WO2002081686A2 - Nucleic acids binding a neuropeptide - Google Patents

Abstract

The invention relates to nucleic acids which bind the neuropeptide Y, in addition to the use thereof.

Description

 Neuropeptide binding nucleic acids

description

The present invention relates to nucleic acids capable of binding to neuropeptide Y and various uses thereof.

State of the art

Neuropeptide Y

The neuropeptide Y was first isolated from porcine brain by Tatemoto in 1982 (Tatemoto et al, 1982). Together with the pancreatic polypeptide (PP) and the peptide YY (PYY), NPY belongs to the family of pancreatic polypeptides. A common feature of these structurally related, tyrosine-rich peptides is a sequence of 36 amino acids with a C-terminal tyrosinamide residue (cf. FIG. 1) (Conlon et al, 1991), (Larhammar, 1996). While NPY acts as a neurotransmitter in the central and peripheral nervous system, PYY and PP are mainly hormone-like (Michel et al, 1998). High concentrations of NPY have been detected especially in the brain, especially in the hypothalamus, in the nucleus accumbens and in the amygdala (Chronwall, 1985), (O'Donohue et al, 1985).

The neuropeptide Y is an evolutionarily highly conserved peptide, which suggests an important role in the regulation of fundamental physiological functions. The pig and human NPY differ only in a single amino acid at position 17 (hNPY: methionine, pNPY: leucine). Even the NPY sequence of the ray (Torpedo mamorata), which is very distant in terms of development, is identical to the human NPY in 33 of the 36 positions (Larhammar, 1996).

Structure and function of NPY

The three-dimensional structural model of NPY is based on X-ray crystal structure data for the pancreatic polypeptide of turkey (aPP) (Glover et al, 1983), on comparative secondary structure studies of NPY and aPP using circular dichroism (Tonan et al, 1990) and on 2-D NMR analyzes ( Saudek and Pelton, 1990), (Darbon et al, 1992). This model largely agrees with the structure of Allen and co-workers generated by molecular modeling (Allen et al, 1987).

An N-terminal polyproline helix (AS 1-8) is connected antiparallel via a ß-turn (AS 9-14) with a -helix (AS 15-32). While the two helices are closely associated by hydrophobic interactions, the C-terminus is relatively free to move. This hairpin-shaped motif, referred to as the "PP" fold, is characteristic of all members of the pancreatic polypeptide family (Monks et al, 1996).

NPY is important in controlling a large number of physiological and pathophysiological processes: The NPY release leads, for example, to an increase in food intake (Inui, 1999), (Loftus et al, 2000), to an inhibition of lipolysis, to an increase in the activity of lipoprotein lipase and thus to increase fat storage (Ingenhoven and Beck-Sickinger, 1999). NPY modulates within the central nervous system (CNS) the increase in the secretion of luteinizing hormone and insulin and the reduction in the secretion of growth hormones (Kalra et al, 1992), (McDonald et al, 1985), (Moltz and McDonald, 1985). Based on these findings, NPY has been assigned an important role in the pathophysiology of obesity and diabetes. In the peripheral nervous system, NPY has a direct vasoconstrictive effect or potentiates the vasoconstrictive effects of noradrenaline and angiotensin II (Maturi et al, 1989), (Wahlestedt and Hakanson, 1986), (Lundberg et al, 1987). NPY also enhances memory performance (Flood et al, 1989) and regulates the circadian rhythm of mammals (Hall et al, 1999). Comparative studies with NPY deficient and NPY overexpressing mice showed that the experimental alcohol consumption of mice is inversely proportional to the NPY level in the brain. Animals with normal NPY expression had a lower preference for alcohol and were more sensitive to its hypnotic properties (Thiele et al, 1998).

The involvement of the NPY in the control of various physiological processes mechanistically requires a complex signal transduction to generate selective control signals in the individual effector organs. It is assumed that the different receptor subtypes can be selectively activated by binding different NPY conformations and specific amino acid side chain residues of the neuropeptide hormone (Beck-Sickinger, 1996). Neuropetid Y receptors

There are currently six NPY receptor subtypes, five of which are already cloned (Yl, Y2, Y4, Y5 and Y6). Pharmacological studies suggest the existence of another receptor called Y3 (Cabrele and Beck-Sickinger, 2000). The NPY receptors belong to the family of G protein-coupled, heptahelical membrane receptors (Michel, et al, 1998). The receptors are localized in a wide variety of tissues, with particularly high expression densities for blood vessels, kidneys, pancreas, intestine and brain being characteristic (Ingenhoven and Beck-Sickinger, 1999). The activation of the NPY receptors leads to inhibition of the adenylate cyclase in almost all examined tissues and cells and thus to the accumulation of cyclic AMP (cAMP) (Beck-Sickinger, 1996). In addition, further signal responses are described, such as the inhibition of Ca ⁺⁺ channels in neurons (Walker et al, 1988), the modulation of K ⁺ channels in cardiomyocytes (Bleakman et al, 1991) and the release of Ca ⁺⁺ from intracellular Save (Perney and Miller, 1989). Historically, the receptors that can only be activated by the holopeptide NPY have been referred to as Yl receptors, while those that are also activated by C-terminal NPY fragments, such as. B. NPY 13-36 and NPY 18-36, activatable receptors of the Y2 class have been assigned.

Due to the involvement of NPY in numerous central and peripheral effects, the development of receptor-subtype-specific active substances is of particular importance. A prerequisite for this is a detailed characterization of the different receptor subtype-ligand complexes.

Characterization of the receptor-ligand interaction

In general, structural statements regarding the activation of G protein-coupled receptors are very difficult. The recently resolved first X-ray crystal structure of a G protein-coupled receptor, rhodopsin, was a milestone on the way to elucidating the interaction of receptor and ligand for this special case (Palczewski et al, 2000).

Due to the homology between the different G protein-coupled receptors, it is also possible to draw conclusions about the functional domains of NPY receptors. However, little is known about the molecular mechanisms of signal transmission, the precise binding sites and the specific NPY-NPY receptor subtype interactions. de- Exact characterization requires the use of alternative investigation methods. Indirect methods, such as mutagenesis within the transmembrane regions and the extracellular loops of the receptors, have elucidated a large number of the structure-function relationships of NPY with the different receptor subtypes (Cabrele and Beck-Sickinger, 2000). The development of anti-receptor antibodies should provide further insight into the structural relationships between receptor and ligand (Eckard et al, 1999). Although the large immunoglobulins can specifically bind to a receptor subtype, they are often unable to competitively inhibit the natural ligand. Binding of the antibody to the receptor can trigger a signal transmission that normally only takes place after activation by a hormone or neurotransmitter (Strosberg, 1992). Modifications to the primary and, consequently, the secondary and tertiary structure of the natural ligand NPY also provided further insights into the interactions between receptor and ligand. The systematic exchange of the 36 amino acids of the NPY for alanine (so-called alanine scan) as well as various amino acid deletions and amino acid substitutions led to the identification of amino acids important for the ligand-receptor interaction (Cabrele and Beck-Sickinger, 2000).

Both the N and the C terminus of the peptide are required for the interaction of the Y1 receptor with the NPY, while in the case of the Y2 receptor short fragments of the C terminus (e.g. NPY 18-36) represent the binding domain for NPY. In addition to the holopeptide NPY, the Y5 receptor can also be activated by partially N-terminally deleted variants (e.g. NPY 2-36) of the NPY. A promising approach to clarify the physiological and pathophysiological role of NPY is the development of highly affine and specific non-peptide antagonists as "pharmacological tools".

The Yl receptor

The mRNA of the Yl receptor was detected in the brain (hippocampus, thalamus, amygdala and cortex) as well as in the peripheral organs such as the heart, kidney, spleen and lungs. Activation of the Yl receptor triggers a long-lasting vasoconstriction and increases the effects of other vasoconstricting substances such as angiotensin II and noradrenaline (Buschauer, 2000). In addition to other receptor subtypes, the Yl receptor also seems to play an important role in regulating food intake (Inui, 1999). The effects of anxiolysis and sedation mediated by the central nervous system are Receptor modulated (Heilig et al, 1993). The pharmacological profile of the Yl receptor is characterized by a high affinity for NPY (IC ₅₀ : 0.2 nM) and PYY (IC ₅₀ : 0.7 nM) as well as for the NPY mutant NPY [P ³⁴ ] with an IC ₅₀ - Characterized value of 0.5 nM. In this NPY mutant, the glutamine side chain residue at position 34 was substituted by a proline residue, creating a selective Yl receptor agonist. In contrast, a strongly reduced affinity of the Yl receptor for N-terminally shortened variants of NPY and PYY was observed. The NPY (NPY 2-36) truncated by an amino acid N-terminal shows a 75-fold worse Yl receptor affinity compared to the NPY. The affinity of even shorter NPY variants with Kd values lies in the micromolar range z. B. NPY 3-36, NPY 13-36, NPY 18-36. The D-arginine derivative BIBP 3226 was described as the first Yl-selective NPY antagonist with an affinity in the nanomolar range (Cabrele and Beck-Sickinger, 2000).

The Y2 receptor

The Y2 receptor is primarily located centrally in the nervous system, although it is found in high density in the hippocampus (Dumont et al, 1992). Numerous effects mediated by it are based on inhibition of neurotransmitter release. By attacking presynaptic Y2 receptors, NPY inhibits the release of glutamate in the CNS and the release of norepinephrine and acetylcholine in the peripheral nervous system. Selective agonists (see Table 1) are truncated NPY peptides, NPY 13-36 (IC ₅₀ : 0.32 nM), NPY18-36 (IC ₅₀ : 0.25 nM) and NPY [Ahx ^{5 "24} ] ( IC ₅₀ : 2 nM) In the centrally shortened NPY variant NPY [Ahx ^{5 "24} ], amino acids 5-24 were replaced by an aminohexanoic acid spacer. It was only recently possible to develop the first selective Y2 receptor antagonist BIIE0246 with an affinity of IC ₅₀ : 3.3 nM (Dumont et al, 2000).

The Y5 receptor

The Y5 mRNA was detected almost exclusively in the brain, especially in areas that are important for appetite regulation (Gerald et al, 1996). The introduction of alanine at position 31 and aminoisobutyric acid (Aib) at position 32 created a highly selective Y5 receptor agonist that does not bind to either Yl or Y2. It is assumed that the biologically active conformation of the C-terminus of NPY at the Y5 receptor is characterized by a special turn structure, which is induced or stabilized by the dipetid Ala-Aib (Cabrele et al, 2000). Before the discovery of the Y5 receptor, it was thought that Yl and a Y-1-like receptor are responsible for the appetite-increasing effects of NPY. Later It was found that the N-terminally truncated NPY derivative NPY 2-36, which has a low affinity for the Yl reteptor but which stimulates close up absorption even more than the NPY, has a high affinity for a new NPY receptor, the Y5 Subtype, has (Buschauer, 2000). The uptake could be inhibited by the administration of selective Y5 antagonists (CGP 71683 A). The observations on Y5 and Yl knock-out mice have questioned the importance of NPY in regulating food intake. Neither Yl nor Y5 receptor-deficient mice lose body weight (Inui, 1999). The involvement of other factors and receptors is discussed.

Table 1 shows a summary of the characteristics of the NPY receptor subtypes. The information in brackets relates to the ICsQ value. The basis for the compilation of this data was the review article by Cabrele 2000 (Cabrele and Beck-Sickinger, 2000)

Tabelle 1 : Charakterisierung der NPY-Rezeptorsubtypen. Die Aufgabe der Erfindung

Table 1: Characterization of the NPY receptor subtypes. The object of the invention

The present invention has for its object to provide an agent that is able to bind to neuropeptide Y and fragments thereof. Furthermore, the invention is based on the object of providing a means which allows differentiation of the different NPY receptor subtypes.

Solution of the task

According to the invention, the object is achieved in a first aspect by a nucleic acid, in particular an isolated nucleic acid, binding neuropeptide Y or a derivative thereof and derivatives of this nucleic acid.

In one embodiment it is provided that the derivative of neuropeptide Y is the C-terminal part of neuropeptide Y, in particular the part comprising at least amino acid positions 24 to 36.

In a second aspect, the object is achieved by a nucleic acid, in particular isolated nucleic acid, which discriminates in terms of its binding behavior between the members of the pancreatic polypeptide family.

In a third aspect, the object is achieved by a nucleic acid, in particular isolated nucleic acid, which inhibits the interaction between the neuropeptide Y receptor Y2 and a ligand of the neuropeptide Y receptor Y2.

In a preferred embodiment it is provided that the ligand is selected from the group comprising NPY, hPP and BIIE 0246.

In a fourth aspect, the object is achieved by a nucleic acid, in particular isolated nucleic acid, which binds to different conformations of neuropeptide Y. In a fifth aspect, the object is achieved by nucleic acid, in particular isolated nucleic acid, binding to the conformation of neuropeptide Y, which activates the neuropeptide Y receptor Y2.

In an embodiment of the various aspects of the present invention, it is provided that the nucleic acid comprises a sequence which is selected from the group comprising SEQ H> NO: 1-18 and 44-64 and their derivatives.

In a further embodiment it is provided that the nucleic acid is one that is selected from the group comprising DNA, RNA, polynucleotides, oligonucleotides, aptamers, apazymes and intramers.

In one embodiment of the nucleic acids according to the invention it is provided that the derivatives of the nucleic acid comprise at least one modified connecting group, preferably at least one modified connecting group in the sugar phosphate backbone and / or at least one modified sugar residue and / or at least one modified base or one or more combinations thereof.

In a preferred embodiment of the nucleic acids according to the invention it is provided that the modified base is selected from the group consisting of 5-fluoroaracil, 5-bromouracil, 5-chloroaracil, 5-ioduracil, hypoxanthine, xanthine, ethenoadenosine, 4-acetylcytosine, 5- ( Carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thio-uridine, 5-carboxymethylaminomethyluracil, dihydrouracil, ß-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylguanine 2,2-dimethylguanine, 2-methyladenine, 2-ethylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, ß-D-mannosylqueosine, 5 ' -Methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, methyl ester, uracil-5-oxyacetic acid, pseudouracil, queosin, 3-thiocytosine, 5-methyl-2-thiouracilil, 2-thiouracil , 5-methyluracil, uracil-5-oxyessi methyl acid, uracil-5-oxyacetic acid, 3 (3-arnino-3-N-2-carboxypropyl) uracil and 2,6-diaminopurine. In a further embodiment of the nucleic acids according to the invention, it is provided that they have at least one modification or label at the 5 'and / or 3' end, the modification and / or label preferably being selected from the group which comprises the biotin group; the digoxygenine cluster; Fluorescent dyes, in particular fluorescein and rhodamine; psoralen; Contains thiol group (s), amino group (s), ethylene glycol group (s) - or cholesteryl group (s).

In a still further embodiment of the nucleic acids according to the invention it is provided that the modified group of compounds is selected from the group of grapes, the phosphomono- or phosphodithioates, alkylphosphonates, arylphosphonates, phosphoroamidates, phosphate triesters, preferably P (O) -alkyl derivatives; Compound groups in which oxygen atoms in the bridge between the sugars formed by the phosphate group are replaced by other bonds, preferably NH, CH ₂ or SP compounds, more preferably 3'-NHP (O) - (O ^" ) O-5 'phosphoramidates; dephosphointemucleotide compounds, preferably acetamidate, carbamate compounds or peptide nucleic acids (PNA).

In one embodiment of the nucleic acids according to the invention it is provided that the modified sugar residue is selected from the group consisting of 2'-azido-2'-deoxy, 2'-amino-2'-deoxy, 2'-fluoro-2'- Deoxy-, 2'-chloro-2'-deoxy, 2'-O-methyl, 2'-O-allyl, 2'-C-fluoromethyl grappa and / or modifications.

In a sixth aspect, the object is achieved by using a nucleic acid according to the invention for the manufacture of a medicament.

In a seventh aspect, the object is achieved by the use of a nucleic acid according to the invention for the manufacture of a medicament for the treatment of diseases or conditions in which an inhibition of the interaction between the neuropeptide Y or a derivative thereof and a neuropeptide Y receptor leads to a change in the Disease or condition.

In an eighth aspect, the object is achieved by using a nucleic acid according to the invention, in particular for the production of a medicament, for reducing the effectiveness. tion of neuropeptide Y in an organism, in particular in a human or animal organism.

In a ninth aspect, the object is achieved by the use of a nucleic acid according to the invention, in particular for the manufacture of a medicament, for reducing the rank of food intake, for promoting lipolysis, for decreasing the activity of lipoprotease, for decreasing rank for fat storage, for decreasing the secretion of luteinizing hormone , to reduce the secretion of insulin, to increase the secretion of growth hormone, to expand the vascular system, to reduce the vasoconstrictor effect of norepinephrine, to reduce the vasoconstrictor effect of angiotensin and to influence the circadian rhythm in mammals.

In a tenth aspect, the object is achieved by using a nucleic acid according to the invention to characterize the interaction topology of the interaction between neuropeptide Y and neuropeptide Y receptor at the molecular level.

In an eleventh aspect, the object is achieved by using a nucleic acid according to the invention for the detection and / or comparison of the receptor activation, in particular the receptor activation in the brain and very particularly the activation of neuropeptide Y receptors.

In a twelfth aspect, the object is achieved by using a nucleic acid according to the invention for the detection of neuropeptide Y or a derivative thereof.

In a thirteenth aspect, the object is achieved by using a nucleic acid according to the invention for the purification of neuropeptide Y or a derivative thereof.

In a fourteenth aspect the problem is solved by an antagonist for a neuro peptide ^¬ Y receptor, particularly for the neuropeptide Y Y2 receptor.

In a Ausführangsform is contemplated that the antagonist is a nucleic acid according to the invention comprises or is ^¬. In a fifteenth aspect, the object is achieved by a receptor mimetic for a neuropeptide Y receptor, in particular the neuropeptide Y receptor Y2.

In a preferred embodiment it is provided that the receptor mimetic comprises or is a nucleic acid according to the invention.

In a sixteenth aspect, the object is achieved by using a nucleic acid according to the invention for screening compounds, the compounds being selected from the group comprising agonists of a neuropeptide Y receptor and antagonists of a neuropeptide Y receptor.

In a preferred embodiment, the compounds are low molecular weight compounds.

In a seventeenth aspect, the object is achieved by a compound obtainable from the use of the nucleic acids according to the invention for screening compounds.

In an eighteenth aspect, the object is achieved by using a neuropeptide Y or a derivative as the target molecule in the context of an in vitro selection process.

In a nineteenth aspect, the object is achieved by a method for identifying and isolating nucleic acids which are able to bind to a target molecule, comprising the following steps:

incubating the target molecule or a part thereof with a large number of nucleic acids, preferably a nucleic acid library, the nucleic acids having different sequences,

Selection and isolation of those nucleic acids which are able to bind to the target molecule or a part thereof,

optionally amplifying the isolated nucleic acids and repeating the first two steps; and optionally determining the sequence and / or binding specificity of the isolated nucleic acids,

wherein the target molecule is neuropeptide Y or a derivative thereof.

In a twentieth aspect, the object is achieved by the uses according to the invention of one or more of the nucleic acids according to the invention, preferably in a complex with a neuropeptide Y or a derivative thereof, for the rational design of compounds, in particular agonists and antagonists of the neuropeptide Y receptor, preferably of inhibitors. The inhibitors are preferably low molecular weight inhibitors.

The present invention is based on the surprising finding that it is possible to produce nucleic acids which bind to neuropeptide Y or a derivative thereof. It is particularly noteworthy here that the nucleic acids according to the invention are able to discriminate between the different receptor sub-types within the group of the neuropeptide Y receptors. The various receptor sub-types are those as indicated in Table 1, the disclosure content of which is included here. Discrimination is to be understood here in particular to mean that the individual nucleic acid according to the invention relates to the compounds to be discriminated, i.e. the different conformations of neuropeptide Y or its analogs, has a different affinity. The difference between such affinities is typically 1 nmolar to 5 mmolar.

In addition, the nucleic acids according to the invention have a number of surprising properties which also allow a wide range of possible uses. One of these properties can be seen in the fact that discrimination takes place in the sense that the nucleic acids according to the invention can distinguish between the members of the pancreatic polypeptide family. In addition to the neuropeptide Y, this polypeptide family also includes the pancreatic polypeptide and the peptide YY.

Another surprising property of the nucleic acids according to the invention can be seen in the fact that the nucleic acids according to the invention specifically the interaction between new ropeptide Y, or a derivative thereof, and the receptor for the neuropeptide Y, also referred to herein as the neuropeptide Y receptor. The type of influence is an inhibition. It is particularly noteworthy here that the interaction also takes place specifically between the neuropeptide Y or a derivative thereof and the neuropeptide Y receptor subtype Y2.

It was also particularly surprising to discover that the nucleic acids according to the invention can distinguish between the different conformations which can be ingested by neuropeptide Y. The presence of different conformations also appears to be responsible for the binding specificity of neuropeptide Y to the different receptor subtypes. With the nucleic acids according to the invention, the inventors have succeeded in generating nucleic acids which specifically interact with that conformation of the neuropeptide Y and form a complex which is required for a high-affinity interaction with receptor subtype Y2. As a result of this conformation specificity of the nucleic acids according to the invention, the nucleic acids according to the invention can be viewed and used as mimetics of the neuropeptide Y receptors and in particular of the neuropeptide Y receptor Y2. This immediately results in the use of the nucleic acids according to the invention in test or detection systems in which neuropeptide Y receptors and in particular neuropeptide Y receptor Y2 can be used, for example when screening for agonists and antagonists of neuropeptide Y, but also in the areas of therapy and research.

A derivative of neuropeptide Y is to be understood here to mean in particular a derivative which is capable of binding to a neuropeptide Y receptor. In addition, the term is also intended to encompass those molecules which comprise part of the amino acid sequence of neuropeptide Y, in particular a sequence of 5 consecutive amino acids of the natural sequence of neuropeptide Y.

Nucleic acids or nucleic acid ligands in the sense of this invention preferably comprise DNA, RNA or chemically modified forms of DNA or RNA. More preferably, the nucleic acid ligands belong to one of the classes of aptamers, aptazymes, ribozymes, intramers, Spiegelmers, natural nucleic acid ligands or are naturally occurring protein-binding nucleic acids. Particularly preferred are the Aptazymes, intramers and Ap ^¬ are Tamere, of the three turn, especially the group of intramers. Natural nucleic acids are also to be understood here as nucleic acids, be it as a starting materials (partially or completely) for in vitro selection methods or as nucleic acids according to the invention which have a sequence which is contained in the transcriptome of a cell or an organism, the term transcriptome here being understood to mean in particular the entirety of the nucleic acids expressed. If natural nucleic acids are used, it is particularly preferred if natural libraries, such as cDNA fragments of transcribed R As, are used instead of randomized libraries in selection processes.

Vitro selection is to be understood here to mean in particular the process by which the nucleic acid ligands according to the invention, preferably from the grappa of aptamers, intramers, aptazymes or ribozymes, more preferably from the grappa of aptamers and aptazymes, by in vitro selection from combinatorial libraries can be isolated from nucleic acids with different sequences.

For the purposes of this invention, aptamers (Ellington and Szostak, Nature 346 (1990), 818-822) are understood to mean single-stranded nucleic acid ligands which are isolated from combinatorial libraries of randomized nucleic acids by in vitro selection. This process is sometimes referred to as SELEX ("systematic evolution of ligands by exponential enrichment") (Tuerk and Gold, Science 249 (1990), 505-519). These aptamers consisting of ssDNA or ssRNA have very high affinities and specificities for their antigens. To date, nucleic acid ligands for metal ions, organic compounds, peptides, proteins, or even complex structures such as viruses and cells have been isolated (review article: Gold et al., Annu. Rev. Biochem. 64 (1995), 763-797; Ellington and Conrad, Biotechnol. Annu. Rev. 1 (1995), 185-214; Famulok, Curr. Opin. Struct. Biol. 9 (1999), 324-329, Hofmann, 1997). The high potential of the technology lies in the in vitro process of aptamer selection. The nucleic acid ligands are enriched from combinatorial libraries of up to 10 ¹⁵ individual sequences by repeated cycles of contact with the antigen, separation of all non-binding nucleic acids and enzymatic amplification of the molecules interacting with the target molecule.

Aptazymes in the sense of the present invention are hybrid molecules of ribozymes and aptamers and accordingly consist of a catalytic and a ligand-binding nucleic acid domain. The activity of the ribozyme is regulated, ie activated, by the binding of the ligand to the ligand-binding nucleic acid domain (allosteric center) or inhibited. Aptazyme technology is known to the expert and is described, for example, in Soukup and Breaker, Curr. Opin. Stract. Biol. J0 (2000), 318-325. Aptazymes can be modularly constructed from a ribozyme part and the allosteric centimeter by rational design or can be isolated de novo by in vitro selection. For example, an aptazyme was developed from a hammerhead ribozyme, a small catalytic motif that can cleave RNA sequences in a sequence-specific manner (Forster and Symons, Cell 49 (1987), 211-220; Haseloff and Gerlach, Nature 334 (1988), 585-591 ), and an aptamer domain that specifically binds to ATP, so that the binding of ATP led to an allosteric inhibition of catalytic activity (Tang and Breaker, Chem. Biol. 4 (1997), 453-459, Tang and Breaker, Nucleic Acid Res. 26 (1999), 4222-4229). On the other hand, aptazymes could also be isolated by in vitro selection from combinatorial libraries of different nucleic acid sequences which contained a novel binding domain for a ligand (Robertson and Ellington, Nat. Biotechnol. 17 (1999), 62-66, Koizumi et al, Nat Stract. Biol. 6: 1062-1071 (1999). Aptazymes in the sense of this invention can, for example, be those which contain a ligand binding site for NPY. Such aptazymes can be produced on the basis of the nucleic acids according to the invention by building them modularly with a ribozyme part and the allosteric centimeter. A schematic representation of an aptazyme is shown in Figure 15

In addition to what has been said above, natural nucleic acid ligands are also to be understood here as meaning those nucleic acids, nucleic acid ligands or parts thereof, ie nucleic acid motifs, which are encoded as such in the genomes of organisms and have the ability to bind to a target protein. Examples include the HIV-Tar RNA, which binds to the HIV Tat protein, or the HIV-RRE-RNA, which binds to the HIV-REV protein, among many others. A large number of such natural nucleic acid ligands are known to the person skilled in the art. So far unknown nucleic acid ligands for certain target proteins can also be isolated from combinatorial libraries of natural nucleic acids of different sequences, which represent, for example, the genomic RNA of an organism, an organ or a cell, by in vitro selection. The preparation of such combinatorial libraries is known to the person skilled in the art and is described, for example, in Singer et al., Nucleic Acid Res. 25 (1997), 781-786. In this way it was possible, for example, to isolate genomic DNA sequences which bind the transcription factor TFIIIA from Xenopus laevis (Kinzler and Vogelstein, Nucleic Acid Res. 17 (1989), 3645-3653). Another experiment failed mRNA fragments can be isolated from the transcriptome of the bacterium E. coli ligands for the bacteriophage MS2 protein (Shtatland et al, Nucleic Acids Res. 28 (2000), E93). Surprisingly, new, previously unknown interactions were found, for example with the mRNAs of the rffG gene or the ebgR gene. This shows that new nucleic acid ligands for proteins can be isolated from libraries that are constructed from the transcriptome of an organism.

The nucleic acids according to the invention can be administered directly by means of all galenical techniques and compositions which comprise one or more of the nucleic acids according to the invention in their various embodiments. Examples include solutions, powders, tablets, gels and the like. The nucleic acid according to the invention is typically produced and / or administered together with a stabilizer, adjuvant, pharmaceutically acceptable carrier, preservative, diluent, carrier or a combination thereof. Methods for introducing the nucleic acids according to the invention into cells are known to the person skilled in the art.

The therapeutic use of the nucleic acids according to the invention is based on the surprising properties of the nucleic acids according to the invention disclosed herein. The nucleic acids according to the invention can be contained individually or in combination with a wide range of active ingredients in corresponding medicaments. As a result of the specific interaction of the nucleic acids according to the invention with neuropeptide Y, these can be used therapeutically to influence diseases or conditions in which the inhibition of the interaction between neuropeptide Y and a neuropeptide Y receptor, in particular neuropeptide Y receptor Y2, leads to a change in the disease or drive the state. Such diseases or conditions which are based on this mechanism or which are to be treated or changed via this mechanism are known to those skilled in the art. Examples in this connection are: hypertension, stress (Grouzmann, E. et al; Journal of Clinical Endocrinology and Metabolism, Vol. 68 (4), p. 808 - 813), neuroblastomas (Grouzmann, E. et al; Journal of Clinical Endocrinology and Metabolism, Vol. 68 (4), p. 808-813), Phaochromocytome (Grouzmann, E. et al; Journal of Clinical Endocrinology and Metabolism, Vol. 68 (4), p. 808-813) , endocrine pancreatic tumors Waeber, G. et al. Peptides, vol. 16 (5), pp. 921-929. 1995), human pituitary tumor (Grouzmann, E. et al. Regulatory Peptides 75-76 (1998) p. 89-92), hypertension, unstable angina, heart attack, adiprositas, bulimia, anxiety, epilepsy, depression, memory and memory.

The nucleic acids according to the invention can be used in the diagnostic field like antibodies in a large number of diagnostic applications (review article: Hesseberth et al, J Biotechnol 74 (2000), 15-25; Brody and Gold, Biotechnol 74 (2000), 5-13 ; Jayasna Clin Chem 45 (1999), 1628-1650; Osbome et al., Curr Opin Chem Biol I (1997), 5-9). By binding the nucleic acids according to the invention to the target structure, i.e. the NPY, this can be marked. If the nucleic acid according to the invention bears a label, this and thus the complex of NPY and nucleic acid according to the invention can be detected. Suitable labels are known to those skilled in the art and include, among others, those selected from the group consisting of radioactive labels, labels with fluorescent dyes, labels with groups such as biotin or digoxygenin, or labels with enzymes such as beta-galactosidase. Suitable fluorescent dyes for labeling nucleic acids are known to those skilled in the art and include i.a. AMCA-X, fluorescein, rhodamine, Cy3, Cy 5, Cy 5.5, HEX, D-AMCA, tetramethylrhodamine or Texas Red. Detection of the said complex with other nucleic acid / protein complexes can be done, for example, by FACS (Ringquist and Parma, Anal Chem. 70 (1998), 3419-3425), fiber-optical microarray sensors (Lee and Walt, Anal Biochem 282 (2000), 142-146), capillary electrophoresis (German I, Anal Chem 70 (1998), 4540-4545), intermolecular force measurements (Moy et al., Science 265, (1994), 257-259) or fluorescence microscopy.

Alternatively, the nucleic acids according to the invention, the NPY or both can be labeled with fluorescent dyes and the interaction between the nucleic acid and the NPY can lead to a measurable change in the fluorescence signal. Such changes in fluorescence can also be based on a change in the intrinsic fluorescence of the NPY after binding of one of the nucleic acids according to the invention. Also a change in the intrinsic fluorescence of the change of NPY may lead itself, for example, which can be used for the measurement signal and whose Verändemng due to binding of the nuc ^¬ leinsäuren by introducing a Trp-point mutation. Another example of fluorescence-based detection is the binding of thrombin to anti-thrombin aptamers labeled with fluorescent dyes by measuring the change in fluorescence anisotropy (Potyrailo RA et al, Anal Chem 70 (1998), 3419-25). Up to 0.7 amol Thrombin can be detected. In another embodiment, one interaction partner, the nucleic acid or the protein is labeled with a fluorescence-quenching grappe (quencher), the other is labeled with a fluorophoric grappe (donor), the interaction between the two interaction partners leading to the quencher quenching the fluorescent emission of the donor. If the interaction takes place, this can be measured by reducing the fluorescence signal. This method, also known as FRET ("fluorescence resonance energy transfer"), is known to the person skilled in the art and is also used for the detection of protein / protein interactions (Sorkin A. et al, Curr Biol. 10 (2000), 1395-1388; Latif R. and Graves P., Thyroid. 10 (2000), 407-412, 14: Buranda T. et al, Cytometry 37 (1999), 21-31). The person skilled in the art is familiar with the principles of the detection of interactions between molecules by means of fluorescent labeling and the various design options for the detection by single or multiple fluorescent labeling of one or both interaction partners.

Likewise, the complexes comprising the nucleic acids according to the invention and the NPY can be detected on suitable sensors which are coated with one of the two binding partners. The detection can take place here via the increase in mass after the binding of the second binding partner. For example, complexes of RNA aptamers and the enzyme 2'-5'-oligoadenylate cyclase or RNA aptamers and the NS3 protease of the hepatitis C virus were detected by so-called SRP ("surface plasmon resonance") analyzes (Hartmann et al., J Biol Chem 273 (1998), 3236-3246; Hwang et al., Biochem Biophys Res Commun 279 (2000), 557-562). Other suitable analysis methods, such as quartz crystal microbalance ("Qartz Crystal Microbalance", QCM) (Kosslinger et al., Biosens Bioelectron 7 (1992), 397-404; Hengerer et al., Biosens Bioelectron 14 (1999), 139-144) are also known to the person skilled in the art.

In a special embodiment, the interaction between the protein and the nucleic acid ligand, in particular an aptazyme, can be detected by a catalytic event. The NPY-recognizing nucleic acid domain is linked to a ribozyme domain in such a way that the binding of the NPY allosterically influences the activity of the ribozyme domain, ie activates or inhibits it. The construction of signaling aptazymes is known to the person skilled in the art and is described in the literature (Hesselberth et al, J Biotechnol 74 (2000), 15-25; Marshai and Ellington, Nat Stract Biol 6 (1999), 992-994; Soukup and Breaker, Trends Biotechnol 17 (1999), 469-476). Examples of ribozyme domains found in aptazyme constructs have been used are effector-activated ribozyme ligases (Robertson and Ellington Nucleic Acids Res 28 (2000): 1751-1759) or Hammerhead ribozymes (Soukup et al., J Mol Biol 298 (2000), 623-632). In the case of ribozyme ligases that self-link to an oligonucleotide, the active ribozyme domain can be detected via reverse transcription PCR with a primer complementary to the linked oligonucleotide (Robertson and Ellington, Nat Biotechnol 17 (1999), 62-66). Active hammerhead domains can be detected by cleaving a substrate oligonucleotide, for example in a FRET assay (Jenne et al, Angew. Chem. 111 (1999), 1383-1386). The oligonucleotide to be cleaved is marked with a fluorescent (donor) and a fluorescent quenching (quencher) group. In the unsplit state, the donor and quencher are in the immediate vicinity and the light emitted by the donor is absorbed by the quencher. After the cleavage, the donor is released and the fluorescence now freely emitted by it can be detected.

Particularly advantageous applications of the nucleic acids according to the invention result from the differential interaction of the aptamer DP3 disclosed herein with receptor subtype-specific conformations of the NPY, which enable the use of the nucleic acids according to the invention to characterize the interaction topology of the NPY-NPY receptor interaction at the molecular level.

The Grouzmann et al. Monoclonal antibodies against various NPY epitopes described can discriminate between NPY and the other members of the pancreatic polypeptide family with an affinity of 3.8 x 10 ^"8 M to 2.5 x 10 ^{" 10} M (Grouzmann et al., 1992). A selectivity of these antibodies towards distinct, biologically active NPY conformations has not been described so far. Because of their size, antibodies also have the disadvantage of possible steric effects on the ligand-receptor interaction. However, the nucleic acids according to the invention can be a differentiated tool for studying the receptor activation. This can be used, for example, in in vivo studies or in experiments on brain sections: In areas of the brain where several NPY receptor subtypes are expressed, selective inhibition of the Y2 receptor subtype by DP3 could better understand its importance in the context of the function of the other receptors. As can be seen from what has been said above, the distinct properties of the nucleic acids according to the invention can often be used as an alternative to antibodies, in particular in the field of diagnosis, additional possibilities for differentiation being provided by the increased specificity or selectivity of the nucleic acids according to the invention towards antibodies.

The following uses and diagnostic application examples for the nucleic acids according to the invention are mentioned here: tension headache (Ashina, M et al Pain 83 (1999) 541-547), stress (Grouzmann, E. et al; Journal of Clinical Endocrinology and Metabolism, Vol . 68 (4), p. 808-813), neuroblastomas (Grouzmann, E. et al; Journal of Clinical Endocrinology and Metabolism, Vol. 68 (4), p. 808-813), phaeochromocytome (Grouzmann, E. et al; Journal of Clinical Endocrinology and Metabolism, Vol. 68 (4), p. 808-813), endocrine pancreatic tumors Waeber, G. et al. Peptides, vol. 16 (5), pp. 921-929. 1995), human pituitary tumor (Grouzmann, E. et al. Regulatory Peptides 75 - 76 (1998) p. 89 - 92) and in conditions or diseases in which the CNS-mediated effect of neuropeptide Y is in the foreground (influencing the Hormone secretion (corticosterone, aldosterone, LH, insulin and vasopression increase, somatotropin decreases); central influence on cardiovascular functions, increased intake of food, improvement of cognitive performance, anticonvulsant effect, analgesia, anxiolysis, sedation) and in those in which the peripheral effects in the foreground (vasoconstriction (blood pressure rises), cardiodepression, potentiation of various transmitter effects, e.g. intensification of the vasoconstriction triggered by noradrenaline and angiotensin II, presynaptic: inhibition of the release of neurotransmitters, e.g. of noradrenaline and co-transmitter (NPY , ATP) from sympathetic neurons, influencing the intestinal function (secretion decreases), Adipose tissue: lipolysis and thermogenesis decrease (uncoupling protein decreases), kidney: reduction of the global filtration rate, but diuretic and natriuretic effects (renin decreases). It is obvious to the person skilled in the art that the applications of the nucleic acids according to the invention shown here in connection with the diagnosis also extend to the therapeutic area, in particular when the effect of the neuropeptide Y is causal.

In order to use the nucleic acids according to the invention such as DP3 as a detection module for scientific but also diagnostic applications, alternative labeling strategies are necessary in addition to the radioactive labeling of the aptamers. A fluorescence mark Kierang (eg fluorescein) at the 5 'end of DP3 via a guanosine 5'-phosphorothioate group ((Burgin and Pace, 1990)) did not restrict the binding behavior to NPY in the in vitro binding analyzes (data not shown). As a result, the fluorescent dye at this position should not affect the functional conformation of the aptamer. Steric competition of the binding of DP3 to NPY by the fluorescent dye is also not likely.

Furthermore it could be shown in the work that DP3 is characterized by a high specificity within the pancreatic polypeptide family. Although the human pancreatic polypeptide (liPP) has a 50% sequence homology to NPY, it was not complexed by DP3. This sequence specificity is with regard to a diagnostic application of the nucleic acids according to the invention both in immunohistochemical analyzes and for the quantification of NPY, which in endocrine tumors, for. An increased concentration in the plasma can be found, for example, in the pheochromocytoma (Grouzmann et al., 1994; (Grouzmann et al., 1998). Drolet and co-workers have already used fluorescent aptamers to determine protein concentrations in an ELISA-like assay (ELONA , enzyme-linked oligonucleotide assay) (Drolet, et al., 1996).

Likewise, the nucleic acid ligands according to the invention, such as antibodies, can be used in ELISA applications known to the person skilled in the art. An example of the use of an aptamer in an ELISA application is the specific detection of the VEGF protein using anti-VEGF aptamers (Drolet et al., Nat Biotechnol 14 (1996), 1021-1024; Kato et al., Analyst 125 ( 2000), 1371-1373). The nucleic acids according to the invention can also be used for the detection of NPY on chips as carrier materials. The use of nucleic acid ligands in such formats has been described, for example, in Brody et al. (Mol Diagn 4 (1999), 381-388). Suitable sample materials include body fluids such as blood, urine, lymph fluid, vaginal fluid, cerebrospinal fluid, punctures, stool, biopsies, cellular samples and digestions thereof. This results in the specific detection of NPY in the samples to be examined.

The nucleic acids according to the invention can also be used for these purposes by means of so-called kits. In the present case, such a kit comprises at least one of the nucleic acids according to the invention, which has optionally already been labeled with one of the brands described above. markings are provided. Other embodiments include other reagents, such as positive and negative controls. Negative controls can be designed in such a way that a nucleic acid is contained in the kit that does not bind to NPY or certain forms thereof, as are also described here, or does not modulate their function, and in particular does not inhibit them. In a further embodiment, an NPY can be added as a positive control to which the nucleic acid according to the invention also present in the kit binds. In one embodiment of the kit, both the nucleic acid according to the invention and the NPY can be expressed either individually or together in a cell, preferably expressed or in an expressible form. In other embodiments, the kit may further include buffers, wash solutions, and the like.

The nucleic acids according to the invention can be used to form a complex with the NPY described herein, in addition to the therapeutic and diagnostic area, also in the technical or preparative area. Due to their high specificity and stability, a purification of natural NPY from various biological materials by an affinity-chromatographic process is conceivable. The RNA molecules could be biotinylated at the 5 'end via a thiophosphate group and thus coupled to streptavidin columns as an aptamer affinity matrix.

In these purification procedures, the nucleic acids according to the invention can be coupled to carrier materials such as, for example, Sepharose, agarose, or magnetic particles. These affinity carrier materials can then be used for the preparative purification of NPY. Corresponding protocols such as affinity precipitation, analogous to immunoprecipitation, or affinity chromatography are known to the person skilled in the art. Protocols for analog immunoprecipitations are described, for example, in Ausubel et al. (Current Protocols in Molecular Biology (1991), Wiley Interscience, New York) or Sambrook et al. (Molecular cloning - a laboratory manual, ^2nd Ed (1989), Cold Sprin Harbor Laboratory Press, Cold Spring Harbor.). After application of the sample and binding of the interaction partner, the material can be freed from unspecific binding partners with suitable washing solutions. The specifically bound interaction partner, ie the NPY, is then eluted and optionally quantified. Typically, high salt concentrations, detergents such as SDS, chaotropic agents such as urea or guanadinium hydrochloride are used as eluents or elution conditions. Furthermore, the protein can be obtained by heat denaturation Nucleic acid ligands, typically between 60 ° C. and 95 ° C., or denaturing the structure of the nucleic acid ligands by complexing divalent ions, for example by EDTA. Protocols that can be used to resolve the interaction between the nucleic acid ligands and the target protein to be purified are known to the person skilled in the art. The above-mentioned samples can be used as a sample. In addition, owing to the preparative character of this embodiment, the sample can also be a cultivation medium for cells which form the respective binding partner, with or without cells (in the case of the export of the binding partner), or a disruption of cells. A protocol for the purification of human L-selectin expressed in CHO cells by means of a specific aptamer via affinity chromatography has been described by Romig and co-workers (Romig, J Chromatogr B Biomed Sei Appl 731 (1999), 275-284).

Another application for the nucleic acids according to the invention is the use thereof for the rational design of small molecules, in particular inhibitors. The structure of the complex of the nucleic acids according to the invention and the neuropeptide Y is determined by means of techniques known to the person skilled in the art, such as NMR or X-ray analysis.

Another form of using complex formation between one of the nucleic acids according to the invention and NPY takes place in the screening methods according to the invention, which are described below.

For example, libraries of substances, preferably of low molecular weight substances, can be searched for substances which, by binding to one of the two interaction partners, ie NPY or at least one of the nucleic acids according to the invention, block the interaction of the two interaction partners. Analog screening systems have been developed, for example, for screening inhibitors of protein / protein interactions. Thus, small-molecule inhibitors for the interaction between cyanovirin-N and the HIV protein gpl20 (McMahon et al., J. Biomol. Screen 5 (2000), 169-176) or small-molecule inhibitors for the interaction between peptides in a phage library of the tyrosyl- H. influenza tRNA synthase (Hyde-DeRuyscher et al, Chem. Biol. 7 (2000), 17-25). Molecules, ie more precisely inhibitors, which displace the nucleic acid sequences of NPY according to the invention could be functionally equivalent to those according to the invention Be nucleic acids. In addition to the use of the nucleic acids according to the invention as inhibitors, they can thus also be used to identify compounds from a library or assign a specific function or use to the members of this library, namely that they have an action analogous to the nucleic acids according to the invention and the interaction between Modulate NPY and Y2 receptor, inhibit more precisely. In addition to this use, the nucleic acids according to the invention, like the members of the library identified in the screening method according to the invention, can also be valuable guide structures and thus serve to develop novel therapeutic agents. In these screening systems, the interaction between the nucleic acids according to the invention and the NPY can, as already described, be detected using fluorescence-based methods (for example FRET) or using aptazyme contracts. When the nucleic acids according to the invention are displaced by a substance present in the library, a signal is generated. The substance identified in this way binds to the same interaction center as the nucleic acids according to the invention. As a result, the identified substance with regard to binding is a functional analog of the nucleic acid according to the invention and, with increased probability, is a substance that can inhibit the downstream signal transduction cascade by binding to the active conformation of the NPY. An advantage of this screening system is that neither the substance library nor the target protein has to be labeled. As a result, the process can be considerably rationalized. In addition, the properties of the substances contained in the library and of the protein are not changed by the markings that are usually introduced retrospectively in conventional processes. Another advantage is that the screening is focused on the interaction center, since all substances that bind to other areas of the protein are not detected. This increases the chances of identifying substances with the desired inhibitory effect.

In a further aspect of the invention, this also relates to those compounds which are obtained by such a process or to which a certain property can be assigned by such a process and in particular their use for the manufacture of medicaments, the medicaments preferably being intended for those diseases whose pathogenicity mechanism is based on the screening process or its conception. Examples and figures

The invention is explained below on the basis of examples, figures and the sequence listing, from which further features, embodiments and advantages of the various aspects of the invention result. It shows:

1 shows the primary structure of neuropeptide Y;

2 shows the selection target NPY, which is N-terminally biotinylated and immobilized on streptavidin agarose; 3 shows the course of the NPY selection up to the sixth selection cycle;

4 shows the course of the NPY selection from selection cycle 6c;

Fig. 5 shows a binding study of the selected RNA library to NPY after cycle

12 compared to the unselected pool RNA; 6 shows the sequences determined from the enriched pool of the NPY selection and consensus sequences derived therefrom; Fig. 7 binding curves of representative aptamer sequences to NPY;

8A shows the sequences of the synthetic peptides neuropeptide Y, random NPY and the human pancreatic polypeptide; 8B shows the dissociation constants determined for various aptamers;

9 shows the desorption profile of the aptamer DP3 in the presence of NPY immobilized on streptavidin agarose; Fig. 10 Stability of 2'-amino-2'-deoxypyrimidine (2'NH ₂ ) DP3-RNA (A) and unmodified 2 'hydroxy (2'OH) DP3-RNA (B) in fetal calf serum;

11 shows the binding of the NPY aptamer (DP3) to various, immobilized

NPY variants;

12 shows the competition of the specific binding of 3H-NPY to G protein-coupled transmembrane receptor subtypes;

13 shows a model of the biologically active NPY conformation on the Y2-

Receptor;

14 shows the result of a study on the binding of unselected and selected nucleic acid pools to NPY; and

15 shows the binding behavior of various selected clones as a function of

Amount of available NPY as the target molecule of the aptamers. Example 1: In vitro selection of neuropeptide Y (NPY) specific RNA molecules and their functional characterization

The course of the in vitro selection

In the first selection margin, a radioactively labeled 2'-amino-pyrimidine-RNA library of 3.75 nmol (approx. 2.3 genome copies) with the N-terminally biotinylated and immobilized NPY (see FIG. 2) was obtained after separation the pre-column binder (streptavidin agarose binder) by the preselection. It can be assumed statistically that two candidates from each sequence are represented in the initial pool. The non-binding RNA molecules were removed by washing with 100 column volumes of selection buffer. Subsequently, the 0.34% (based on the total amount of radioactively labeled RNA) of the functional sequences bound to NPY was determined under denaturing conditions (7M urea, 3 mM EDTA). In the following selection margins (cycles 2 to 6) both RNA concentration (500 pmol = 3.3 μM) and the NPY concentration (26 μM) were kept largely constant. A detailed overview of the elution results and the incubation and washing conditions of the first six selection cycles is shown in FIG. 3, the relative proportion of bound RNA in [%] being plotted for the individual selection cycles (abscissa). An unspecific binding of the nucleic acid libraries to the underivatized column material (see guard column [%]) could not be detected in the course of the entire selection, as can be seen in Tables 2 and 3, as can the detailed conditions of the selection and preselection and their results (SV = column conditions) (Tables 2 and 3 are shown as a list under the arrow diagrams in Figs. 4 and 5.)

After six selection cycles, there was a clear accumulation of 23.8% of NPY-binding RNA molecules (FIG. 3, cycle 6, FIG. 4, cycle 6a). With the aim of selecting as affine aptamers as possible in this selection cycle, the selection cycle 6a was repeated in two parallel approaches with the RNA library generated in cycle 5 under different, more stringent conditions:

If the peptide concentration used was reduced to 5 μM, the percentage decreased Proportion of bound RNA to 10% (parallel approach 6b). Under even more stringent conditions with an ionic strength of 300 mM NaCl in the incubation batch, an increased wash volume from 100 to 650 column volumes and a wash buffer mixed with 300 to 750 mM NaCl and 1% Triton X-100 (Fig. 12 6c), a drastic reduction was shown the NPY-RNA ligand to 0.95% (parallel approach 6c). These very affine binders were eluted and used in the 7th selection cycle.

In the following selection margins (cycle 7-12) the selection pressure was gradually increased with the background of selecting as specific NPY aptamers as possible. For example, the non-specific competitor heparin, a negatively charged glucosaminoglycan, was added to the binding approach from cycle 7 (final 1 μg / μl), the washing volumes were increased and both NaCl (300mM, 500mM and 750mM) and Triton X100 (1% ) added. Furthermore, the NPY concentration was gradually reduced to 2.5 μM (cycle 9 to 12). The further course of the selection (6c-12) and the detailed selection conditions are shown in FIG. 5 and the table below, which is also referred to as Table 3 herein. Analogous to cycle 6, the 8th selection cycle was carried out with two parallel approaches. The ninth selection cycle was continued with the RNA library generated in cycle 8b. Due to the more stringent selection conditions, the proportion of NPY-binding RNA molecules decreased from approx. 8% in the 7th cycle to less than 2% in cycle 9 to 12. The amino acid sequences of peptides I and III are as follows:

Peptide I NH ₂ -WNNDNPLFKS ATTTVMNPKFAES-COOH (SEQ.ID.No. 19)

Peptide III NH ₂ -DLREYRRFEKEKLKSQW-COOH (SEQ.ID.No. 20)

Aprotinin (0.2 μg / μ), leupeptin (0.2 μg μ) and Pefabloc (0.5 mM) were used as protease inhibitors (inhibitors) in cycle 9-12 (SV = column volume, FCS = fetal calf serum) ,

In order to ensure binding of the selected RNA molecules to non-immobilized NPY, the target peptide was incubated with the enriched pool for 1 hour at 37 ° C. in solution and only then on streptavidin agarose for 15 minutes at 37 ° C. immobilized. Through the use of derivatized precolumn material (cycle 7 to 9), immobilized with synthetic peptides, which are characterized by different isoelectric points of pI 6, l (peptide I) and a pl 10 (peptide III) (cf. material) RNA sequences binding due to non-specific peptide interactions are eliminated. Since no increased proportion of bound nucleic acids was detectable up to the ninth cycle in the case of the precolumn material (FIG. 4), preselection was dispensed with in the last three rounds.

In order to further increase the stringency of the selection and to bring the in vitro conditions as close as possible to the conditions "in vivo", the ninth to twelfth selection mouths were carried out in the presence of 10% FCS. The proportion of NPY-binding RNA ligands has thus decreased further. As was shown in a preliminary experiment, no signs of degradation were detectable for NPY in the presence of 10% FCS and the protease inhibitors aprotinin, pefabloc and leupeptin for more than 2 hours. A renewed enrichment under FCS conditions could not be achieved in the following selection cycles. The selection was ended after the 12th cycle.

The binding of the selected pool to NPY

Before the cloning rank and sequencing of the selected ligands, the binding behavior of the enriched RNA library from cycle 12 and the unselected pool RNA was examined in the presence of NPY. The individual binding reactions were composed of different peptide concentrations (50 nM-7 μM) and a constant RNA concentration (1 nM), which was kinased at the 5 'end.

To denature the ribonucleic acids, the batches in binding buffer without peptide were heated to 90 ° C. for 1 min, immediately cooled on ice, MgCl ₂ (1 mM) was added, the mixture was centrifuged briefly and the peptide was then mixed in carefully. Incubation was carried out for 1 h at 37 ° C under schwa ^¬ chem agitation (7,000 rpm) in a Thermomixer. Bound and unbound RNA were separated by nitrocellulose filtration and the radioactivity remaining on the filter membranes (0.45 μm, Millipore) was quantified using the phosphorimager (STORM860).

The dissociation constants (Kd values) for the individual representative aptamer sequences at the NPY were determined using the following equation:

q = (f / 2R _t ) = (P _t + R _t + Kd - [(P _t + R _t + Kd) ² -4P _t R _t ] ^{1 2} (Equation 1) q is the proportion of RNA bound in equilibrium. P _t and R _t are the total peptide and RNA concentrations, respectively. f represents the efficiency of RNA-peptide complexes retained on the nitrocellulose filter (Jellinek et al, 1994).

The binding study shown in FIG. 5 shows a comparison of the binding behavior of the selected RNA library to NPY after cycle 12 with that of the unselected RNA. The relative proportion of complexed RNA [%] is shown depending on the NPY concentration [μM] used.

It is clear from the binding curves shown in FIG. 5 that the enriched RNA library from cycle 12 shows a substantially higher affinity for NPY than the unselected pool. While the curve of the selected library had a saturation range at an NPY concentration of 5 μM, no significant retention on the nitrocellulose membrane could be detected for the unselected pool. For this reason, it was not possible to determine the dissociation constants for the unselected pool RNA. A dissociation constant of approx. 500 nM was calculated for the selected pool RNA.

Cloning and sequencing of the selected pool

After the selection has been made, there are various RNA molecules in the enriched pool which have different binding properties for the target peptide NPY. In order to identify and characterize these individual sequences, the RNA library was cloned after the 12th cycle. By reverse transcription of the eluted RNA and subsequent PCR amplification of the cDNA obtained, the restriction sites necessary for the cloning rank could be introduced by the primers PM39-5 'and PM-25-3'. After restriction digestion of the PCR product with Hind III and Bam HI, the ds DNA was ligated into the analogously treated vector pGEM-4Z (Promega). The ligation products were transformed into E. coli, propagated and the plasmid DNA for sequencing was isolated from individual E. coli clones.

Sequencing of 30 clones (Sanger, et al., 1977) resulted in sequences that could be divided into two families (I, II) due to their almost identical primary structure. In addition, among the sequenced clones were candidates who had no homology to the quenzfamihen or have each other. These sequences, which occur only once, are generally referred to as orphans (FIG. 6).

The sequences according to the invention which can be assigned to the various families and the resulting consensus sequences according to the invention, generally referred to herein as sequences according to the invention, are as follows and are shown again in FIG. 6:

Family I

Clone sequence

DPll (ll) CAAGGCCGGGTTAGGTGGGACCGTGATCTGATGTGGCGTG (SEQ.ID.No 1)

DPI CAAGGCCGGGTTAGGCGGGACCGTGATCTGATGTGGCGTG (SEQ.ID.No 2)

DP27 CAAGGCCGGGTGAGGTGGGACCGTGATCTGATGTGGCGTG (SEQ.ID.NO 3)

DP12 (2) CAA.GGCCGGGTTAGGTGGGACCGTGATCTGATGTGACGTG (SEQ. ID .No 4)

DP28 (2) CAAGGCCGGGTTAGGGGGGACCGTGATCTGATGTGGCGTG (SEQ.ID.No 5)

DP2 CAAGGCCGGGTTAGGTGGGACGTGATACTGATGTGGGGAG (SEQ.ID.No 6)

DP5 (2) CAAGGCCGGGTTAGG-GGGACCGTGATCTGATGTGGCGTG (SEQ.ID.No 7)

DP20 CAAGGCCGGGTTAGG-GGGACCGTGATCTGTAGTGGCGTG (SEQ.ID.No 8)

DP26 (2) CAAGGCCGGGTTAGGTGGGAC-GTGATCTGATGTGGCGTG (SEQ.ID.No 9)

DP22 CAAGGCCGGGTTAGGTGGGACTGTGATTTCGATGTGGCTG (SEQ.ID.No 10)

DP4 CAAGG GGTTAGG-GGGACCGTGATCTGATGTGGCGTG (SEQ.ID.No 11)

Consensus sequence:

CAAGG FFHGG TKAGG XGGGA CX KR W YTS DWKKK RSS G (SEQ.ID.No 12) where F is either C or a nucleotide deletion at the site, H either G or a nucleotide deletion at K site either G or T , X is either G, C or T or a deletion by one

Nucleotide at W either A or T Y either C or T

S either C or G

D either A or G or T

R means either A or G. Family II

DP3 CAGCAGGAGGGCCGGCGTTAGGGTTAGCGAGCCGATTGAA SEQ.ID.No. 13)

DP8 CAGCAGGAGGGCCGGCGTTAGGGTTAGCGAGCCGAT-GAA SEQ.ID.No. 14)

Consensus sequence:

CAGCAGGAGGGCCGGCGTTAGGGTTAGCGAGCCGATLGAA SEQ. ID. No. 15)

where L means either T / U or a deletion by one nucleotide at the site.

orphans

DP14 CTGCCAAAGGTTGGCCATGTGTGGGGGAAGCTCCAACGT- SEQ. ID .No .16) DP19 GCCACCAATGCGCACCCACCCAGACACGTCAAGTCAAGT- SEQ. ID .No .17) DP25 GGATACTTAGCGTAAGTNAGTGGCTGGGGGGTGACGAAG- SEQ. ID .No .18)

The sequences according to the invention shown in FIG. 6 represent only the originally randomized region 40 N of the nucleic acid library used without the constant primer binding sites. The number of identical sequences is indicated in the brackets. The 2'-amino-2'-deoxycytidine and 2'-amino-deoxyuridine residues are designated as C or T. N shows a nucleotide residue that cannot be clearly determined. Mutations and deletions (-) within a family are shown in bold, which always refer to the first sequence shown.

The sequencing showed that the diversity of the pool was greatly minimized after 12 selection edges at the NPY. The majority (approx. 80%) of all selected clones belong to sequence family I. The members of this highly conserved family differ only in point mutations and deletions. The most common family I sequence is clone DP11. The second sequence family consists of only two sequences that differ in one position (see Fig. 6).

The nucleic acid sequences according to the invention further comprise those sequences which are in the sequences according to SEQ. ID.No. 1 to 18 are contained, in particular those which have a length of at least 10, 15, 20, 25, 30, 35 or 40 nucleotides. Example 2: Determination of the dissociation constant of individual sequences on NPY by nitrocellulose filter binding studies

In order to determine the affinity of representative sequences on NPY, nitrocellulose filter binding studies were carried out. The target peptide NPY was incubated in increasing concentrations with the individual, radioactively labeled RNA sequences. Bound RNA was separated from unbound RNA via nitrocellulose filtration and the radioactivity remaining on the filter membranes was quantified as in the preceding examples. By adding 20 μg streptavidin per batch, the retention of the short peptides on the filters should be improved.

It could be shown that all tested sequences except DP 14 and DP 19 (orphans) can complex the free peptide in solution, although aptamers against the immobilized NPY were generated in the first selection cycles. FIG. 7 shows the proportion of NPY-bound RNA which was retained on the filter as a function of the NPY concentration. The following assignment applies: Family I DP4 (open square) and 11 (filled square), Family II DP3 3 (filled circle), orphan 25 (filled triangle), orphan 19 (open triangle), pool (open circle). The fraction of the bound ³² P -labeled RNA is shown as a function of the NPY concentration used, the lines representing the fit of the individual data points for each aptamer in accordance with a monophase binding reaction. The results from two independent determinations (standard deviation) are included in each curve.

The dissociation constants (Kd), which describe a monophase reaction (cf. Eq. 1), could be determined from the kinetics of the individual sequences. Kd values of 470 nM and 410 nM were calculated for the most frequently occurring sequence DP 11 and for the clone DP4, which also belongs to family I. Ligand DP3 from family II shows the highest NPY affinity with a dissociation constant of 370 nM. From this grand, DP3 was used for further investigations. A Kd value of 25 μM was determined for DP25, an affinity for NPY almost 70 times lower than that of DP3. The dissociation constants for the sequences DP 14 and DP 19 could not be determined because only 10% of the entered set RNA at a concentration of 7 μM NPY were retained on the filter. The unselected pool RNA shows a low binding of 4% at an NPY concentration of 7 μM, which cannot be quantified in terms of a monophase reaction.

Example 3: Specificity of DP 3 within the pancreatic polypeptide family

In a preliminary experiment, an in vitro filter retention assay showed that both aptamer DP3 and DP 11 did not bind the completely randomized NPY. In order to investigate the specificity of DP3 within the pancreatic polypeptide family, its binding ability to the pancreatic polypeptide (PP), which is a member of the pancreatic polypeptide family along with the neuropeptide Y (NPY) and the peptide YY (PYY), was tested in a nitrocellulose filter assay. A common feature of the structure-related, tyrosine-rich peptides is a sequence of 36 amino acids with a C-terminal tyrosinamide as shown in the introduction. The hPP was not bound at a concentration of 10 μM of DP3, although NPY and hPP are 50% sequence homologous, as can be seen from FIG. 8, which in part A shows the sequence of the synthetic peptides neuropeptide Y (NPY), random NPY ( rNPY) and the human pancreatic polypeptide (hPP) (homologous amino acid residues of NPY and hPP are highlighted in bold, CONH ₂ represents the carboxy-terminal amidation) and in part B shows the determined dissociation constants (nM) of the in vitro selected aptamers (nd = undetectable) , In a further experiment, the hPP was immobilized on streptavidin agarose (30 μM) via its N-terminal biotin residue and incubated with radioactively labeled RNA under selection conditions (corresponding to cycle 5). No interaction between DP3 and the hPP could be detected here either.

Example 4: Determination of the dissociation constants of the aptamer DP 3 on NPY by analytical affinity chromatography

In the filter retention assay it was determined that the aptamer DP3 is characterized by the best affinity for NPY in comparison to the other sequences examined. Since incomplete retention of the peptide complexes can influence the Kd values, the dissociation constant of DP3 should be verified by analytical affinity chromatography. 300 μl NPY streptavidin agarose material (120 μM) was filled into a 1 ml syringe without air bubbles, which was sealed with glass wool at the bottom. Care should be taken to prevent the column material from running dry. The affinity matrix was first equilibrated in the binding buffer

K ₂ HPO ₄ 0.9 mM

NaH ₂ PO ₄ 5 mM

NaCl 70 mM

MgCl ₂ I mM

KC1 1.3 mM

Heparin 1 μg. ul

and ensured the optimal pH of 7.6 and the MgCl concentration of 1 mM. Then 50 pmol of [α- ³² P] -GTP-labeled RNA were applied to the derivatized streptavidin agarose in a volume of 50 μl binding buffer, in which it was denatured and renatured. In order to ensure the sharpest possible peak separation, the column was prepared with the derivatized Sepharose material with a small diameter of 5 mm and a filling height of 6 cm. The mixture was then washed with binding buffer until no radioactivity could be detected on the column. The run was collected in fractions from 250 ul to 1000 ul binding buffer and the radioactivity amount for each fraction was determined in the scintillation counter. The elution profiles were then plotted by adding the absolute radioactive values (y-axis) against the corresponding elution volume (x-axis). The first wash fractions, which contained non-binding RNA, were not taken into account. The elution profiles were used to determine the elution volume (V) at which half of the aptamers bound to NPY (L) could be eluted again from the affinity matrix.

The dissociation constants were calculated according to the following equation (Dean, 1995):

Equation 1 _κd _ (V - Vm) x [L] V - Vo

[L] = concentration of the peptide immobilized on the affinity matrix. V = The elution volume of the ligand corresponds to the washing volume with binding buffer, in which half of the ligand bound to the affinity matrix is again eluted from the column.

V ₀ = total penetrable volume of the column. It was determined on the basis of the elution volume of non-specific RNA molecules (Pool Mic Pmodl-40N).

V _m = the gel exclusion volume was determined using the elution tip of a Sepharose

Matrix of non-penetrating molecule (dextran blue) determined.

Kd = dissotation constant of the ligand (RNA aptamer) to the target molecule (peptide) immobilized on the matrix. The following applies: the smaller the aptamer dissociation constant, the later the nucleic acid ligands will elute.

Since a saturation range is achieved (Fig. 9), it was possible to determine the elution volume at which half of the ligand bound to the affinity matrix was again eluted from the column using the binding buffer described above. The elution volumes determined in duplicate and the concentration of the immobilized peptide were ideal for calculating the dissociation constants. According to equation 2, DP3 results in a Kd value of 300 nM. This Kd value is comparable to the Kd value determined by the filter retention experiment (370 nM) for DP 3. Based on this result, it can be assumed, on the one hand, that the retention of the RNA-peptide complexes on the nitrocellulose filter was quantitative, and, on the other hand, that the binding behavior of DP3 to the immobilized ligand is almost identical in comparison to the ligand in solution. Interaction with the unselected RNA library (Mic Mod 40N) and the NPY-derivatized column material could not be detected.

Example 5: Stability of the 2'-amino-2'-deoxy nucleic acids

Since the biological activity of the aptamer DP3 was to be investigated in competition studies in a cell culture model, the stability of DP3 in MEM medium was which contains 10% fetal calf semen (FKS). The radioactively labeled RNA was incubated at 37 ° C. with the medium for 48 hours and analyzed after various times. (60 pmol 2'NH ₂ or 2'OH RNA were incubated in 200 μl MEM buffer with 10% FCS for 24 or 48 hours at 37 ° C. A 20 μl aliquot was taken from each reaction mixture after the corresponding times removed and applied to a 15% PAA gel.)

10 shows the autoradiograms for 2'-amino-2'-deoxy DP3 (A) and for 2 'hydroxy DP3 RNA (B). It could be shown that the stability of the 2'-amino-2'-deoxy modified RNA aptamer DP3 in 10% FCS is significantly increased compared to the unmodified RNA with the same primary sequence. FIG. 10 A shows that no degradation can be detected for the 2'-amino-2'-deoxy DP3 RNA within 12 hours. Only after 48 hours can part of the 2'-A_nmo-2'-Deoxy DP3 RNA degradation be detected. In contrast, the unmodified DP3-RNA molecules show a very low stability in 10% FCS (FIG. 10 B). After 5 min they are completely broken down into shorter fragments, after 10 min only the radioactive labeled nucleotides can be seen on the lowest running front of the gel. The unselected MicMod40N pool has sufficient stability for the cell culture experiments in which cells are incubated with the RNA for 90 min (Blind, 2000). These observations are in good agreement with previous studies of 2'-modified RNA libraries in biological media (Lin, et al., 1994).

Example 6: Identification of the minimal aptamer binding site on NPY

In order to be able to define the binding region of DP3 at the NPY more precisely, various biotinylated NPY fragments (7 μM) were incubated with the radioactively labeled RNA ligand DP3 (50 pmol). The biotinylated peptides were present in the immobilized state in order to avoid precipitation problems, such as those that occurred with some peptide fragments in previous experiments in solution. The various NPY peptides used are shown in Fig. 11, with the following assignment:

1 to 7, 9, 10: NPY variants with different N- and C-terminal amino acid elements 11 to 14 substitutions of the amino acid arginine by alanine at different positions in the C-terminal region, amino acid mutations are given in square brackets and highlighted in bold in the peptide sequence.

8: Y2 receptor agonist, characterized by the substitution of the central amino acids 5-24 with an aminohexanoic acid linker. 15: Yl receptor agonist, 16 Y5 receptor agonist, Aib = aminoisobutyric acid

The amino-terminal Biotinyliemng and the carboxy-terminal Amidierang are characterized by Biotin and -CONH ₂ . The relative binding affinities of the different peptide variants are divided into three classes compared to the NPY: - 0-10% binding compared to the NPY control, + 11-80% binding compared to the NPY control, ++ 81-100% binding in Comparison to the NPY control. The amino acid residue (R33), which is essential for the interaction with the aptamer DP3, is highlighted.

It could be shown that both the N-terminus of NPY (AS 1-10) and the central amino acid section (AS 5-24) are not necessary for the interaction with the aptamer (Fig. 11 Nos. 1 to 8). In contrast, the following dissociation constants for DP3 were determined for the C-terminal NPY variants in a filter binding study: NPY 18-36: 1160 nM, NPY [Ahx ^{5 "24} ]: 710 nM and NPY 13-36: 760 nM, which is an approx. Corresponds to 2-3 times poor affinity compared to the total NPY (Fig. 11 Nos. 8 to 10), so the C-terminus represents the interaction area between NPY and DP3.

To investigate the extent to which the basic arginine side chains influence the electrostatic interaction between the negatively charged RNA molecule and the positively charged peptide motif, four arginines in the C-terminal region were substituted by alanine residues. It was shown that arginine at position 33 is essential for the interaction with the aptamer. In contrast, an exchange of the arginine at position 19 to the alanine does not affect the interaction with the RNA. Binding to the NPY derivative with arginine substitution by alanine at position 25 is reduced by 50%. The arginine residue 35 plays a minor role in the formation of aptamer NPY complexes: Despite substitution by the uncharged alanine, about 3/4 of the RNA used is still bound in a binding experiment (cf. FIG. 11). These results allow the following conclusions to be drawn: The C-terminal NPY region represents the epitope which is bound by DP3, DP3 having a high specificity with regard to an amino acid residue, arginine 33. A point mutation at this point (R33A) leads to a complete loss of peptide recognition.

The investigations also show that the more shortened C-terminal fragments NPY 18-32, NPY 18-34, NPY 18-28, NPY 25-34, NPY 25-36 (Fig. 19, No. 3 to 7) , which reflect the postulated interaction domain, have no or only a very weak interaction with DP3 as drank NPY contracts.

Example 7: Interaction of aptamer DP 3 with specific NPY receptor agonists

One method of characterizing the NPY receptor subtype interaction was the development of specific receptor subtype agonists (cf. Table 1). In analogy to this, the binding behavior of the aptamer DP3 to various NPY agonists should be investigated.

The substitution of gin 34 after proline produces a highly selective Y1 receptor agonist (Fuhlendorff et al, 1990), which is no longer bound by the Y2 receptor. Cabrele et al. were able to show that the dipeptide Ala31-Aib32 favors a conformation which is preferentially bound by the Y5 receptor (Cabrele, et al., 2000). Both modifications affect the interaction with DP3. While no binding could be detected on the NPY mutant Pro 34 (Yl receptor agonist) (see Fig. 11, No. 15), the interaction with the Ala31 Aib32 NPY mutation is reduced by approx. 50% compared to the interaction of DP3 and NPY (see Fig. 11, No. 16). In contrast, DP3 has a high affinity for the centrally shortened Y2 receptor agonist NPY [Ahx ^{5 "24} ] (see Fig. 11, No. 8) (Beck et al., 1989). The N-terminally shortened NPY Variants (cf. FIG. 11, No. 9 and 10) which are only bound by the Y2 receptor, but not by the Y1 or Y5 receptor, are bound by DP3 almost as well as the entire NPY.Aptamer DP3 shows binding behavior to NPY is similar to that of the Y2 receptor, and these clues will be further investigated in the following by receptor competence studies. The selective inhibition of NPY / NPY receptor interaction by DP3

The inhibition of NPY binding to individual receptor subtypes by DP3 was investigated on neuroblastoma cell lines which endogenously express either the hYl receptor (SK-NM-C cells) or the hY2 receptor (SMS-KAN cells). BHK cells (ATCC No. CRL-1632), which were stably transfected with the Y5 receptor (rY5), were used for competition on the Y5 receptor. Clone DP 3 showed the highest affinity within the analyzed sequences in the in vitro investigations to determine the dissociation constant and was therefore used for the cell culture studies. The unselected pool RNA, which has no in vitro affinity for NPY, was used as a control approach.

The results of the competition studies are summarized in FIG. 12, more precisely the competition of the specific binding of ³ H-NPY to G protein-coupled transmembrane receptor subtypes Yl (A), Y2 (B), Y5 (C) as a function of the RNA Concentration (aptamer RNA (filled circle), unselected pool RNA (unfilled triangle)). It can be seen that the specific binding of NPY to the investigated receptor subtypes is increasingly inhibited with increasing concentration of DP3. This effect cannot be observed in the presence of the unselected RNA pool. The course of the competition curve at the Yl and Y5 receptor differs significantly from that of the competition curve at the Y2 receptor: The competition between DP3 and the Y2 receptor for the NPY can be described by monophasic kinetics. The IC ₅₀ values determined indicate the concentration of DP3 at which half-maximum inhibition of the binding of NPY to a receptor subtype is achieved. For the competition of the binding of NPY to the Y2 receptor, an IC ₅₀ value of 73 + 25 nM was calculated. In contrast, the competition curves at the Y5 and Yl receptors are characterized by a flat, biphasic course. The determined IC ₅₀ values for Yl and Y5 with 328 nM ± 141 (Yl) and 416 nM + 63 nM (Y5) are significantly higher than the IC ₅₀ value for DP3 at the Y2 receptor. From the competition curves (Fig. 12, A and C) it is also clear that at a DP3 concentration of 1-10 nM about 10-20% of the binding of NPY to the Yl or Y5 receptor is inhibited. A complete competition of the complex formation of NPY and the receptor subtypes (Yl, Y5) is only achieved at a DP3 concentration of 1000 nM.

The corresponding Kj values could be calculated from the calculated IC ₅₀ values, taking into account the Kd value for NPY for the respective receptor subtype (cf. Table 4). Demzu- consequently, a Kj value of 1.3 nM shows an approximately 40-200 times higher DP3-dependent inhibition of the NPY / Υ2 receptor interaction than the inhibition of NPY binding to the Y1 and Y5 receptors, although the Y2 Receptor has the highest affinity for NPY.

Receptor IC ₅₀ (nM) Kd (nM) Kj (nM)

Yl 328 + 141 0.18 50 + 21 Y2 73 + 25 0.02 1.3 + 0.5 Y5 416 + 63 1.7 262 ± 40

Table 2: IC ₅ o values for the receptors were determined from the competition studies of DP3 in the cell culture system. The individual Kd values for NPY binding to the receptor subtypes Yl, Y2 and Y5 are taken from the literature (Cabrele and Beck-Sickinger, 2000). The Kj values for DP3 were calculated using the following equation 2: K ₍ = IC ₅₀ [(l + L) Kd ^"1 ] ^{" 1} , where L is the concentration of ³ H NPY used (for Yl and Y5 [1 nM] , for Y2 [2 nM]).

Example 8: The selection and in vitro characterization of NPY-specific aptamers

The neuropeptide hormone NPY is involved in a variety of physiological control processes in the human organism. Complex ligand-receptor interactions on different G-protein-coupled NPY receptor subtypes are necessary for the differential regulation of different target organs by a peptide.

In the context of the present invention, the stabilized 2 'amino-modified RNA aptamers were selected in vitro, which can bind the neuropeptide Y with high specificity in order to create a selective tool for characterizing the NPY-NPY receptor subtype interaction. Sequences of frequently selected structure motifs bind highly affinitely

In binding studies with NPY, dissociation constants from 370 nM to 470 nM were determined for the sequence families I and II (DP3, DP4 and DP11). In contrast, the simple clones have a lower NPY affinity (see FIG. 7). The majority of the selected and analyzed aptamers are able to bind the free peptide, although these were generated in an affinity-chromatographic selection process with immobilized NPY. For DP3, quantitatively comparable dissociation constants were determined both in the filter binding assay and in analytical affinity chromatography (cf. FIG. 9). This observation is an indication that NPY can adopt a similar conformation in the soluble or immobilized state and that the potential binding domains are accessible to the aptamers in both cases.

The affinity of the NPY aptamers for their target molecule is comparable to that of previous peptide selections. For the HIV-1REV aptamer, for example, a Kd value of 19 nM (Xu and Ellington, 1996), for the substance P aptamer of 190 nM (Nieuwlandt, et al., 1995), for the CD18-cyt peptide aptamer of 500 nM (Blind, et al, 1999) and a Kd of 900 nM (Williams, et al., 1997) for the vasopressin aptamer. The HIV-1 REV aptamers show the highest affinity among the anti-peptide aptamers due to the high proportion of positively charged arginine residues (10 of the 17 amino acids in total) of the HIV-1 REV peptide, which is also a natural RNA binding partner.

Since the aptamer DP 3 has the highest NPY affinity compared to the other representative sequences examined, DP3 was used for the further characterization of the NPY-aptamer interaction as well as for the subsequent NPY-NPY receptor subtype interaction.

The full C-terminus of NPY is mandatory for receptor and DP3 binding

In order to map the binding region of DP3 to NPY, different subunits of NPY (N- and C-terminally shortened NPY variants) and mutant variants of NPY were examined (see Fig. 11). According to our binding study, the full C-terminus, in particular the amino acid arginine at position 33, is required for the interaction with the DP3 aptamer. The C-terminal epitope of NPY also represents the NPY interaction domain for the interaction with all receptor subtypes (see Fig. 2). Only biologically active NPY forms are bound by DP3. NPY fragments that only consist of parts of the C-terminus are neither complexed by DP3 (Fig. 19, entries 1-7) nor by an NPY receptor subtype (personal message A. Beck Sickinger): The C-terminal contract NPY 18-34 are missing the two terminal amino acids R35 and Y36 compared to the NPY 18-36 contract. The NPY 18-34 contract shows no binding to DP3 or to an NPY receptor, while the N-terminally truncated NPY fragment NPY 18-36 is a selective Y2 receptor agonist (IC ₅₀ = 0.25 nM). The affinity of DP3 to NPY 18-36 is also comparable to the affinity of DP3 to NPY. The complete C-terminus of the NPY is mandatory for the interaction with the receptors, but not sufficient. It is believed that there are various biologically active conformations of NPY that can selectively activate certain NPY receptor subtypes (Beck-Sickinger, 1996).

DP3 and the Y2 receptor show comparable binding behavior to selective NPY variants

An established method for studying the selective binding and activation of the different receptor subtypes by NPY is the investigation of selective receptor agonists and their binding behavior to NPY receptor subtypes (see introduction), (Cabrele and Beck-Sickinger, 2000). The selective agonists known to date from the literature are exclusively NPY peptide variants.

After it was shown that DP3 can bind a selective Y2 receptor agonist (NPY 18-36) with high affinity, the binding behavior of DP3 to other receptor subtype-specific NPY variants was examined.

The substitution of isoleucine by leucine at position 31 of the NPY and in particular the replacement of the glutamine side chain residue at position 34 with the tumor-inducing amino acid proline leads to a specific Y1 receptor agonist (Fuhlendorff, et al., 1990). Interaction of DP3 with the Yl -specific NPY [L ³¹ P ³ ] mutant could not be detected the. The Y5-specific NPY mutant Ala31-Aib32 is also only bound by DP3 with reduced affinity (see Fig. 11).

In the selective Y2 receptor agonist NPY [Ahx ^{5 "24} ], 4 N-terminal amino acids are linked to the C-terminal NPY fragment NPY 25-36 via an aminohexanoic acid linker. While the isolated C-terminal fragment NPY 25-36 is neither biologically is still bound by DP3, the centrally deleted NPY variant is both a selective Y2 receptor agonist (Y2 = IC ₅₀ 2nM, Yl = IC ₅ o> 4 μM, Y5 = IC ₅₀ 795 nM) and a binding partner of DP3 (Beck, et al, 1989) It is discussed that the biologically active NPY conformation, which is selectively bound by the Y2 receptor, forms a hairpin-like structure, with the N and C terminus being spatially close together ( See Fig. 2. The C-terminus represents the functional region (binding region), the N-terminal region being the structural component that stabilizes the correct orientation of the C-terminus (Cabrele and Beck-Sickinger, 2000).

The binding studies with the NPY variants, specific for individual receptor subtypes, show that DP3 is characterized by a selective binding behavior to NPY similar to the Y2 receptor subtype (cf. Table 5).

NPY variant Yl receptor Y2 receptor Y5 receptor aptamer-DP3

NPY + + + + + + + +

NPY 18-34 - - - -

NPY 13-36 - + + - -H-

NPY 18-36 - + + - + +

NPY [Ahx ^{5 "24} ] - + + - + +

NPY [L ³¹ P ³⁴ ] + + - + + -

NPY [A ³¹ , Aib ³² ] - - + + +

Table 3: Binding behavior of NPY and NPY variants to the Yl, Y2 and Y5 receptor and the aptamer DP3 in comparison. Binding affinities: ++ Affinity from the NPY receptor subtypes or from DP3 to an NPY variant is comparable to the affinity for NPY; + the affinity is reduced by 50% compared to the NPY; - no binding is detectable

Mutants: NPY 13-36: and NPY 18-36: N-terminal shortened NPY variants, NPY 18-34: N- and C-terminal shortened NPY variants, NPY [Ahx ^{5 "24} ]: substitution of the central amino acids 5 -24 by an aminohexanoic acid linker, NPY [L ³¹ P ³⁴ ]: substitution of the amino acids at position 31 from isoleucine to leucine and at position 34 from glutamine to proline, NPY [A ³¹ , Aib ³² ]: substitution of the amino acids at position 31 from isoleucine to alanine and at position 32 from tyrosine to aminoisobutyric acid.

The binding affinities for NPY and for the NPY variants to the individual NPY receptors are taken from the publication Cabrele & Beck-Sickinger (2000) (cf. Tab. 1).

The selective competition of the binding of NPY to the Yl, Y2 and Y5 receptor by DP3

As a result of the selective in vitro binding behavior of DP3 to NPY variants comparable to the Y2 receptor, the question arises: Is the binding behavior of NPY to its various NPY receptor subtypes (Yl, Y2 and Y5) differentially modulated by DP3 in the cell culture model?

In the competition studies between aptamer, NPY and NPY receptor subtype carried out in the cell culture model, the lowest Ki value for DP3 was determined for the Y2 receptor (KJ: Y2 = 1.3 + 0.5). The competition of NPY on the Yl and Y5 receptors by DP3 was lower by a factor of 40 (Yl) to 200 (Y5) compared to competition on the Y2 receptor (KJ: Y1 = 50 ± 21, KJ: Y5 = 262+ 40). The unselected pool RNA showed no inhibition of NPY binding in any receptor subtype (Fig. 12, Table 4).

Not only the inhibition constant of DP3 in the presence of the Y2 receptor, but also the course of the curve in the presence of the Y2 receptor differs from the competition on the Yl and Y5 receptors. While a biphasic curve is characteristic of the Y1 and Y5 receptors, a monophasic curve is evident on the Y2 receptor. For the Yl and Y5 receptors, an inhibition of the binding of the NPY to the receptor by DP3 could be shown even at low concentrations (1-5 nM) (cf. Fig. 12). NPY binding by DP3 does not lead to a significant increase in the inhibition of the NPY-NPY receptor interaction when the aptamer concentration is increased. Only at a DP3 concentration of 50-100 nM does the inhibition of NPY receptor binding increase again significantly. Such a biphasic curve shape can be described mathematically by a receptor binding model based on two binding sites.

For the Y2 receptor, on the other hand, there is a monophasic curve, which corresponds to a receptor binding model based on a binding site. The attempt to compete on the Y2 receptor lacks the aptamer's initial high-affinity binding behavior at the NPY, which characterizes the DP3 concentration range of 1-5 nM at the Yl and Y5 receptors.

This suggests that NPY exists in different conformations that differentially activate certain receptor subtypes. The aptamer DP3 binds to the different conformations of the NPY with different affinities. Thus, the NPY conformation, which activates the Y2 receptor, is a conformation bound by DP3 with a relatively high affinity. On the other hand, two active NPY conformations can be postulated for the receptors Y1 and Y5, one of DP3 highly affine and a second of DP3 very low affinity-bound NPY conformation.

The comparison shows the strongest overall inhibition of the aptamer DP3 on the Y2 receptor and the strongest interaction of the aptamer with the NPY conformation activating the Y2 receptor. It cannot be clarified whether these different conformations are induced by the Y2 receptor and then preferentially recognized by the aptamer or induced and / or stabilized by the aptamer itself. Ultimately, on the basis of the competition studies alone, it cannot be ruled out that the different inhibition of the aptamer in the binding of the NPY to its receptors is solely due to different NPY conformations and not to the structural characteristics of the receptor subtypes themselves in the sense of a "low and high affinity binding site" is due.

It is clear from the results of the receptor binding studies together with the results of the trials with truncated and mutated NPY variants that DP3 is a specific NPY ligand with mimetic Y2 receptor properties. This was the first time with the help of a selective NPY nucleic acid ligands are confirmed that the neuropeptide Y can be present in different biologically active conformations in vivo, the Y2 receptor-specific NPY conformation differing from the conformation which can activate the Y1 or Y5 receptor.

Arginine33 of the NPY is essential for aptamer binding

In the context of fine mapping experiments to characterize the minimal aptamer binding site at NPY, four positively charged arginine residues in the C-terminal region were substituted by alanine. It was shown that the arginine residue at position 33 is essential for the DP3-NPY complex bond. Substitution by alanine leads to complete loss of aptamer binding.

13 shows a model for the biologically active NPY conformation at the Y2 receptor, which is competed for by the aptamer: the C-terminal binding region for the aptamer DP3 to NPY is red (red, in black and white representation, lighter gray: for Binding essential) is highlighted, whereby the color gradation shows the corresponding meaning of the individual amino acids for the complex formation of DP3 and NPY (black: AS not involved in the binding).

A number of viral proteins (e.g. HIV-Rev, HIV-TAT) contain arginine-rich epitopes, which represent the binding region for the natural nucleic acid ligands. Structural analysis of the RNA-peptide complexes by means of NMR spectroscopy has succeeded in elucidating the molecular interactions between the arginine residues of the peptides and the RNA aptamers and in defining a typical arginine binding motif. The peptides are located in the "major groove" of RNA. Specific interactions through hydrogen bonds between the guanine residues of the RNA and guanidinium groups of the arginine side chains of the peptides stabilize the complex. In many cases, ligand binding requires an expansion of the "large groove" of the RNA helix, which is achieved, for example, by purine-purine base pairs. The RNA aptamers, which are unstructured in solution, are characterized by a defined architecture in the presence of the peptide (Hermann and Patel, 2000). For the HIV-1 Rev complex it was shown, for example by NMR analysis, that the structure of the peptide is also changed as a function of the nucleic acid ligand after the RNA binding. The HIV-1 REV peptide adopts an α-helical conformation after binding of a Rev aptamer (sequence family I), while the same peptide takes on an unfolded structure in complex with another Rev aptamer (sequence family II). For the MS2 coat protein it could be shown by X-ray crystal structure analysis that the protein has no conformational differences in comparison to the freely available MS2 coat protein despite the binding of a natural viral RNA or aptamer RNA. In this example, an induced fit mechanism only changes the structure of the RNA (Valegard et al., 1990), (Convery, et al., 1998).

In order to obtain qualitative information regarding the change in the secondary structure of aptamer DP3 and NPY after complex formation, CD spectroscopic analyzes were carried out.

Example 9: Selection of aptamers binding to neuropeptide Y.

Basically similar to the procedure described in the preceding examples, further aptamers against neuropeptide Y (NPY) were selected in accordance with the following process steps.

selection Criteria

The following buffer was used as binding buffer: 20 mM Tris pH 7.5, 100 mM NaCl, 5 mM MgCl ₂ . The incubation time was 7 minutes at 22 ° C. The target molecule used was synthetic, biotinylated NPY which was coupled to beads coated with streptavidin (Magnetic Dynabeack Dynal). The pool used, ie the nucleic acid library used as the starting library, had the following structure: 5'- GGGATAGGATCCACATCTACGTATTA N30 TTCACTGCAGACTTGACGAAGCTT-3 ', N30 for 30 randomized positions with a balanced ratio of the four bases A, C, G and T of 1: 1: 1: 1 stands. 5'-GATAATACGACTCACTATA GGG ATA GGA TCC ACA TCT ACG T-3 'was used as the primer for the amplification and 5' -AAG CTT CGT CAA GTC TGC AGT GAA-3 'for the 3' end. Transcription in RNA and reverse transcription in cDNA was carried out according to protocols known to the person skilled in the art. The conditions for carrying out the PCR are: 45 sec at 94 ° C, 60 sec at 50 ° C and 90 sec at 72 ° C. The amplification for rounds 1-6 consisted of 20 PCR cycles or for the rounds 7 - 11 out of 16 PCR cycles. Non-binding RNA sequences were separated by washing with 500 μl binding buffer. The bound sequences were eluted using 51 μl H ₂ O for 3 min at 98 ° C. The entire selection was carried out over 11 selection cycles.

Analysis of the enriched pools

The purification of the RNA formed in 50 μl transcription batches and labeled with ³³ P-GTP was carried out using the Qiaquick nucleotide removal kit according to the manufacturer (Qiagen). Binding studies were carried out for 1 h at 37 ° C using an aliquot of 1 µl of the purified RNA in each binding assay.

The result of the binding test is shown in Fig. 14. A total of three pools were examined with regard to their binding behavior to the target or to the beads used for the selection without target. NCIH RndO represents the unselected pool, NCIH Rnd6 the pool obtained after 6 rounds of selection and NCIH Rndl 1 the pool obtained after 11 rounds of selection. It can be established that after 11 selection edges there is a considerable accumulation of aptamers that specifically bind to the target, which, although to a lesser extent, can already be observed after 6 rounds of selection.

The result of a sequence analysis of the selected aptamers is summarized in the table below.

Table 4: Aptamers selected against NPY

In a further embodiment, the above nucleic acids (SEQ ID No. 44-64) can contain at least one of the constant parts of the sequence of the starting library, i. H. fully or partially include the constant portion of the 5 'end (5' -GGGATAGGATCCACATCTACGTATTA) and / or the 3 'end (TTCACTGCAGACTTGACGAAGCTT-3').

It is within the scope of the usual skills of a person skilled in the art to shorten these sequences or nucleic acids in order to determine the - minimal - part of the molecule or the sequence required for binding to the target molecule.

Furthermore, FIG. 15 shows, on the basis of various selected clones, the binding behavior as a function of the amount of target offered, ie NPY. It can be seen that, in the present case, clone NC1H11C29, for example, binds only slightly better than the starting pool, referred to in FIG. 15 as N30 Ellington Pool. The references cited here are as follows in detail:

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The features of the invention disclosed in the above description, the claims, the drawings and the sequence listing can be essential both individually and in any combination for realizing the invention in its various embodiments.

Claims

Expectations 1. Nucleic acid, in particular an isolated nucleic acid, binding neuropeptide Y or a derivative thereof and derivatives of this nucleic acid. 2. Nucleic acid according to spoke 1, characterized in that the derivative of neuropeptide Y is the C-terminal part of neuropeptide Y, in particular the part comprising at least amino acid positions 24 to 36. 3. Nucleic acid, in particular isolated nucleic acid, which discriminates in terms of its binding behavior between the members of the pancreatic polypeptide family. 4. Nucleic acid, in particular isolated nucleic acid, which inhibits the interaction between the neuropeptide Y receptor Y2 and a ligand of the neuropeptide Y receptor Y2. 5. Nucleic acid according to spoke 4, characterized in that the ligand is selected from the Grappe, which comprises NPY, hPP and BIIE 0246. 6. Nucleic acid, in particular isolated nucleic acid, binding to different conformations of neuropeptide Y. 7. Nucleic acid, in particular isolated nucleic acid, binds to the conformation of neuropeptide Y, which activates the neuropeptide Y receptor Y2. 8. Nucleic acid according to one of claims 1 to 7, characterized in that the nucleic acid comprises a sequence which is selected from the group comprising SEQ ID NO: 1-18 and 44-64 and their derivatives. 9. Nucleic acid according to one of claims 1 to 8, characterized in that the nucleic acid is one which is selected from the group comprising DNA, RNA, polynucleotides, oligonucleotides, aptamers, aptazymes and intramers. 10. Use of a nucleic acid according to one of claims 1 to 9 for the manufacture of a medicament. 11. Use of a nucleic acid according to one of claims 1 to 9 for the manufacture of a medicament for the treatment of diseases or conditions in which an inhibition of the interaction between the neuropeptide Y or a derivative thereof and a neuropeptide Y receptor leads to a change in the disease or of the state leads. 12. Use of a nucleic acid according to any one of claims 1 to 9, in particular for the manufacture of a medicament, for reducing the effect of neuropeptide Y in an organism, in particular in a human or animal organism. 13. Use of a nucleic acid according to any one of claims 1 to 9, in particular for the manufacture of a medicament, for reducing food intake, for promoting lipolysis, for reducing the activity of lipoprotease, for reducing fat storage, for reducing the secretion of luteinizing hormone , to reduce the secretion of insulin, to increase the secretion of growth hormone, to enlarge the blood vessels, to reduce the vasoconstrictor effect of norepinephrine, to reduce the vasoconstrictor effect of angiotensin and to influence the circadian rhythm in mammals. 14. Use of a nucleic acid according to one of claims 1 to 9 for characterizing the interaction topology of the interaction between neuropeptide Y and neuropeptide Y receptor at the molecular level. 15. Use of a nucleic acid according to one of claims 1 to 9 for the detection and / or comparison of the receptor activation, in particular the receptor activation in the brain and very particularly the activation of neuropeptide Y receptors. 16. Use of a nucleic acid according to any one of claims 1 to 9 for the detection of neuropeptide Y or a derivative thereof. 17. Use of a nucleic acid according to one of claims 1 to 9 for purifying neuropeptide Y or a derivative thereof. 18. Composition, in particular pharmaceutical composition, comprise a nucleic acid according to one of Claims 1 to 9, a vector according to Claim 10 and / or a cell according to one of Claims 11 to 13 together with a suitable carrier material. 19. Antagonist for a neuropeptide Y receptor, in particular for the neuropeptide Y receptor Y2. 20. Antagonist according to claim 19, characterized in that the antagonist comprises a nucleic acid according to one of claims 1 to 9. 21. Receptor mimetic for a neuropeptide Y receptor, in particular the neuropeptide Y receptor Y2. 22. Receptor mimetic according to claim 21, characterized in that the receptor mimetic comprises a nucleic acid according to one of claims 1 to 9. 23. Use of a nucleic acid according to any one of claims 1 to 9 for screening compounds, in particular low molecular weight compounds, the compounds being selected from the group comprising agonists of a neuropeptide Y receptor and antagonists of a neuropeptide Y receptor. 24. Connection available from use according to address 23. 25. Use of a neuropeptide Y or a derivative as a target molecule in the context of an in vitro selection process. 26. A method of identifying and isolating nucleic acids capable of binding to a target molecule comprising the following steps: Incubating the target molecule or a part thereof with a multiplicity of nucleic acids, preferably a nucleic acid library, the nucleic acids having different sequences, Selection and isolation of those nucleic acids which are able to bind to the target molecule or a part thereof, optionally amplifying the isolated nucleic acids and repeating the first two steps; and optionally determining the sequence and / or binding specificity of the isolated nucleic acids, characterized in that the target molecule is neuropeptide Y or a derivative thereof. 27. Use of a nucleic acid according to any one of the preceding claims, preferably in a complex with a neuropeptide Y or a derivative thereof, for the rational design of compounds, in particular of agonists and antagonists of the neuropeptide Y receptor, preferably of inhibitors and preferably of low molecular weight inhibitors.