 (2x) and A016 (2x), A 'is the core region of aptamer G017, B is the core region of aptamers A036, A021 and A041, and C is the core region of aptamer A015 (2x), A026, A028, A034, G012, 0009 (2x), G033, G047, A025, G023 and A042 (only 2 bp in P1), and under D the core region of the aptamers A006, A012, A007 and G015. The letter Y in the sequence stands for a pyrimidine base (U or C), R stands for a purine base (A or G).

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (15)

RNA aptamer or alleles and / or derivatives thereof, characterized in that it binds specifically to lipopolysaccharide-binding protein (LBP). RNA aptamer according to the preceding claims, characterized in that it has in the core region a conserved single-stranded region which is bounded by 2 helices, said gene 2 helices are part of a three-helix junction. RNA aptamer according to the preceding Claims, characterized in that the three-helix junction region of the Represents nucleic acid molecule, in which 3 helices meet P1, P2 and P3 together. RNA aptamer according to the preceding Claims, characterized in that the single-stranded region containing the helices P2 and P3 connects, consisting of 4 to 5 nucleotides that are part of a Consensus sequence are. RNA aptamer according to the preceding Claims, characterized in that the RNA aptamers are modified, in particular 2'-fluoro-pyrimidine carry. RNA aptamer according to the preceding Claims, characterized in that the consensus sequence motif in the single-stranded region YGCUUCY (Y: C or U) or RUUUCY (R: A or G, Y: C or U). RNA aptamer according to the preceding Claims, characterized in that the helices P3 and P1 by 0 to 3 single Nucleotides, preferably 2'-fluoro-pyrimidine radicals connected are. RNA aptamer according to the preceding Claims, characterized in that the helices P1 and P2 are replaced by 1-6 single-stranded nucleotides, preferably 2'-fluoro-pyrimidine radicals, connected are. Process for the preparation and / or isolation of a RNA aptamer, specifically the lipopolysaccharide-binding protein (LBP) binds, characterized by the steps a) Provision a complex mixture of DNA sequence variants (randomized DNA pool) by chemical DNA synthesis. b) production of a complex mixture of transcript sequence variants from the randomized DNA pool. c) Selection for 2'-fluoro-pyrimidine-modified RNA molecules (2'-F-Y RNA), the specifically the lipopolysaccharide binding protein (LBP) or partial sequences tie from it. d) Amplification of the selected 2'-fluoro-pyrimidine-modified RNA molecules (2'-F-Y-RNAs). Process for the preparation and / or isolation of a RNA aptameres according to claim 9, characterized in that the in vitro selection in 8 rounds of selection takes place, where in selection round no. 1 in the preparative T7 Sequenase reaction a T7 promoter bearing primer having the nucleotide sequence 5'-TAATACGACTCACTATAGGGAATTCGAGCTCGGTACC-3 'is used. Process for the preparation and / or isolation of a RNA aptamer, according to claim 9, characterized in that in the selection rounds 2 to 4, 7 and 8 is used for the selection of Ni-NTA agarose. Process for the preparation and / or isolation of a RNA aptameres according to claim 9, characterized in that in the selection rounds 5 and 6 used for selection Ni-NTA Magnetic Agarose Beads. Process for the preparation and / or isolation of a RNA aptameres according to claim 9, characterized in that for the reverse transcription of the selected RNA is a primer with the nucleotide sequence 5'-CCAAGCTTGCATGCCTGCAG-3 'is used. RNA aptamer of any one of claims 1 to 8, obtainable by a method according to claims 9 to 13th Use of an RNA aptamer from any of the foregoing claims or a mixture for the manufacture of a medicament for treatment and or diagnosis of patients with sepsis.