DE10122862A1 - New nucleic acid that binds to gonadotropin releasing hormone, useful for treating and diagnosing overproduction of hormones, e.g. endometriosis - Google Patents

Abstract

Nucleic acid (I) that binds to gonadotropin releasing hormone (GnRH), especially the human peptide, is new. Independent claims are also included for the following: (1) preparing and/or identifying nucleic acids (Ia), particularly (I), that bind to GnRH; (2) preparing L-nucleic acid (Ib) that binds to GnRH having the natural configuration; (3) complexes of GnRH and (I); (4) kit for detecting GnRH that comprises (I); and (5) screening for GnRH agonists.

Description

The present invention relates generally to GnRH-binding nucleic acids and methods for the production and / or identification of nucleic acids that bind to GnRH, method for the production of L-nucleic acids that bind to GnRH, various uses of on GnRH-binding nucleic acids and compositions comprising GnRH-binding Nucleic acids.

The peptide hormone GnRH I (gonadotropin releasing hormone, gonadoliberin), hereinafter also referred to as GnRH, is a decapeptide which is formed in the hypothalamus and the secretion of the gonadotropins LH (luteinizing hormone) and FSH (follicle stimulating hormone) by the pituitary gland stimulated (Carolsfeld J., et al. (1999) Endocrinology, 141/2: 505-512; McLachlan RI, et al. (1996) J. of Endocrinology. 148: 1-9) ( Fig. 1). GnRH is secreted from the hypothalamic neurons in a pulsatile form and then binds to its receptor on the cell surface of the pituitary gland. The ligand-receptor complex is internalized, which leads to a release of FSH (Beck A., et al. (1985) DNA 4: 76) and LH, which in turn stimulate the formation of sex hormones such as estrogen, progesterone or testosterone. Sex hormones influence the proliferation and maturation of cells (Naor Z., et al. (1998) Frontiers in Neuroendocrinology, 19; 1-19, Welt CK, et al. (1999) J. Clin. Endocrinol. Metab. 84 (6): 2163-2169; Vizcarra, JA, et al. (1997); Endocrinology 138: 594-601).

As a result of a pathological overproduction of sex hormones, excessive cell regeneration occurs and thus abnormal tissue growth ( Fig. 1). Examples of benign gynecological diseases using GnRH analogs such as buserelin that prevent the overproduction of sex hormones are endometriosis (von der Linden PJ Q, 1996, Human Reproduc. 11, Supplement 3, 53-65) and uterine fibrosis (Cramer, SF (1998) Arch Pathol. Lab Med 122: 1045-46, Morini, A. et al., (1993) Minerva Ginecol 45 (10): 455-465). An example of the use of GnRH antagonists such as Cetrorelix (Asta Medica) is in vitro fertilization (IVF) (Jennings, 1996). In addition, GnRH antagonists are used to suppress the growth-promoting effects of the body's own sex hormones even in malignant diseases such as breast cancer and prostate cancer.

The mechanism of action of the GnRH agonists is based on the downregulation of the GnRH Concentration. The GnRH agonists bind more strongly to the GnRH receptor than the GnRH itself. All receptors are thus occupied by agonists and then internalized. Ent speaking, FSH and LH production is increased at short notice at the beginning of therapy device (English "flow up"). After all receptors are internalized, GnRH can no longer bind to free receptors and is down-regulated and consequently also LH and FSH. GnRH agonists are not available orally and are therefore often used as depot injections abreicht. In prostate cancer, treatment with agonists leads to Im during this time power.

In contrast to the GnRH agonists, GnRH antagonists also bind to the GnRH Receptor, but the flow up described in the agonists does not occur, which immediately leads to a reduced distribution of FSH and LH and the release of these in turn Hormones leads. GnRH antagonists like Cetrorelix have the advantage that as a result of them underlying other mechanism of action observed in GnRH agonists There is no "flow up" of FSH and LH, making therapy "more manageable". Similar  like the GnRH agonists, the GnRH antagonists are not available orally. The Appli cation of the GnRH antagonists by injection is associated with considerable costs. Ent GnRH antagonists are currently only available in in vitro fertilization (IVF) Application.

The present invention thus lies in a further aspect of the invention The task is to provide a means that binds specifically to GnRH.

The present invention is based on the object in a still further aspect tel provide, reduce the release of gonadotropins and consequently also reduce the release of sex hormones. It is a task in particular the present invention to provide such means that compared to the means according to the prior art are associated with fewer side effects.

Finally, in another aspect, the present invention is based on the object Alternatives to the GnRH agonsites and GnRH- To provide antagonists.

In another aspect, the present invention is therefore based on the object de to provide a means that be known from GnRH agonists according to the prior art did not show "flow-up" of FSH and LH and at the same time it can be administered orally.

According to the invention, the object is achieved in a first aspect by a nucleic acid, that binds to GnRH.

According to the invention, the object is achieved in a second aspect by a nucleic acid, which binds to GnRH, but not to Buserelin.

In one embodiment of the nucleic acids according to the invention it is provided that the Nucleic acid is an L-nucleic acid.

In a further embodiment of the nucleic acids according to the invention, that the nucleic acid is selected from the group comprising DNA and RNA.  

In a still further embodiment of the nucleic acids according to the invention, the procedure is preliminary hen that the binding constant of the nucleic acid according to the invention at room temperature 1 µM or less, preferably 100 µM or less, and more preferably 50 µM or less. In a very particularly preferred embodiment, the bin is constant at room temperature as little as about 15 nM. The length of the nucleic acids is about 50 to 100 nucleotides in these embodiments.

In a still further embodiment of the nucleic acids according to the invention, the procedure is preliminary hen that the binding constant of the nucleic acid of the invention at 37 ° C 1 µM or less, preferably 300 µM or less and more preferably 100 µM or less ger is. In a very particularly preferred embodiment, the binding con is was as little as about 30 nM at 37 ° C.

In one embodiment of the nucleic acids according to the invention it is provided that these have a length which is selected from the group, the lengths with 15 to 150 nucleos tides, 20 to 120 nucleotides, 30 to 120 nucleotides, 40 to 100 nucleotides, 40 to 70 Nucleotides and 50 to 70 nucleotides.

In a particularly preferred embodiment of the nucleic acids according to the invention provided that the nucleic acid is selected from the group consisting of SEQ.ID.NO.1 to SEQ.ID.No includes.

In a further preferred embodiment, GnRH is human GnRH

In a third aspect of the invention, the object is achieved by a method for producing and / or identifying nucleic acids which bind to a target molecule, comprising the steps

• a) generating a heterogeneous population of nucleic acids;
• b) contacting the population of step a) with the target molecule;  
• c) separating the nucleic acids that do not interact with the target molecule kicking;
• d) optionally separating the nucleic acids alternating with the target molecule effect; and
• e) optional sequencing of the nucleic acids that are in alternation with the target molecule have interacted,

where the target molecule is GnRH.

In a preferred embodiment, the method relates to the production and / or identification fication of nucleic acids according to the invention.

In a preferred embodiment it is provided that the subsequent step following step c)

• a) Amplification of the nucleic acids that interact with the target molecule are kicking

is carried out.

In a further preferred embodiment it is provided that steps b) to d) be repeated.

In a still further embodiment it is provided that the GnRH is human GnRH.

In a fourth aspect of the invention, the object is achieved by a method for producing L-nucleic acids which bind to a target molecule which occurs in the natural configuration and which comprises the following steps:

• a) generating a heterogeneous population of D-nucleic acids;
• b) contacting the population of step a) with the optical antipode of the target molecule;  
• c) separating the D-nucleic acids that are not with the optical antipode of the Target molecule has interacted;
• d) Sequencing of the D nucleic acids with the optical antipode of the target molecules have interacted; and
• e) Synthesis of L-nucleic acids in sequence with those in step d) for the sequences determined D-nucleic acids are identical,

wherein the target molecule is L-GnRH and the optical antipode of the target molecule is D-GnRH.

In one embodiment, the following step is inserted after step c):

• a) Amplification of the D-nucleic acids with the optical antipode of the target molecules have interacted.

In one embodiment, steps b) to e) are repeated.

In a further embodiment it is provided that the heterogeneous population of nuc flax acids comprises one of the nucleic acids according to the invention.

In a still further embodiment it is provided that the target molecule in addition to GnRH Buserelin is used.

In a preferred embodiment, the GnRH is human GnRH or corresponds to the Se GnRH sequence of those of the human GnRH.

In a further embodiment it is provided that the method is intended for the manufacture position and / or identification of the nucleic acids according to the invention.

In a fifth aspect of the invention, the object is achieved by use at least one of the nucleic acids according to the invention for the manufacture of a medicament.  

In one embodiment it is provided that the medicament for the treatment of crane kungen is or serves or is determined, which is due to overproduction of a hormone which is selected from the group consisting of the Gonadotropins, GnRH, LH, FSH and Sexual includes hormones.

In a preferred embodiment it is provided that the sex hormone is selected from the group that includes estrogen, progesterone and testosterone.

In a further embodiment it is provided that the disease is selected from the group that includes malignant and benign diseases.

In a preferred embodiment it is provided that the disease is selected from the group that includes endometriosis, uterine fibroids, breast cancer and prostate cancer.

In a sixth aspect, the object is achieved by using at least one the nucleic acids according to the invention for the preparation of an agent, preferably one Medication, for contraception in men

In a seventh aspect, the object is achieved by a composition comprising at least one of the nucleic acids of the invention and one pharmaceutically acceptable len carrier.

In an eighth aspect, the task is solved by complex comprising GnRH and min at least one of the nucleic acids according to the invention.

In a ninth aspect, the object is achieved by using at least one of the Nucleic acids according to the invention for the detection of GnRH.

In a tenth aspect, the object is achieved by methods for screening for a GnRH agonist, which comprises the following steps:

• - providing a candidate GnRH agonist,
• Providing at least one of the nucleic acids according to the invention,  
• - Provision of a test system that signals when a GnRH agonist is present generated, and
• - Determine whether the candidate GnRH agonist is a GnRH agonist.

In an eleventh aspect, the problem is solved by using one of the inventions nucleic acids according to the invention for producing an agent for in vitro fertilization. before preferably, the agent is a drug.

Finally, in a twelfth aspect, the task is comprehensively covered by a kit at least one of the nucleic acids according to the invention dissolved.

In a preferred embodiment of all aspects of the present invention GnRH human GnRH or the sequence of the GnRH corresponds to that of the human GnRH.

The present invention is based on the surprising finding that it is possible To produce nucleic acids that bind specifically and with high affinity to GnRH. As a result This binding behavior can make these nucleic acids both therapeutic and diagnostic or preparative purposes. The therapeutic application Availability lies in the fact that the nucleic acids according to the invention are GnRH-specific Represent antagonists. Due to their specific, system-inherent properties they do not have the disadvantages and side effects common to the GnRH antagonists and analogues on. So in particular occur with the GnRH agonists and GnRH antagonists observed and known in the prior art disadvantages. In particular, the joins flow-up was not observed using the prior art agents.

With the availability of the nucleic acids according to the invention, new agents become available for the first time provided that intervene in central neuroendocrinological control mechanisms. there there is a paradigm shift compared to the means known in the prior art shape that the agents, here the nucleic acids according to the invention, are now not on the recipe gate and downstream control loops such as signal transduction and the like is directly influenced, but GnRH specifically bound and thus from the phy ecological events is removed. The particular therapeutic applications of the  nucleic acids according to the invention are thus not limited to the indi specifically disclosed herein cations, but extend to all those indications where GnRH is direct or indirectly involved. The participation of GnRH can be done within the normal, d. H. non-pathological, as well as in the context of pathological conditions or events ge. As long as a binding event involving GnRH is involved, can be used in the context of the present invention by means of the nucleic acid according to the invention acids are influenced, because preferably by forming a complex from GnRH and at least one of the nucleic acids according to the invention a participation of GnRH is no longer possible in the respective event, the GnRH thus from the Ge happen to be removed or intercepted.

In addition, the invention is based on a further surprising finding on the basis that nucleic acids are disclosed for the first time with the nucleic acids according to the invention and have been made available that are able to between GnRH and Buserelin discriminate. The only difference between Buserelin and GnRH is that they contain amino acids position 6 an exchange takes place and buserelin ver compared to GnRH by one amino acid is shortened. GnRH is also very conserved in all mammals. There are individual Ami Differences in acidity B. in chicken (amino acid positions 6 and 7) and salmon (amino acid positions 7 and 8) (Carolsfeld, J., et al., 2000).

This property of the nucleic acids according to the invention allows a differentiation from Buserelin and GnRH, especially in the field of diagnostics. For example, with an antibody that does not distinguish between Buserelin and GnRH Total titers can be determined from GnRH and Buserelin and then using the he nucleic acids according to the invention the relative proportion of GnRH in the by the antibody certain total titers can be determined. Subsequent subtraction can result in the Titer determined by Buserelin and thus the course of the Buserelin Therpie determined and be monitored

The nucleic acids according to the invention are also to be understood here as meaning those which are homologous to the sequences substantially disclosed herein. Under essentially ho Such sequences or nucleic acids are to be understood molog, their homolo  based on the primary sequence 70% and preferably 80% and before is usually 90%.

Furthermore, all those nucleic acids are also intended as nucleic acids according to the invention are understood to include parts of the nucleic acid sequence disclosed herein, so far these parts are involved in the binding of GnRH.

The nucleic acids according to the invention can either be D-nucleic acids or L- Nucleic acids are present. The nucleic acids according to the invention are preferably in the form of L-nucleic acids. Furthermore, it is possible that one or more parts of the nucleus acids are present as D- and one or more parts of the nucleic acids as L-nucleic acids. The term part of the nucleic acid is to be understood as little as a nucleotide. Such nucleic acids are referred to herein as D, L nucleic acids. It is therefore in the Within the scope of the present invention, the nucleic acids according to the invention are part of a are longer nucleic acid, this longer nucleic acid consisting of several parts, at least part of which is one of the nucleic acids according to the invention. The other part of the This longer nucleic acid can either be a D-nucleic acid or an L-nucleic acid be trained. This other part of the longer nucleic acid can have different functions have onen. A possible function is that an interaction with other Mo reading is enabled.

L-nucleic acids are understood here to mean those nucleic acids which consist of L-nucleotides, are preferably made up entirely of L nucleotides.

D-nucleic acids are understood here to mean those nucleic acids which are derived from D- Nucleotides, preferably made up entirely of D nucleotides.

The configuration of the nucleic acids according to the invention as L-nucleic acid is from particularly advantageous for a number of reasons. L-nucleic acids are the Enantiomers of the naturally occurring D-nucleic acids. D nucleic acids are however in aqueous solutions and especially in biological systems or biological Samples not very stable due to the widespread use of nucleases. The course before upcoming nucleases, especially those in animal cells, are not able to  Break down nucleic acids. This increases the half-life of the L-nucleic acids in such systems quite significantly, including in an animal or human body. The lack of degradability of the L-nucleic acids also prevents that degradation products are formed under the influence of nucleases, which in turn are undesirable May have side effects. This aspect distinguishes L-nucleic acids from practical all other active ingredients, such as those associated with the therapy of diseases gene can be used that defi by a pathological overproduction of GnRH belong to the nested form.

Furthermore, it is within the scope of the present invention that the inventive Nucleic acids, regardless of whether they are present as D- or L- or D, L-nucleic acids, as DNA or as RNA, can be double or single-stranded. Typischerwei The nucleic acids according to the invention are single-stranded nucleic acids ren, but the secondary structures defined by their primary sequence and thus can also develop tertiary structures. In the secondary structure there are a large number of nucleic acids according to the invention also double-stranded sections. The fiction However, moderate nucleic acids can also be double-stranded in the sense that two complementary strands are hybridized with each other. This can lead to stabilization Sization of the nucleic acids lead, which is particularly advantageous if the nuclein acids in the D-form, d. H. in the naturally occurring form.

The nucleic acids according to the invention can be modified. Such modification can refer to the individual nucleotides of the nucleic acids and are in the Technology well known. Examples of such modifications can be found e.g. B. in Kusser, W. (2000) J Biotechnol, 74: 27-38; Aurup, H. et al. (1994) Nucleic Acids Res, 22, 20-4; cum mins, L.L. et al. (1995) Nucleic Acids Res, 23, 2019-24; Eaton, B.E. et al. (1995) Chem Biol, 2, 633-8; Green, L.S. et al., (1995) Chem Biol, 2, 683-95; Kawasaki, A.M. et al., (1993) J Med Chem, 36, 831-41; Lesnik, E.A. et al., (1993) Biochemistry, 32, 7832-8; Miller, L.E. et al., (1993) J Physiol, 469, 213-43.

The determination of the binding constants can basically be done using the so-called called equilibrium dialysis, as described in the examples. A  Another possibility for determining the binding constants is to use them a so-called Biacore device, which is known to the experts in the field.

Using equilibrium dialysis resulted for the nuc according to the invention linseic acids the following ranges of binding constants. At room temperature and one A length of the nucleic acid of 100 nucleotides according to the invention resulted in a binding constant range from 48 to 105 nM. When shortening the nucleic acid according to the invention For lengths between 50 and 67 nucleotides, binding constants from 14 to 55 were obtained nM observed. Temperatures of about 21 ° C. are understood here below room temperature the.

At a temperature of 37 ° C and a length of the nucleic acid of the invention of 99 or 100 nucleotides resulted in a binding constant range of 27 to 300 nM. at Shortening the nucleic acids according to the invention to a length of 50 to 67 nucleotides binding constants of 88 to 1000 nM were found.

The individual values were as follows, the names of the individual nucleic acids being further explained in the examples:
The binding constant of the 100 nt RNA Aptamer A10 is 92.5 ± 12 nM at room temperature. The shortened variant (50 nt, D- and L-RNA) has a binding constant of 200-500 nM. At 37 ° C the binding constant for the D- and L-RNA is approx. 1 µM. The binding constant of the 99 nt DNA aptamer 42 is 55 ± 7 nM at room temperature and 230 ± 70 nM at 37 ° C. The binding constant of the shortened D variant (60 nt) is 43 ± 7 nM at room temperature and that of the shortened L variant (60 nt) is 49 ± 6 nM at room temperature. The binding constant of the 100 nt long DNA aptamer A1 is 32 ± 5 nM at 37 ° C. The binding constant of the D variant shortened to 67 nt is 18 ± 4 nM and for the L variant 17 ± 3 nM at room temperature and 97 ± 9 nM at 37 ° C.

The method disclosed herein for the production and identification of nucleic acids that bind to GnRH and in particular the other features and properties disclosed herein are based on the so-called SELEX process, which is the subject of the US  U.S. Patent 5,475,096. The exact execution of the SELEX procedure is the specialist known in the field.

Typically, the individual is amplified as part of the SELEX process nucleic acids binding to the target molecule using the polymerase chain Reaction. it can be effected by a suitable reaction that the poly merase has an increased error rate, which leads to a change in the primary sequence of the bin leads the nucleic acid. As a result of this change, new Se is generated sequences that show a changed binding behavior compared to the starting sequences, for example an increased affinity or specificity. It is within the scope of the present Er finding that the sequences according to the invention completely or partially from such parts of the nucleic acid library used as starting or starting material originate from the randomized range of the individual members of the nucleic acid library. However, it is also within the scope of the present invention that the Se sequences completely or partially from such parts as the starting or starting material Nucleic acid library used come from the non-randomized range of the individual members of the nucleic acid library. Such a non- Randomized area is, for example, the area that acts as a binding site for the Amplification primer is used.

The method for the production of L-nucleic acids, which is also disclosed here Bind GnRH is based on the procedure of Fürste et al., Which is the subject of the internati onale patent application WO 98/08856. The exact implementation of this procedure is known to those skilled in the art.

In the context of the present invention, GnRH is used as the target molecule. In the optical antipode in the context of the present invention is the enantio mere of the naturally occurring GnRH, d. H. the D-amino acid GnRH.

The use of buserelin in such a procedure is said to discriminate increase the selection process between specific and non-specific binding to GnRH  hen. This selection is based on the consideration that be from the selection pool Those nucleic acids that bind Buserelin in addition to GnRH are already to be removed.

The various uses of the nucleic acids according to the invention result from whose property as GnRH antagonists and the participation of GnRH in the various those diseases, as already explained above. In the following, endometriosis, Uterine fibrosis (leiomyomas), prostate cancer and breast cancer diseases are shown in which GnRH plays a role and in this respect by means of the nuclein according to the invention acids can be treated and / or diagnosed.

Endometriosis is a disease in which endometrial tissue is excluded half of the uterus, e.g. B. in the ovaries, fallopian tubes, intestine, rectum, bladder, vagina after is pointed. Endometriosis occurs in 5-15% of all premenopausal women and will influenced by menstrual cycle. Endometriosis manifests itself in its light form abdominal pain, bleeding, inflammation, in its severe form due to damage Reproductive organs, impaired bladder or bowel function. Endo Metriosis leads to subfertility in 30-40% of all cases.

Treatment is either through surgery, hormone therapy, or treatment with GnRH analogues. The GnRH analogs inhibit and reduce estrogen production Ren thus the tissue growth of the endometrium. Because estrogen for the maintenance of the kno Chen mineral density (BMD) is important, is in many therapies with GnRH analogues in ge wrestle doses of estrogen added ("add-back" therapy).

In endometriosis, GnRH agonists and no antagonists are used because they are long-term a suppression of sex steroid biosynthesis from the beginning of controlled ovarian Hyper stimulation is required.

In the context of the present invention, the agents according to the invention can thus be combined men can be used with other pharmaceutically active ingredients. In the case of En dometriosis, for example, together with estrogen.  

A further use of the nucleic acids according to the invention can be in the context of Be act and / or diagnose uterine fibrosis (leiomyomas). This is what it is about are benign tumors of the uterine muscles in 25-40% of all premenopausal women. Leiomyomas affect about 2.45% of all women and 8.30% of women around 50th uterine fibrosis may be inheritable since it is 3, 4 times more common in women with a positive family relationship layer occurs. The growth of leiomyomas decreases with treatment with progesterone, during pregnancy and during menopause. The symptoms are: diffuse abdominal Pain, anemia due to heavy menstrual bleeding and infertility. Leiomyo me go to 35% for chromosome abnormalities (deletion in chromosome 7 and trans location in 12).

Current forms of treatment are hysterectomy (more than 200,000 cases a year) or Fibroid resection and hormone therapy. The dose is given as part of hormone therapy of GnRH analogs: e.g. B. Decapeptyl (= Triptorelin, Ferring), whereupon the volume the fibroids are reduced, possibly due to the reduction in the concentration of pro gesteron and estradiol.

A further use of the nucleic acids according to the invention can be in the context of Be act and / or diagnose prostate cancer. For example, Abare is currently lix (GnRH antagonist) is used. For the treatment of advanced prostate cancer Leuprogel, a GnRH agonist is used.

A further use of the nucleic acids according to the invention can be in the context of Be act and / or diagnose breast cancer.

Breast cancer is the most common form of cancer in women in North America and Europe, (5 million Women in the next 10 years, leading cause of death for women between 40 and 55 years ren). In many cases, breast cancer is inoperable. If there are micrometastases, sys Therapy tries to delay the growth of metastases. The type of system mix therapy (chemotherapy or endocrine therapy) depends on the age, the status of the Menopause and the presence or absence of hormone receptors. Another The form of therapy is oophorectomy, i.e. H. surgical castration in premenopausal cases Women to block estrogen production. As part of the chemo currently practiced  therapy, four cytostatics are applicable: cyclophosphamide, 5'-fluorouracil, methotrexate and Doxorubicin (Adriamycin). Newer drugs are: Taxole (Paclitaxel, Docetaxel) and Topoisomerase inhibitors.

About 2/3 of all patients show an objective response. With complete response is the survival rate is 5-15 years. Side effects include gastrointestinal effects, tired known kidney and liver damage.

In anti-estrogen therapy, tamoxifen (anti-estrogen) is administered to the estro gene receptor binds and thus blocks the estrogen effect and leads to a significant extension reduction in symptom-free time. As a side effect in premenopausal patients ovarian cysts appear.

As part of a therapy using GnRH analogs, Zoladex (= Goserelin, GnRH antagonist, Astra Zeneca) s. c. given as a deposit. The response rate be contributes 21-44% in premenopausal women. Hot showers and side effects occur decreased libido.

Another application of the nucleic acids according to the invention is the use as Pill for the man.

When developing a target molecule in male contraception, among other things, that Inhibition of GnRH and thus the suppression of LH and FSH secretion

Contraceptives such as gossypol, ketoconazole, spironolactone, acetazolamide and other ver bonds have already been examined. They all had toxic side effects.

For the new compound triptolide (triptolidecan) and related compounds, the from Tripterygium wilfordii were shown to be fully grown complete infertility in rats.

Under the influence of Cetrorelix, a GnRH antagonist, an inhibition of the Spermatogenesis in rhesus monkeys are shown. A disadvantage of the hormonal male  Contraception is the long-lasting effect of two to three months. With the fiction Nucleic acids are another means of male contraception as a result of their Mechanism of action ready.

There is yet another possible use of the nucleic acids according to the invention themselves in the context of in vitro fertilization.

In IVF, the ovaries are hyperstimulated by the administration of exogenous gonadotropins. because this leads to an increase in the number of oocytes (usually 1-5). The oocytes are removed before ovulation and fertilized in vitro. The time of removal of the Oocytes are critical to the success of IVF.

In 30-40% of all cases, the endogenous LH concentrations rise and lead to a ver early ovulation, to a reduced effectiveness of the endogenous hormones and to a loading damage to immature oocytes. As a result, there are fewer pregnancies and increased dropout rates.

A number of connections are currently in use, proving that the one with the Therapeutic approach pursued according to the nucleic acids of the invention works An example represents Cetrorelix (Asta Medica). The flare-up effect typical of agonists becomes full constantly avoided. During the first generation of the antagonists due to serious problems histamine-induced allergic reactions, Cetrorelix and Ganirelix are representatives the latest generation of GnRH antagonists is able to completely overcome these problems avoid. Medication with GnRH antagonists leads to a significant shortening of The rapiezeit.

The use of buserelin (Suprefact) reduces LH production which in turn reduces Lowers testosterone levels in the body. The hormone level increases in the first days or weeks for now. Possible side effects are: hot flashes, loss of libido during treatment, breast swelling, weight gain, fatigue

In general, the use of GnRH agonists in this area reveals a number of Disadvantages such. B. hot showers, vaginitis, loss of bone density, after stopping the  Agonist administration: renewed fibroid growth. This is offset by the following advantages: Decrease in uterine volume, fibroid size and fibrosis vascularization.

The disadvantages associated with GnRH antagonists in this area are not apparent in the sufficient bioavailability, which occurs particularly with flexible peptidomimetics. The advantages as opposed to: Real competitors, dose-dependent effects; the for The agonist typical flare-up effect is completely avoided and no heat showers.

The application or use of the nucleic acids according to the invention can be within the scope the manufacture of a medicament take place, here the nucleic acid according to the invention ren, optionally together with other pharmaceutically active compounds themselves act as pharmaceutically active agents. Such drugs include in the Usually at least one pharmaceutically acceptable carrier. Such a carrier For example, a solvent such as water, a buffer, starch, sugar or gelatin the like. Such carriers are known to those skilled in the art.

A further use of the nucleic acids according to the invention can consist in that these themselves are the starting product for drug development. Among other be there are two basic options. On the one hand, the screening of connections libraries, preferably libraries of low molecular weight Compounds, and the rational design of drugs based on the inventions nucleic acids according to the invention.

In the case of the rational construction of active ingredients, it can be based on the inventions nucleic acids according to the invention a structure, preferably a three-dimensional structure, can be derived, which then in the design, d. H. Sequence of nucleic acids that flows differ from the sequence of the nucleic acids specifically disclosed herein or those which are obtainable by the methods according to the invention, but nevertheless differ as before show the disclosed binding behavior of the nucleic acids according to the invention. In Another step in the rational construction of active substances is the one at GnRH dende - three-dimensional - structure mimicked by chemical groups that differ are of nucleotides or nucleic acids.  

In the case of screening compound libraries, the procedure is as follows: for example, by means of competitive tests known to the experts, suitable GnRH Analogs, GnRH agonsites or GnRH antagonsites can be determined. Such one For example, the test could be structured as follows: The Spiegelmer is connected to a fixed phase coupled. To identify GnRH analogs, labeled GnRH can be used as a test to admit. A potential analogue would displace the GnRH molecules from the Spiegelmer, which would be accompanied by a reduction in the signal caused by the marking.

A cell culture assay could be used for screening for agonists or antagonists as described in the examples herein, particularly in such Test format functionality can be ascertained, but in fact you would have to do as below described using the cell culture assay. With the Spiegelmer you can only go to Bin screening.

In the kit according to the invention it can be provided that this one or more of the comprises nucleic acids according to the invention. The kit can also have one or more positive or include negative controls. A positive control can, for example, GnRH before preferably in liquid form. A negative control can, for example, be Busere lin, preferably in liquid form. Furthermore, the kit can have one or more Include buffers. The individual components can be in dried or lyophilized form or in solution in a fluid. The kit can include one or more vessels that may contain one or more of the components of the kit.

The invention is now based on the following figures and examples, as well as the sequence tokolls further illustrates which further advantages, features and execution forms of the invention result. It shows

Fig. 1, the system of the endocrine control of the testis and ovary by GnRH and the possible on the basis of this system genesis of prostate and breast cancer,

Fig. 2 shows the result of an RNA selection against GnRH to Thiolsepharose 4B affinity material at room temperature,

Fig. 3 shows the result of a DNA-selection against GnRH to thiopropyl sepharose 6B affinity material at room temperature,

Fig. 4 shows the result of sequence analysis of the cloned DNA from the RNA selection on Thiolsepharose 4B at room temperature,

Fig. 5 shows the result of sequence analysis of the cloned DNA from the DNA-selection at room temperature thiopropylsepharose 6b and the result of Se quenzanalyse the cloned DNA from the evolutive optimization at 37 ° C to thiopropylsepharose 6b

Fig. 6 shows the result of the determination of the binding constant for one of the nucleic acids according to Invention (A10 RNA, SEQ.ID.No.44) at room temperature by equilibrium dialysis,

Fig. 7 shows the result of chemical and enzymatic analysis of one of the nucleic acids to the invention OF INVENTION (RNA A10, SEQ.ID.No.47),

Fig. 8 shows the result of the determination of the binding constant for one of the nucleic acids according to Invention (SEQ.ID.No.57) at room temperature by equilibrium dialysis,

Fig. 9 shows the result of the determination of the binding constant for one of the nucleic acids according to Invention (SEQ.ID.No.57) at room temperature by equilibrium dialysis,

Fig. 10 shows the result of chemical and enzymatic investigation of any of OF INVENTION to the invention nucleic acids (DNA 42 SEQ.ID.No.15),

Fig. 11 shows the result of the determination of the binding constant for one of the nucleic acids according to Invention (SEQ.ID.No.43) at room temperature by equilibrium dialysis,

Fig. 12 shows the result of the determination of the binding constant for one of the nucleic acids according to Invention (SEQ.ID.No.43) at room temperature by equilibrium dialysis,

13 shows the result of the determination of the binding constant for one of the nucleic acids according to Invention (SEQ.ID.No.77) at 37 ° C analysis. By Gleichgewichtsdia,

Fig. 14 shows the result of the determination of the binding constant for one of the nucleic acids according to Invention (SEQ.ID.No.144) at room temperature by equilibrium dialysis,

Fig. 15 shows the result of the determination of the binding constant for one of the nucleic acids according to Invention (SEQ.ID.No.144) at room temperature by equilibrium dialysis,

Fig. 16 shows the result of the sequence analysis of the cloned DNA from the reselection of 30% mutant clone A1,

Fig. 17 is a result of Ca ²⁺ measurements on GnRH receptor-containing cells by using two of the nucleic acids (SEQ.ID.No.57 SEQ.ID.No.43 and) according to the invention,

Fig. 18 shows another result of Ca ²⁺ measurements on GnRH receptor-containing cells using two of the inventive nucleic acids (SEQ.ID.No.57 SEQ.ID.No.43 and),

Fig. 19 shows the result of a sequence analysis of the cloned DNA from the 37 ° C-RNA selection,

Fig. 20, the binding to the target molecule variants of clone A1,

Fig. 21, the binding to the target molecule variants of clone A10,

Fig. 22, the binding to the target molecule variants of clone A42,

Fig. 23 shows the result of the sequence analysis of the RNA selection of example, and

Fig. 24 the result of the sequence analysis from the 37 ° C-RNA selection of Example

The various sequences disclosed herein can be assigned to the sequences of the sequence listing as follows:

The following should also be noted about the individual sequences:

• - SEQ.ID.No.45 to 56 represent binding variants of clone A10.

In the context of the present invention, the following nucleic acid (sequences) are particularly preferred:
SEQ.ID.No.44,
SEQ.ID.No.57,
SEQ.ID.No.15,
SEQ.ID.No.43 and
SEQ.ID.No.144

At this point, the following should be noted about the individual figures:
The results shown in FIGS. 6 to 15 and 17 and 18 result from the respective investigation using the following sequences:
The sequences shown in FIG. 20 (comprising parts a), b), c) and d)) and FIG. 22 (comprising parts a) and b)) are DNA sequences which are described in FIGS sequences Fig. 21, Fig. 23 and Fig. 24 shown contrast to RNA sequences.

FIG. 23 shows the same sequences as FIG. 4, the sequences shown in FIG. 23 additionally comprising the primer binding sites. The sequences were obtained as part of an RNA selection on thiol sepharose 4b at room temperature. FIGS. 4, 19 and 23 thus relate to the results of the RNA selection. Here, FIG. 21, the result of the Verkür wetting of the different clones obtained or binding sequences.

The upper part of FIG. 5 represents the result of the DNA selection at room temperature, FIG. 22 showing the shortened and stabilized sequences resulting therefrom (binding variants of clone 42). The lower part of FIG. 5 shows a sequence analysis of the cloned DNA from the evolutionary optimization at 37 ° C. on thiopropylsepharose 6b. The shortenings (of the binding variants of clone A1) are shown in FIG. 20.

example 1 Production of the peptides used for the selection

The D and L peptides were standardized by Jerini Bio Tools (Berlin, Germany) according to the f-moc standard Solid phase synthesis produced. Some of the unmodified peptides were (Amers ham Buchler GmbH) on tyrosine for the purposes of equilibrium dialysis. The peptides had the following sequences and modifications.

D-peptides

GnRH I: all D (pyrGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂

)
Molecular weight: (amide): 1182.6 g / mol
GnRH I-Cys: all D (pyrGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-Cys-NH ₂

)
Molecular weight (amide): 1286.5 g / mol
³

H-GnRH I: all D (pyrGlu-His-Trp-Ser- [ ^3rd

H] Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂

)
Molecular weight: (amide): 1182.6 g / mol
PyrGlu = pyroglutamate = 5'Oxo-L-proline
Molecular weight: 129.11 g / mol

L-peptides

GnRH I: all L (pyrGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂

)
Molecular weight: (amide): 1182.6 g / mol
³

H-GnRH I: all L (pyrGlu-His-Trp-Ser- [ ^3rd

H] Tyr-Gly-Leu-Arg-Pro-Gly-NH ₂

)
Molecular weight: (amide): 1182.6 g / mol

Example 2 Preparation of the oligonucleotides

The oligonucleotides were synthesized using the standard phosphoramidite method (NOx xon Pharma AG, Berlin, Germany). Start pools and the primers were then washed purified electrophoretically (8% PAA / 8 M urea).

ssDNA and RNA template, PCR primer for selection

RNA selection

ssDNA template

PCR primer

For the RNA start pool, the synthesized single-stranded DNA is amplified by PCR and then transcribed into a new RNA library of 120 bases in length by T7 transcription. The RNA molecules were radiolabelled internally during the T7 transcription by incorporation of ³² P-UTP.

DNA selection

ssDNA Start Pool

PCR primer

X = Glen Research Spacer Phosphoramidite 18
Primer B: 5'-CCAAGCTTGCATGCCTGCAG-3 '
Primer Seq: 5'-GGGAATTCGAGCTCGGTACC-3 '

Primers A and B were used to amplify the DNA for DNA selection. In contrast to primer B, primer A additionally contains a 20 nucleotide long poly-T Tail connected via a polyethylene glycol linker with the Pri mer region A was connected.

The resulting PCR product consisted of a double strand with two polynucleotide chains of different lengths (100 nt and 120 nt), of which, after strand separation via denaturing gel electrophoresis, the shorter strand, internally radiolabeled by incorporating ³² P-dCTP, could be used for selection ( Williams KP, et al. (1995) Nucl. Acics. Res. 23/20: 4220-4221).

Example 3 In vitro selection column material

The C-terminal cysteine-modified D-GnRH can be reversibly modified on thiol groups Sepharose can be immobilized by cleavage of 2-thiopyrides between the Thiol group of the peptide and 1-thiol-2-hydroxypropyl group of the matrix disulfide bridges to be knotted.

For the RNA selection, thiol sepharose 4B (Pharmacia) was used as the column material and for the DNA selection Thiopropylsepharose 6B (Pharmacia) was used.

buffer conditions

The optimal buffer conditions for the selections were determined by preliminary tests averages.

These were for RNA selection:
10mM Hepes pH 7.4; 137mM NaCl, 2.7mM KCl, 2mM CaCl ₂ , 1mM MgCl ₂ , 0.005% Tritone X-100
and for DNA selection:
20 mM Tris pH 7.4; 137 mM NaCl; 5 mM KCl; 2mM CaCl ₂ 1mM MgCl ₂ ; 0.005% Triton X-100

In vitro selection

An RNA or DNA library with a complexity of 10 ¹⁵ molecules each was brought into contact with the target D-GnRH immobilized on thiol sepharose. The oligo nucleotides were previously denatured for 5 minutes at 95 ° C. and then renatured for 15 minutes at room temperature. In order to suppress non-specific binding to the column material, a column with the same column volume of unmodified Sepharose was connected upstream of the D-GnRH-modified Sepharose column from the second selection round. The most favorable buffer conditions under which it was possible to separate non- and less strongly binding oligonucleotides were tested in preliminary experiments.

The guard column was washed with n column volumes of selection buffer, removed, and then the main column was washed with n column volumes of selection buffer.

The D-GnRH-binding oligonucleotides were isolated by elution with the free one D-peptide. After the dissociation of the nucleic acid / GnRH complexes were selected RNAs reproduced by reverse transcription PCR and in the subsequent T7 Transcription rewritten in a new RNA library. The DNAs were obtained by PCR amplified and the shorter strand by denaturing gel electrophoresis of the longer one Strand separated and cleaned.

In this way, a library enriched in GnRH-specific binders was created. By repeating the steps of attachment, selection and dissociation several times the diversity of the newly generated oligonucleotide banks decreased, and in addition the average affinity for GnRH increased with each cycle.  

Selection at 37 ° C

In order to select more thermostable RNA and DNA binders, a selection was made in parallel 37 ° C carried out.

For this purpose, a combi was used for RNA selection using conventional solid-phase synthesis Native DNA oligonucleotide library generated, the two constant primer binding region NEN and contained a segment of 60 bases, in which each base position by appropriate Mix the individual phosphoramidites to 85% of the sequence of the GnRH binder A10 ent spoke and was randomized to 15%.

To optimize GnRH binding at 37 ° C, the one carried out at room temperature DNA selection resumed in the second round. The following rounds were made selected at 37 ° C. In addition, the stringency was checked from the third 37 ° C selection round increased washing of the main column, from the fourth round onwards by 2.5 times the excess Competitor (GnRH-binding RNA oligonucleotide) and from the fifth round by Verrin reduction of the peptide excess in the peptide elution (five-fold instead of ten-fold excess) elevated.

Example 4 Equilibrium dialysis

To determine the binding properties of the individual identified RNA aptamers and DNA aptamers were used in the equilibrium dialysis method. For this purpose, triti relabeled D-GnRH in one of two equal-sized dialysis chambers, which are given by a semipermeable membrane from the respective unlabelled aptamer in the second Chamber was separate. The pore size of the membrane was chosen so that the peptide was good was able to diffuse between the chambers while the aptamers were retained. Dialysis was continued until an equilibrium was reached (approx. 24 h). to finally, equal volumes of the respective sample were taken from both chambers. In the event of a binding of the D-GnRH to the aptamer, one could be found in the aptamer chamber greater concentration of labeled peptide can be detected than in the chamber without the aptamer. Based on the radioactivity determined in the two chambers, it was possible  the percentage of D-GnRH that is attributable to the respective aptamer had bound.

The equilibrium dialysis method was also used to determine the binding constant (K _d ) of the D-GnRH binding. For this purpose, the RNA or DNA in concentrations of 100 nM to 5 µM was equilibrated against a solution with an infinitesimally small concentration of D-GnRH and the respective percentage binding was determined. The data obtained was finally fitted using the Grafit program using non-linear regression methods based on the standard binding equation for 1: 1 stoichiometry.

Example 5 Biacore measurements

In order to validate the quality of the measurement method used and thus the determined binding constant and to gain insight into the binding kinetics, the RNA and DNA aptamers were additionally examined on a Biacore device (Biacore 2000, Freiburg, Germany). For this purpose, the aptamers were biotinylated and coupled to a streptavidin-coated sensor chip. D-GnRH was then routed via this chip. Optics determined the plasmon resonance of the surfaces, which in principle corresponded to the mass that had bound to the sensor chip at each point in time (RU = pg / mm ² ). By varying the amount of D-GnRH used, the binding constant could be determined at room temperature.

The binding constant for the L-nucleic acids, also referred to herein as Spiegelmers was also determined. For this purpose, biotin-coupled L-GnRH was added to the strepta vidin-coated chip surface coupled.

In order to test the specificity, the connection of the Spiegelmers to different hypo thalamus hormones compared to the connection to GnRH examined.  

Example 6 reselection

In order to elucidate the structure of DNA clone A1, a new selection with a based on the sequence of the A1 clone 30% randomized pool at 37 ° C. For this purpose, a combinatorial DNA was Oligonucleotide library generated, the two constant primer binding regions and a Seg ment of 60 bases, in which each base position by mixing the 70% of the individual phosphoramidites corresponded to the sequence of the GnRH binder A1 and 30 % was randomized.

The selection should provide information about the structure, which bases are variable and therefore can be exchanged or deleted.

Example 7 Evidence of the effectiveness of Spiegelmers in cell culture

The effectiveness and specificity of the Spiegelmers was demonstrated in the cell culture assay. For this purpose, cells were used which express the human GnRH receptor in their cell membrane. When the receptors are stimulated, there is an intracellular Ca ²⁺ release. Calcium release is detected with an intracellular fluorescent dye (Fluo-3). The signal appears a few seconds after stimulation of the receptors and can be measured per well either as a fluorescence peak (more or less steep) or as an existing background due to intracellular calcium. GnRH is added as a stimulus. The Spiegelmer (RNA 50mer, DNA 60mer) was pre-incubated in a concentration of 1 e ^-6 mol / l to 1 e ^-10 mol / l with 2 e ^-9 mol / l GnRH for 20 minutes before it was added to the cells has been. In parallel, in a further approach the RNA or DNA level was then added to the cells, then GnRH. Typically, receptor antagonists are tested in this assay format.

The specificity of the Spiegelmers was also tested. This was done in one approach GnRH the peptide buserelin, which differs from the GnRH sequence in an amino acid separates, incubated with the Spiegelmer. As a control for all experiments, one was known ter antagonist used.  

Example 8 Results of the in vitro selection RNA selection

With the help of affinity chromatography, a library of 10 ¹⁵ RNA molecules was able to enrich RNA molecules in the course of 7 selection cycles, which bind the target molecule not only in the immobilized state but also very specifically in solution could. The PCR products obtained in the 6th and 7th round of selection were ligated into plasmids using standard methods and multiplied in E. coli and then sequenced (FIG. 2).

DNA selection

In the DNA selection, specifically binding DNA molecules could be enriched within 7 rounds. The DNA eluted with peptide in the last round was amplified with primer B and primer Seq, so that DNA strands of the same length were formed as a PCR product. The double-stranded DNA could then also be cloned and sequenced as described above ( FIG. 3).

Sequence analysis of the selections RNA selection

The sequences of the clones obtained were determined and compared with one another in order to determine similarities at the primary and secondary structure level. The RNA structure analysis showed that 32 of 55 clones sequenced were completely identical (sequence A10, see FIG. 4). 13 clones differed from the leader sequence only by point mutations or single insertions. 5 other sequences were not homologous to the main sequence A10.

DNA selection

The DNA was cloned as described above and 48 clones were sequenced. All 48 clones were evaluable. Once 15, twice 10, once 4 and 5 identical sequences and five individual sequences could be identified. However, the individual sequence groups showed no longer homologous sequence motifs with one another (see FIG. 5).

Example 9 Binding properties of aptamers and Spiegelmers

All different 100 base long RNA and DNA variants were GnRH binding at room temperature and at 37 ° C by means of equilibrium dialysis.

RNA selection

By varying the amount of D-GnRH used, a binding constant of 90 nM for clone A10, which resulted from the RNA selection, could be determined at room temperature ( FIG. 6). A binding RNA sequence of 48 nucleotides was initially determined by shortening.

The selected sequence of the 100mer A10 (the 48 base long binding region is underlined) reads:

where the sequence of the binding region is as follows:

For the structure elucidation, the 48mer (base 56 to 104) was examined enzymatically with S1 or T2 nuclease and chemically with DMS or kethoxal. Fig. 7 shows a summary of the results. S1 and T2 digestion of certain bases showed that these were unprotected or single-stranded. By combining these results with the structure folding program of mfold Zuker 3.0 (Zuker, M & Stiegler, P. 1981. Nucleic Acids Res. 9, 133-148; Zuker, M. 1989. Methods Enzymol. 180, 262-288.) an 18 base long helix was postulated at the beginning of the molecule, which could also be supported by substitutions within the helix (helix 1).

Furthermore, the chemical and enzymatic tests enabled a UAAA loop Positions 29 to 32 can be identified within the 48m (loop). This loop forms that End of a 6 base long helix with a looped G (Base 22-38) (Helix 2).

To stabilize the secondary structure, the first two AT base pairs were converted into GC base pairs (base 2 to 5 and 45 to 48). In addition, an additional GC base pair (items 1 and 50) was inserted, resulting in the following sequence (50 bases):

This sequence was also made in the enantiomeric form. The binding constants for the aptamer (589 nM; FIG. 8) and for the Spiegelmer (500 nM; FIG. 9) were determined by equilibrium dialysis.

DNA selection

In the selected DNA aptamers, several sequences showed affinity at room temperature against D-GnRH. The binding constant for clone 42 (99mer) was 80 nM Room temperature. This aptamer was able to study its minimal bin domains can be reduced from 100 to 68 bases.

The selected sequence of the 99mer clone 42 (the 68 base long binding region is underlined) is as follows:

and the sequence of the binding region as follows:

To further elucidate the secondary structure, the 99mer was investigated chemically (G, A, T) and enzymatically (S1 nuclease) in the presence and absence of peptide and without. He results are summarized in Fig. 10. These data, together with the mfold Zuker 3.0 DNA structure folding program (SantaLucia J. Jr. 1998. Proc. Natl. Acad. Sci. USA. 95 (4): 1460-1465), supported the following structural motifs. A loop GAAAG from base 55 to 61 was identified. The fact that this loop region could also be chemically modified in the presence of GnRH suggests that this region is not expected to participate in the binding. This region could therefore be shortened and stabilized. Bases A39 to A52 were deleted and replaced by a GTAA loop (loop), which was closed by an additional pair of GC bases, for stabilization. The TA base pairs in the helix were also replaced by GC base pairs (Helix 2). The inversion of the first CG base pair also showed that the molecule begins with a helix of at least 6 Watson-Crick base pairs (Helix 1). There is also an additional region C35 to G48 that is unpaired and is not involved in peptide binding. The G-rich region appears to be very conserved and involved in GnRH binding, since deletions in this section of the sequence always led to loss of binding.

The final sequence is as follows:

The binding constants of the 60-mer (Spiegelmer and aptamer) were determined by means of equilibrium dialysis (Figs. 11 and 12). A K _d value of 100 nM at room temperature and a constant of 1 µM at 37 ° C were determined.

Selection at 37 ° C RNA selection

With the help of affinity chromatography, we were able to detect 4 selection cycles len RNA molecules that enrich the target D-GnRH both in the immobilized state as well as free, in solution, could bind in a highly specific way. The in the 3rd and 4th selection round PCR products obtained were inserted into vectors by standard methods and in E. coli cultivated.

DNA selection

In the 10th round of selection, an increase in GnRH-binding DNA at 37 ° C of 13% can be achieved. The DNA was also cloned and sequenced.

Sequence analysis after evolutionary optimization at 37 ° C RNA selection

A total of 72 E. coli clones were sequenced. 44 different DNA sequences were obtained, all of which were very homologous to the starting sequence A10. The sequences were then examined to see if they had mutations in the 48-mer region which was known to be sufficient to bind D-GnRH. 28 of the 44 different sequences had 1 or more mutations in the 48-mer region. The DNAs of the clones in question were transcribed into the corresponding RNAs and then checked for D-GnRH binding ( FIG. 19).

DNA selection

The sequencing of 48 clones of the DNA molecules selected at 37 ° C. revealed 9 different structures, of which a sequence A1 was present 38 times. Two more sequences were provided two and three times, and there were an additional five individual sequences. Apart from a sequence, the sequences do not resemble any of the DNAs selected at room temperature. Again, no structural similarities between the clones could be found ( Fig. 5).

Example 10 Binding properties of aptamers and Spiegelmers RNA selection

The selected second generation GnRH binders were obtained by equilibrium dialysis examined for their binding to D-GnRH. The determined binding constants corresponded to the affinity of the - as was to be expected based on the sequence analysis First generation GnRH binder. From further experiments with these aptamers apart from that.

DNA selection

Clone A1 (100mer) showed the best binding in equilibrium dialysis at 37 ° C. ( FIG. 13). A binding curve showed a binding constant of 250-350 nM at 37 ° C.

Selected sequence of clone A1 (the binding region is underlined):

The sequence of the binding region is as follows:

In a variant shortened and stabilized to 67 bases, an improvement in the binding constant to 100 nM could be achieved ( FIGS. 14 and 15).

By deletions of 5 bases each from the 5 'end to the 3' end, the entire mole could be roughly searched for stabilizing regions that are essential for binding. It was found that the first 17 bases at the 3 'end are not necessary for binding. In contrast, no base can be omitted at the 5 'end. The 5-base deletions also identified helix loop regions. The helix identified in this way between base A23 and T28 could be confirmed by exchanging bases T24 → A24 and A28 → T28. In addition, a helix with 6 base pairs (C ₆₆ -G ₆₀ and C ₆₇ -G ₇₁ ) is formed. This could be stabilized using a CGTAAG loop.

The following sequence resulted from the shortening:

Example 11 Result of the reselection

Three variants of the A1 clone were found, which differ only in a few bases. The variants were tested in a shortened form in equilibrium dialysis. In addition, 5 other sequence groups could be identified, which occasionally showed sequence motive homologies to clone A1 ( FIG. 16).

Example 12 Result of the cell culture assays

An IC ₅₀ of 2 e ^-7 mol / l was determined for the RNA-based GnRH-binding nucleic acids and 5 e ^-8 mol / l for the DNA-based GnRH-binding nucleic acids. It made no difference whether the Spiegelmers were pre-incubated with GnRH or added directly to the experimental set. A GnRH antagonist used as a standard in this test system was used as a positive control.

The specificity of the Spiegelmers was also tested. This was done in one approach GnRH the peptide buserelin, which differs from the GnRH sequence in an amino acid separates, incubated with the Spiegelmer.

The specificity of the Spiegelmers could be documented by the experiments with Buserelin, which is not bound by the RNA and DNA Spiegelmer even in high concentrations, in contrast to GnRH. (Figs. 17 and 18)
IC ₅₀ = the concentration at which the Spiegelmers inhibit 50% of the Ca ²⁺ release.

Those apparent in the foregoing description, sequence listing and claims Features of the invention can be used both individually and in any combination the realization of the invention in its various embodiments essential his. The disclosure content of the claims is hereby incorporated by reference.  

SEQUENCE LISTING

Claims (29)

1. Nucleic acid binding to GnRH, preferably to human GnRH. 2. Nucleic acid binding to GnRH and not binding to buserelin. 3. Nucleic acid according to claim 1 or 2, characterized in that the nucleic acid is an L-nucleic acid. 4. Nucleic acid according to one of claims 1 to 3, characterized in that the Nucleic acid is selected from the group comprising DNA and RNA. 5. Nucleic acid according to one of claims 1 to 4, characterized in that the Binding constant of the nucleic acid at room temperature 1 µM or less, more preferred is 100 µM or less, and preferably 50 µM or less.   6. Nucleic acid according to claim 5, characterized in that the binding constant at room temperature is as little as about 15 nM. 7. Nucleic acid according to one of claims 1 to 6, characterized in that the Binding constant of the nucleic acid at 37 ° C 1 µM or less, preferably 300 µM or less, and more preferably 100 µM or less. 8. Nucleic acid according to claim 7, characterized in that the binding constant at 37 ° C is as little as about 30 nM. 9. Nucleic acid according to one of claims 1 to 8, characterized in that the Nucleic acid has a length which is selected from the group consisting of 15 to 150 nucleos tide, 20 to 120 nucleotides 30 to 120 nucleotides, 40 to 100 nucleotides, 40 to 70 nucleotides de and comprises 50 to 70 nucleotides. 10. Nucleic acid according to one of claims 1 to 9, characterized in that the Nucleic acid is selected from the group comprising SEQ.ID.No.1 to SEQ.ID.No.164. 11. A method for producing and / or identifying nucleic acids that bind to a target molecule, in particular nucleic acid according to one of claims 1 to 10, comprising the steps

• a) generating a heterogeneous population of nucleic acids;
• b) contacting the population of step a) with the target molecule;
• c) separating the nucleic acids that do not interact with the target molecule;
• d) optionally separating the nucleic acids which interact with the target molecule; and
• e) optionally sequencing the nucleic acids which have interacted with the target molecule,

characterized in that the target molecule is GnRH, preferably human GnRH. 12. The method according to claim 11, characterized in that following step c), the subsequent step

• a) Amplification of the nucleic acids that have interacted with the target molecule

he follows. 13. The method according to claim 11 or 12, characterized in that steps b) to d) be repeated. 14. A method for producing L-nucleic acids which bind to a target molecule which occurs in the natural configuration and which comprises the following steps:

• a) generating a heterogeneous population of D-nucleic acids;
• b) contacting the population of step a) with the optical antipode of the target molecule;
• c) separating the D-nucleic acids which have not interacted with the optical antipode of the target molecule;
• d) sequencing the D-nucleic acids which have interacted with the optical antipode of the target molecule; and
• e) synthesis of L-nucleic acids which are identical in sequence to those determined in step d) for the D-nucleic acids,

characterized in that the target molecule is L-GnRH and the optical antipode of the target molecule is D-GnRH. 15. The method according to claim 14, characterized in that the following step is inserted after step c):

• a) Amplification of the D-nucleic acids which have interacted with the optical antipode of the target molecule.

16. The method according to claim 14 or 15, characterized in that steps b) to e) are repeated. 17. The method according to any one of claims 11 to 16, characterized in that the he terogenic population of nucleic acids a nucleic acid according to any one of claims 1 to 10 includes. 18. The method according to any one of claims 11 to 17, characterized in that as Target molecule is used in addition to GnRH Buserelin. 19. Use of the nucleic acid according to one of claims 1 to 10 for the production of a drug. 20. Use according to claim 19, characterized in that the medicament for Treatment of diseases caused by overproduction of a hormone that is selected from the group of gonadotropins GnRH, LH, FSH and sex hormones includes. 21. Use according to claim 20, characterized in that the sex hormone is selected from the group consisting of estrogen, progesterone and testosterone. 22. Use according to claim 20 or 21, characterized in that the disease selected from the group comprising malignant and benign diseases, more preferred wisely selected from the group consisting of endometriosis, uterine fibroids, breast cancer and prostate includes takarzinoma. 23. Use of the nucleic acid according to one of claims 1 to 10 for the production an agent, preferably a drug, for contraception in men. 24. Use of the nucleic acid according to one of claims 1 to 10 for the production an agent, preferably a medicament, for in vitro fertilization.   25. A composition comprising a nucleic acid according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier. 26. Complex comprising GnRH and a nucleic acid according to one of claims 1 to 10th 27. Use of the nucleic acid according to one of claims 1 to 10 for the detection of GnRH. 28. A method for screening for a GnRH agonist, characterized by the following steps:

• Providing a candidate GnRH agonist,
• Providing a nucleic acid according to one of claims 1 to 10,
• Provision of a test system which generates a signal when a GnRH agonist is present, and
• - Determine whether the candidate GnRH agonist is a GnRH agonist.

29. Kit for the detection of GnRH comprise a nucleic acid according to one of the claims 1 to 10.