EP1159449A2

EP1159449A2 - Test system for detecting a splicing reaction and use thereof

Info

Publication number: EP1159449A2
Application number: EP00907635A
Authority: EP
Inventors: Christoph Hüls; Bettina Bauer; Claus Simandi; Reinhard Lührmann; Tilmann Achsel; Hans-Peter Vornlocher
Original assignee: Aventis Research and Technologies GmbH and Co KG
Current assignee: Aventis Research and Technologies GmbH and Co KG
Priority date: 1999-03-02
Filing date: 2000-02-25
Publication date: 2001-12-05
Also published as: CA2363431A1; PL355179A1; US6579681B1; HUP0203058A2; DE19909156A1; WO2000052201A2; JP2002537822A; WO2000052201A3; AU2915400A; NZ513908A

Abstract

The invention relates to a test system comprising: (a) one or more identical or different immobilized nucleic acids including at least one nucleic acid capable of splicing; (b) at least one gel-free detection system for detecting a splicing reaction; possibly (c) at least one compound containing splicing components; and preferably (d) suitable detection probes; and possibly (e) other additives.

Description

Test system for the detection of a splice reaction and its use

description

The present invention relates to a test system containing

(a) one or more, identical or different immobilized nucleic acid (s) with at least one splicable nucleic acid,

(b) at least one gel-free detection system for the detection of a splice reaction, optionally (c) at least one composition containing splice components, and preferably

(d) suitable detection probes, and if necessary

(e) other tools

Most of the genes coding for proteins in eukaryotes are in their form in the

Genome interrupted by one or more sequences (introns) not coding for the protein. When the genomic DNA is transcribed into the messenger RNA (messenger RNA = mRNA), these non-coding areas (introns) are transferred to the primary transcript. In order to generate a correct form of the mRNA, this precursor mRNA (pre-mRNA) must be processed.

The pre-mRNA is processed by removing the introns and fusing the coding regions (exons). Only then can a continuously read nucleotide strand be made available for translation in the cytoplasm. The formation of mRNA in eukaryotes therefore requires a so-called splicing process in which the non-coding gene regions (introns) are removed from the primary gene transcript.

The splicing takes place in the core before the mRNA is transported out of the core. It is generally carried out in a two-step mechanism, in each of which a transesterification step is involved (Moore, JM et al., (1993) Splicing of precursors to messenger RNAs by the Spliceosome. In The RNA world, Edited by Gesteland RF , Gesteland, JF, Cold Spring Harbor Laboratory Press, 303-358). The first step generates a free 5 ^' exon and a so-called lariat structure of the intron, which is still connected to the 3 ^' exon. The lariat structure contains a branched RNA, which is esterified by esterifying the 5 ^' end of the intron with a 2 ^' hydroxyl group of a ribose in an adenosine, which is approximately 20-40 nucleotides upstream of the 3 ^' end of the Introns is created. The second catalytic step leads to ligation of the exons and the release of the

Introns. Although no nucleotides are incorporated during these reactions, an energy source, e.g. ATP, is necessary for this catalysis (Guthrie, C.

(1991) Science, 253, 157).

Several factors are involved in the process of mRNA splicing. Two classes of splice factors are currently distinguished. The first class consists of four evolutionarily conserved protein RNA particles (small nuclear ribonucleoprotein particles = snRNPs): U1, U2, U4 / U6 and U5, either one (U1, U2, U5) or two (U4 / U6) contain snRNA components (Moore, JM et al., (1993) supra; Guthrie, (1991) supra; Green, MR (1991). Annu. Rev. Cell Bio .., 7,

559). The second class consists of so far little characterized proteins, which are not firmly bound to the snRNPs and are therefore called non-snRNP splice factors (Lamm, GM & Lamond, AJ (1993) Biochim. Biophys. Acoph. 1173, 247; Beggs , JD (1995), Yeast splicing factors and genetic strategies for their analysis, In: Lamond, Al (ed) PremRNA Processing Landes, RG Company, Texas, pp. 79-95. Krämer, A. (1995), The biochemistry of pre-mRNA splicing. In: Lamond, Al (ed), Pre-mRNA Processing. Landes, RG Company, Texas, pp. 35-64).

The composition of the snRNPs is best examined in HeLa cells (Will, C.L. et al., (1995) Nuclear pre-mRNA splicing. In: Eckstein, F. and Lilley, D.M.J.

(eds). Nucleic Acids and Molecular Biology. Springer Verlag, Berlin, pp. 342-372). At relatively low salt concentrations, at which core extracts from HeLa cells can cause the splicing of pre-mRNA in vitro, the snRNPs lie in a 12S U1 snRNP, a 17S U2 snRNP and a 25S [U4 / U6.U5] tri- snRNP complex. At higher salt concentrations (approx. 350-450 mM) the tri-snRNP complex dissociates into a 20S U5 and a 12S U4 / U6 particle. The U4 and U6 RNAs are base-paired in the U4 / U6 snRNP via two intermolecular helices (Bringmann, P. et al. (1984) EMBO J., 3, 1357;; Hashimoto, C. & Steitz, JA (1984) Nucleic Acids Res., 12, 3283; Rinke, J. et al., (1985) J. Mol. Biol., 185, 721; Brow,

THERE. & Guthrie, C. (1988) Nature, 334, 213).

The snRNPs consist of two groups of proteins. The group of general proteins (B / B \ D1, D2, D3, E, F and G) is contained in all snRNPs. In addition, each snRNP contains specific proteins that are only contained in this. According to the current state of research, the U1 snRNP contains three additional proteins (70K, A and C) and the U2 snRNP contains eleven additional proteins. To date, the 20S U5 snRNP carries nine additional proteins with a molecular weight of 15, 40, 52, 100, 102, 110, 116, 200 and 220 kDa, while the 12S U4 / U6 snRNP carries two additional proteins with a molecular weight of contains approx. 60 and 90 kDa. The 25S tri-snRNP [U4 / U6.U5] contains five additional proteins with a molecular weight of approx. 15.5, 20, 27, 61 and 63 kDa. (Behrens, SE & Lührmann, R. (1991) Genes Dev., 5, 1439; Utans, U. et al., (1992) Genes Dev., 6, 631; Lauber, J. et al., (1996) EMBO J., 15, 4001; Will, CL et al. (1995), supra, Will, CL & Lührmann, R. (1997) Curr. Opin. Cell Biol., 9, 320-328).

The composition of the splice components in Saccharomyces cerevisiae has not yet been examined in detail. However, biochemical and genetic studies indicate that the sequences of both the snRNAs and the snRNP proteins are highly conserved in evolution (Fabrizio, P. et al., (1994) Science, 264, 261; Lauber, J. et al., (1996), supra, Neubauer, G. et al., (1997) Proc. Natl. Acad. Sei. USA, 94, 385; Krämer, A. (1995), supra; Beggs, JD (1995) ; supra, Gottschalk, A. et al. (1998) RNA, 4, 374-393).

In order to form a functional splice complex (spliceosome), the individual components (pre-mRNA, snRNPs and non-snRNP proteins) are brought together in a step-by-step process. This is achieved not only through interactions of the pre-mRNA with the protein-containing components, but also through numerous interactions between the protein-containing components themselves

(Moore, JM (1993) supra; Madhani, HD & Guthrie, C. (1994) Annu. Rev. Genetics, 28, 1; Nilsen, TW (1994) Cell, 65, 115). The sequence of the pre-mRNA carries specific recognition sequences for the different splice components. First, the U1 snRNP binds to the 5 ^' splice via these recognition sequences. Region of the intron of the pre-mRNA. At the same time, an as yet undetermined number of various other factors (eg SF2 / ASF, U2AF, SC35, SF1) attach to this complex and cooperate with the snRNAs in the further formation of the pre-spliceosome. The U2 snRNP particle interacts with the so-called branch site in the intron area (Krämer, A. & Utans, U. (1991) EMBO J., 10,

1503; Fu, X.D. & Maniatis, T. (1992) Proc. Natl. Acad. Be USA, 89, 1725; Kramer, A. (1992) Mol. Cell Biol., 12, 4545; Zamore, P.D. et al. (1992) Nature, 355, 609; Eperon, J.C. et al. (1993) EMBO J., 12, 3607; Zuo, P. (1994) Proc. Natl. Acad. Be. USA, 91, 3363; Hodges, P.E. & Beggs, J.D. (1994) Curr. Biol. 4, 264; Reed, R. (1996) Curr. Op. Gene. Dev., 6, 215). In a final step in the formation of the spliceosome, the [U4 / U6.U5] tri-snRNP and a number of previously unspecified proteins interact with the pre-spliceosome to form the mature spliceosome (Moore, JM et al., (1993) supra).

For the process of splicing, there are various interactions between

Pre-mRNA, snRNAs and sn-RNP solved and new ones formed. It is known, for example, that before or during the first catalytic step of the splice reaction in the interacting structures of U4 and U6, two helices are separated from one another and base pairings are formed between U2 and U6 RNAs through new interactions (Datta, B. & Weiner, AM (1991) Nature, 352, 821;; Wu, JA & Manley, JL (1991)

Nature, 352, 818; Madhani, HD & Guthrie, C. (1992) Cell, 71, 803; Sun, JS & Manley, JL (1995) Genes Dev., 9, 843; , At the same time, the binding of U1 to the 5 ^' splice point is released and the pre mRNA binds to the recognition sequence ACAGAG of the U6 snRNA (Fabrizio, P. & Abelson, J. (1990) Science, 250, 404; Sawa, H. & Abelson, J. (1992) Proc. Natl. Acad. Sei. USA, 89, 11269; Kandels-Lewis,

S. & Seraphin, B. (1993) Science, 262, 2035; Lesser, CF & Guthrie, C. (1993) Science, 262, 1982; Sontheimer, EJ & Steitz, JA (1993) Science, 262, 1989; , The U5 snRNP interacts with its conserved Loop 1 with exon sequences that are close to the 5 ^' and 3 ^' splice points. This process appears to proceed sequentially as the entire splicing process progresses from level 1 to level 2 (M. McKeown, (1992) Annu. Rev. Cell Dev. Biol., 8: 133-155 Newman, A. & Norman , C. (1991) Cell, 65, 115; Wyatt, JR et al., (1992) Genes Dev., 6, 2542; Cortes, JJ et al. (1993) EMBO J., 12, 5181; Sontheimer, EJ & Steits, (1993) su- pre). After the splice reaction has ended, the mature mRNA is released and the spliceosome is dissociated (Moore, JM et al., (1993) supra).

By means of alternative splicing, different mature mRNAs can be formed from one and the same primary transcript, which code for different proteins.

This alternative splicing is regulated in many cases. This mechanism can e.g. can be used to switch from a non-functional to a functional protein (e.g. transposase in Drosophila). It is also known that alternative splicing is carried out in a tissue-specific manner. For example, the tyrosine kinase encoded by the src proto-oncogene in nerve cells by alternative

Splicing synthesized in a special shape.

Incorrectly regulated or performed alternative splicing can lead to various clinical pictures. In patients suffering from Grave ^'s disease, it has been shown that incorrect splicing results in a crucial enzyme (thyroperoxidase) in an inactive form (Zanelli, E. (1990) Biochem Biophys. Res. Comm., 170 , 725). Studies for the disease spinal muscular atrophy indicate that a defective gene product of the SMN gene (survival of motor neurons) leads to a considerable disruption of the formation of the snRNPs. By inhibiting the splicing apparatus of the muscle neurons, there is a paralysis of the

Nerve cells and muscle tissue breakdown (Fischer, U. et al., (1997), Cell, 90: 1023-9; Liu, Q. et al. (1997), Cell, 90: 1013-21; Lefebvre, S et al. (1997) Nat. Genet., 16, 265). In the metastasis of cancer cells, certain alternative splice variants of the membrane-bound molecule CD44 seem to play a decisive role. The CD44 gene contains several exons, of which 10 adjacent exons in a different arrangement are spliced out of the pre-mRNA during mRNA generation. In rat carcinoma cells, it was demonstrated that metastatic variants carry exons 4 to 7 or 6 to 7. With the help of antibodies against the part of the protein encoded by exon 6, the metastasis could be effectively suppressed (Sherman, L., et al.,

(1996) Curr. Top. Microbiol. Immunol. 213: 249-269). Incorrect splicing can lead to pronounced phenotypes of the affected organism. It is known that a point mutation in an intron of the ß-globin can lead to a ß ⁺ thalemia. The point mutation creates an incorrect splice location, which leads to a changed reading frame and to premature termination of the peptide chain (Weatherall, D. & Clegg, JB (1982) Cell, 29, 7;

Fukumaki, Y. et al. (1982) Cell, 28, 585). In Arabidopsis thaliana mutants z. B. a point mutation at the 5 ^' splice of the phytochrome B gene to an incorrect expression of the gene. This change does not remove an intron that contains a stop codon in its sequence. The development of the plants is disturbed because the gene is involved in phytomorphogenesis (Bradley,

J.M. et al. (1995) Plant Mol. Biol, 27, 1133).

So far, only a few works have become known in which an influence on splicing processes in the cell has been described. The generation of mature mRNA can be prevented with the aid of antisera or monoclonal antibodies against components of the splice apparatus (Padgett, RA et al. (1983) Cell, 35, 10; Gattoni, R. et al. (1996) Nucleic Acid Res. , 24, 2535).

The NS1 protein, which is encoded by the genome of the influenza virus, can also interfere with the splicing by binding to the U6 snRNA. The protein binds to nucleotides 27-46 and 83-101 of human U6 snRNA and thus prevents U6 from interacting with partners U2 and U4 during the splicing process (Fortes, P. et al. (1994) EMBO J. , 13, 704; Qiu, Y. & Krug, RM (1995) J. Virol., 68, 2425. Furthermore, the NS1 protein also appears to be exported from the nucleus via binding to the poly-A tail of the mRNA formed (Fortes, P. et al. (1994), supra; Qiu, Y. & Krug, RM (1994), supra.) Similar effects are described by a gene product of the herpes simplex virus type 1 genome. The protein ICP27 could effectively prevent the splicing of a model RNA (β-globin pre-mRNA) in in vitro experiments (Hardy, WR & Sandri-Goldin, RM (1994) J. Virol., 68, 7790) generated from the C-terminal domain of the large subunit of RNA polymerase II, to be able to also intervene in the splicing processes (Yurvey, A. et al. (1996) Proc. Natl. Acad. Be USA, 93, 6975; ; WO97 / 20031). The incorporation of artificial nucleotide analogs (5-fluoro-, 5-chloro- or 5-bromouridine) into the mRNA to be spliced can also lead to an inhibition of the splicing process in vitro (Sierakowska, H. et al. (1989) J. Biol. Chem., 264, 19185; Wu, XP & Dolnick, B. (1993) Mol. Pharmacol., 44, 22).

A number of other studies are concerned with the effect of anti-sense oligonucleotides on splicing. The ratio of two different splice products of the rat c-erb oncogene mRNA (c-erbA-alpha 1 and 2) appears to be regulated by another mRNA, rev-ErbA-alpha. Rev-ErbA-alpha is a naturally occurring anti-sense RNA that pairs with the c-erbA-alpha 2 mRNA but not with the c-erbA-alpha 1 mRNA. The splicing of the c-erbA-alpha pre-mRNA to the c-erbA-alpha 2 mRNA was effectively inhibited by an excess of rev-ErbA-alpha mRNA constructs that were complementary to the 3 ^' splice site (Munroe, SH & Lazar, MA, (1991) J. Biol. Chem., 266 (33), 22083).

It was also possible to show that splicing can also be inhibited by generating anti-sense RNA which bind to the intron sequences of the mRNA to be spliced (Volloch, V. et al. (1991) Biochem. Biophys. Res. Comm ., 179, 1600). Hodges and Crooke were able to show that in the case of weakly recognized splice sites, the binding of oligonucleotides is sufficient to successfully prevent splicing. If, on the other hand, recognized splicing sites are preferably incorporated into the constructs, oligonucleotides are required which can additionally activate RNase H (Hodges, D. & Crooke ST (1995) Mol. Pharmacol., 48, 905). A more detailed analysis of the pre-mRNA sequences required for the splicing showed that 19 nucleotides upstream from the branch point adenosine and 25 nucleotides around the 3 ^' and 5 ^' splice site are suitable sequences for generating antisense RNAs ( Dominski, Z. & Kole, R. (1994) Mol. Cell Biol., 14, 7445). Investigations with antisense molecules have been carried out in particular for the inhibition of viruses. Viruses that attack higher organisms often carry intron-containing genes in their genome. It has been shown that antisense oligonucleotides against the 3 ^' splice site of the immediate early pre-mRNA 4/5 gene of the herpes simplex virus in Vero cells could inhibit the replication of the virus (Iwatani, W. et al. (1996) Drug Delivery Syst., 11, 427). To investigate the splicing mechanism, an mRNA is generally first produced by in vitro transcription. Genetic constructs from viruses, e.g. B. adenoviruses, or cellular structural genes. Such mRNAs contain all the important structural elements that are necessary for the recognition of the mRNA by the spliceosome and the splicing process. In general, the mRNA is radiolabelled so that, after separation on a denaturing urea-polyacrylamide gel, the characteristic band patterns can be used to assess whether a splicing reaction has occurred or in which reaction step a disturbance has occurred. However, test systems of this type are very time-consuming and labor-intensive and are therefore not suitable for the systematic detection of substances which can modulate the splicing.

It was therefore an object of the present invention to find a test system with which a large number of compounds from chemical or natural substance libraries can be investigated in a simple and effective manner for their effect in splicing nucleic acids (so-called high through put scree - ning).

It has now surprisingly been found that a test system with a gel-free detection system is suitable for detecting a splicing reaction, to overcome the disadvantages of the conventional test system described above and is therefore suitable for high-through put screening, for example in a robot system.

The present invention therefore relates to a test system comprising (a) one or more, identical or different immobilized nucleic acid (s) with at least one splicable nucleic acid,

(b) at least one gel-free detection system for the detection of a splice reaction, if necessary

(c) at least one composition containing splice components, and preferably

(d) suitable detection probes, and if necessary (e) other tools.

In order to provide a gel-free test system for the investigation of splicing processes, the nucleic acid to be examined must be immobilized on a solid phase. The nucleic acid can be immobilized, for example, covalently, by introducing certain structural elements, for example aptamers, into the nucleic acid to be spliced and using binding partners for these structural elements, or by hybridization.

For the gel-free test system, it is also advantageous to generate a suitable probe that enables the detection of the splicing that has occurred or the splicing that has not occurred. This probe can be, for example, an oligonucleotide used for hybridization to the nucleic acid to be examined or a binding partner that binds to structural elements introduced into the nucleic acid to be examined.

The gel-free detection system therefore preferably contains at least one probe. In particular, the probe is a nucleic acid which is complementary to the splicable nucleic acid, a low molecular compound which binds the splicable nucleic acid and / or a peptide or protein which binds the splicable nucleic acid.

In a preferred embodiment, the splicable nucleic acid contains at least two exons which are separated by at least one intron.

For example, the complementary nucleic acid is complementary to at least one intron, to at least one exon and / or to at least one transition point from one exon and one intron and / or to the exon / exon boundary that resulted after the fusion of the two exons. The complementary nucleic acid serves as a probe for the detection of a splice reaction. For example, the intron released during the splicing reaction can be detected by means of the gel-free detection system, from which it can be concluded that both sub-steps of the splicing reaction have been completed. Alternatively, a suitable detection system, for example a nucleic acid complementary to a transition point between an exon and an intron, can be used to determine whether the exon has been detached from the intron during the splicing process, which can provide information about whether the first splicing reaction on 5 ' -End of the intron and / or the second splice reaction took place at the 3'-end of the intron. Other forms of evidence are explained in detail below.

In a particularly preferred embodiment, the probe is a low molecular weight compound, for example theophylline, xanthine or an aminoglycoside such as tobramycin. For example, if the splicable nucleic acid or a nucleic acid of the gel-free detection system that is complementary to the splicable nucleic acid contains a so-called aptamer structure, ie. H. a binding sequence for such binding partners (see e.g. Jenison, RD et al. (1994) Science 263, 1425-1429, Hamasaki, K. et al. (1998) Biochem. 37, 656-663 or Kiga, D. et al. (1998) Nucleic Acids Res., 26 (7), 1755 - 1760), the splicing process can be detected particularly easily via the binding partner.

In another particularly preferred embodiment, the binding partner can be a nucleic acid binding protein, in particular an "Iran Responsive Element Binding Protein" (IBP), which recognizes a recognition sequence for a nucleic acid binding protein, in particular an "Iran Responsive Element" (IRE) . Proof of a splicing process that may have taken place is provided here by the

Nucleic acid binding protein.

The described interactions between binding partner and structural element in the nucleic acid (low molecular compound and aptamer, IBP and IRE, oligonucleotide and sequence in the nucleic acid) are also suitable for immobilizing the splicable nucleic acid to be investigated on a solid phase. For this purpose, the binding partner must be anchored to the solid phase in a suitable manner. For example, the binding partner can be covalently bound to the solid phase. Furthermore, the coupling of biotin to the nucleic acid and the use of (strept) avidin bound to the solid phase are suitable for anchoring the nucleic acid. This anchoring can also be achieved, for example, by using antibody / antigen interactions.

In general, the probe contains a label, for example a radioactive label, a label with fluorescent dyes, with biotin, with digoxigenin and / or with antibodies. The label is preferably attached to the ligand, for example to the complementary nucleic acid, to the low molecular weight compound or to the nucleic acid binding protein. In particular, with the help of fluorescent dyes, it can be determined in a simple and rapid manner in automated systems whether, for example, by removing the binding partner of the probe in the nucleic acid during the splicing process, a splicing reaction, for example in the presence of at least one substance to be investigated, can proceed undisturbed.

In a further preferred embodiment, the splicable nucleic acid and the probe-binding nucleic acid are linked to one another. This has the advantage that the splicing reaction can be detected directly, for example, by the release of the probe-binding nucleic acid. In this embodiment, the probe-binding nucleic acid is preferably a nucleic acid that can bind a low-molecular compound, for example a so-called aptamer, and / or a nucleic acid binding protein. Since certain structural elements of the nucleic acids are generally responsible for the binding of such probes, the probe-binding nucleic acid is abbreviated with "SE" for structural element in the following preferred constructs, where "3 ^' region" means a nucleic acid section at the 3 ^' end of the nucleic acid :

1. Constructs with structural element (SE) introduced in the 5'-exon: - T7 promoter— SE— Exoni— Intron - Exon2— 3'Region—

- T7 promoter— Exoni— SE— Exoni --Intron— Exon2— 3'Region—

2. Construct with structural element introduced in the intron:

- T7 promoter— Exoni— Intron— SE— Intron— Exon2— 3'Region-

3. Constructs with structural element introduced in the 3'-exon:

- T7 promoter - Exoni - Intron - Exon2 - SE - Exon2 - 3'Region -

- T7 promoter - Exoni - Intron - Exon2 - SE - 3 'region - - T7 promoter - Exoni - Intron - Exon2 - 3' region - SE - 3'Region— - T7 promoter - Exoni - Intron - Exon2 - 3 'Region - SE -

4. Constructs with combinations of the constructs listed in 1. - 3.

5. Constructs with different structural elements:

- T7 promoter— Exoni— SE1— intron - Exon2— SE2— 3 'region - T7 promoter— Exoni— SE1— Exoni— Intron - SE2— Intron— Exon2— SE3—

3 'region -

The constructs for 1. are used in particular to demonstrate whether the exoni could be separated from the intron sequence during the splicing process. The construct for 2. is used for the direct detection of a separated intron sequence. The

Constructs under 3. are used to demonstrate whether Exon2 was successfully separated from the intron sequence. A combination of the constructs according to FIG. 4. serves to prove the individual intermediate and end products during the splicing process. Exoni generally represents a 5 'from the intron and exon 2 generally represents a 3' from the intron.

The constructs under 5 contain various additional recognition sequences which, on the one hand, can affect different detection systems and, on the other hand, can enable the nucleic acid to be bound to a solid phase via their binding partners. For example, a probe-binding nucleic acid can be introduced for immobilization in the 3 ′ region, and at the same time another probe-binding nucleic acid can be introduced in the exoni to demonstrate the splicing process

(see 5, first construct). In addition, for example, three different nucleic acids can be introduced, which serve on the one hand to immobilize the entire nucleic acid and on the other hand to detect the removed intron and to demonstrate the linkage of exoni to the rest of the nucleic acid (see 5th, second construct). The location of the individual probe-binding nucleic acids listed under 5 by way of example can vary according to the constructs described above under 1-4. In addition, the exact location of the individual probe-binding nucleic acids is variable.

In a further embodiment, the nucleic acid, preferably the splicable nucleic acid, in particular a nucleic acid according to one of the above-described embodiments, is therefore directly covalently or indirectly bound to a solid phase via a structural element and binding partner to the structural element or by means of hybridization.

Direct covalent binding can take place, for example, via the 3 ^' terminal cis-diol group of the ribose backbone of the nucleic acid. For example, an RNA can be bound to hydrazine groups of the solid phase after periodate oxidation of the vicinal 2 ^' , 3 ^' hydroxyl groups of the 3 ^' -terminal ribose. Suitable linkers, such as, for example, biotin or dicarboxylic acid linkers, are also suitable for indirect binding. However, as already stated above, the nucleic acid can also be bound via a binding partner, for example via theophylline or xanthine or an aminoglycoside such as tobramycin and / or via a nucleic acid binding protein such as IBP.

For the immobilization of a nucleic acid on a carrier-bound ligand, the theophylline aptamer Th (K _d = 0.9 μM) is suitable, for example, in the case of theophylline as a carrier-bound binding partner (see, for example, Jenison, RG et al. ( 1994) supra) or, in the case of tobramycin as a carrier-bound binding partner, the minimal version of the tobramycin aptamer To (K = 0.2 μM) (Hamasaki, K. et al. (1998) supra). The sequences of the two aptamers are preferably:

Th: AAGUGAUACC AGCAUCGUCU UGAUGCCCUU GGCAGCACUU (40mer) To: GGCUUAGUAU AGCGAGGUUU AGCUACACUC GUGCUGAGCC (40mer)

The solid phase here is, for example, ceramic, metal, in particular noble metal, glass, plastic or polysaccharides, e.g. an agarose polymer.

Probe-binding nucleic acids, for example aptamers, are also suitable for binding labeled probes. B. the aptamer-containing nucleic acid can be detected and also quantified. For example, tobramycin can be reacted with commercially available, NH ₂ -reactive fluorescent dyes (Wang, Y. et al. (1996) Biochemistry 35, 12338 - 12346). With theophylline, a 1-aminoalkyl or 1-thioalkyl derivative of 3-methylxanthine is preferably produced, which can bind to the aptamer (Jenison, RD et al. (1994), supra).

In accordance with the present invention, the splicable nucleic acid is any

Nucleic acid that can be spliced, preferably an RNA, for example in the form of a so-called pre-mRNA or in the form of a DNA that contains RNA sections. In the event that the RNA is to contain additional probe-binding sequences, as already described in more detail above, it is advantageous if these have at least approximately 25 nucleotides from the respective splice site on both exon sides the intron side is at least about 17 nucleotides from the branch point and / or at least about 7 nucleotides from the 5 'splice site. This generally ensures that the additional probe binding sequences cannot interfere with the splicing reactions.

A splicable nucleic acid suitable for splicing in the human system is, for example, the MINX model pre-mRNA (MINX = miniature wild-type substrates; Zillmann, M., Zapp, ML, Berget, SM, (1988), Mol. Cell. Biol., 8: 814-21). A further probe-binding nucleic acid suitable for detection or immobilization, as already described above, can preferably be introduced into the MINX-encoding DNA, the restriction enzyme interface preferably being retained. As a result, if necessary, in a further cloning step, a further identical or different probe-binding nucleic acid can be incorporated for detection or for immobilization, in order to be able to amplify the fluorescence signals or to strengthen the binding to the solid phase.

To immobilize pre-mRNA, for example, the aptamers Th or To are inserted at the 3 'end of the exon2 of the pre-mRNA and the corresponding binding partner is covalently bound to the solid phase. The corresponding coding nucleic acid sequences are shown in FIGS. 1A and 1B.

Here, for example, the corresponding aptamer sequence is inserted as DNA oligonucleotide into the BamHI site of the encoding Minx DNA, ie. H. between positions 219 and 220 of the corresponding Minx pre-mRNA.

As already mentioned above, probe-binding nucleic acids can also be inserted into the intron structure of the pre-mRNA, this being released by the splicing and thus missing in the immobilized mRNA, for example. Such constructs are therefore suitable for the detection of an inhibition of splicing in the first step, ie. H. Opening the mRNA and lariatization, or in the second step, d. H.

Removal of the lariat. If the splicing process were inhibited, the probe-binding nucleic acids would not be removed from the pre-mRNA and could therefore be detected, for example, after immobilization of the pre-mRNA. Examples of suitable nucleic acid constructs are in the form of their coding

Sequences shown in Figures 2A and 2B.

For this purpose, the corresponding aptamer sequence is, for example,

Oligonucleotide into the PstI site of the Minx coding DNA, i.e. H. between positions 88 and 89 of the corresponding pre-mRNA.

As already described in more detail above, the connection between Exoni and Intron is in the first splicing step through the spliceosome at the 5 'splice point of the

Introns open. Only in the second splicing step is there a covalent link between Exoni and Exon2. The exoni is therefore no longer connected to the mRNA during the first step of the splicing reaction and can therefore be removed from the splicing reaction. In connection with constructs that have, for example, an aptamer structure in the intron, a statement can therefore be made as to whether, for example, inhibition has taken place in the first splicing step. If, for example, two different aptamers, which recognize different probes, are installed at the 5 'end of the exoni and in the intron of the pre-mRNA, both the first splicing step and the second splicing step can be followed in a test system. Examples of suitable nucleic acid constructs are in the form of their coding

Sequences shown in Figures 3A and 3B.

For this purpose, for example, the corresponding aptamer sequence is inserted as a DNA oligonucleotide into the EcoRI site of the coding Minx DNA, ie. H. between positions 9 and 10 of the corresponding Minx pre-mRNA.

For investigations in the yeast system, one can, for example, start from the pre-mRNA for U3 of the yeast (Mougin, A. et al., (1996), RNA, 2: 1079-93) and, for example, suitable aptamers such as e.g. B. incorporate the theophylline or tobramycin aptamers described above. Examples of suitable nucleic acid constructs are shown in the form of their coding sequences in FIGS. 4A to 4C. To produce the nucleic acid construct according to FIG. 4A, for example, a suitable aptamer as DNA oligonucleotide is inserted into the SacII site of the coding U3 DNA, ie between positions 22 and 23 of the pre-U3 RNA.

To produce the nucleic acid construct according to FIG. 4B, for example, a suitable aptamer as a DNA oligonucleotide is inserted into the BstNI site of the coding U3 DNA, ie. H. between positions 105 and 106 of the pre-U3RNA.

The nucleic acid sequence according to FIG. 5A represents an example of a nucleic acid sequence with an "Iran Responsive Element" (IRE), which is used for investigations in

Human system is suitable. The IRE is inserted at the 3 'end of the Exon2 analogous to the aptamers described above.

For investigations in the yeast system, an IRE element can be inserted, for example, at the 3 'end of exon2 of the pre-U3RNA, as shown in FIG. 5B:

Another object of the present invention is therefore a splicable nucleic acid, as exemplified above, and its use for the production of a test system.

To carry out the investigations of the individual splicing reactions with the aid of a test system according to the invention, a composition containing the individual splicing components, preferably "small nuclear ribonoprotein particles (snRNP) components and non-snRNP components, is usually used. In particular, the snRNP components contain U1, U2, U4, U5 and / or U6 proteins. In particular, it is preferred to use appropriate cell extracts, in particular eukaryotic cell extracts, for the examinations. For example, the cell extracts from animal cells, in particular mammalian cells, especially Heia Cells, in particular from cell nucleus extracts from He-La cells or cell extracts from fungi, in particular yeasts, can be obtained by processes which are well known to the person skilled in the art (see examples) generally contain all the important factors for performing splicing in vitro.

To carry out the investigations, it is essential to use other aids such as B. use buffer solutions, stabilizers and / or energy equivalents, especially ATP.

The present invention therefore also relates to a method for producing a test system, in which at least one splicable and immobilized nucleic acid and at least one gel-free detection system as well as optionally at least one composition containing splicing components and optionally further aids are put together. Preferred embodiments of the individual components have already been described in more detail above.

Another object of the present invention is a method for finding an active substance, wherein a) one or more identical or different immobilized nucleic acid (s) with at least one splicable nucleic acid sequence in the presence of at least one substance to be examined and at least one composition containing splice components and if necessary, further aids are incubated under suitable conditions, and b) the splice product which may be formed is detected by means of a gel-free detection system.

Preferred individual components of the method according to the invention have already been described in more detail above.

The active substance can be a pharmaceutically active compound

Fungicide, a herbicide, a pesticide and / or an insecticide, preferably it is an antibiotic. The substance to be examined is generally a natural one occurring, a naturally occurring and chemically modified and / or a synthetic substance. In particular, so-called combinatorial substance libraries can be searched particularly easily and quickly with the methods according to the invention.

In the introduction to the description, it was already pointed out that various diseases can be attributed to a malfunction of the splicing mechanism. The present invention is therefore also suitable for diagnosing a disease.

Another object of the present invention is therefore a method for

Diagnosis of a disease, in which a) one or more, identical or different immobilized nucleic acid (s) with at least one splicable nucleic acid sequence are incubated in the presence of at least one composition containing splicing components and, if appropriate, further auxiliaries under suitable conditions, and b) that possibly formed Splice product is detected using a gel-free detection system.

The diseases to be diagnosed are preferably hereditary diseases,

Cancer and / or viral diseases, in particular Grave's disease, spinal muscular atrophy, ß'-thalassemia, cancer related to the c-hereditary oncogene, hepatitis C infections and / or herpes simplex virus infections. In this case, the composition containing splice components can be, for example, a treated or untreated tissue sample from the patient.

The following figures and examples are intended to describe the invention in more detail without restricting it. The constructs are shown in DNA sequences. The spliceable RNAs can be generated by in vitro transcription as described below. Description of the figures

1A shows the coding nucleic acid sequence in which a Th aptamer was inserted at the 3 'end of the exon2 of a pre-mRNA coding for Minx.

1B shows the coding nucleic acid sequence in which a To aptamer was inserted at the 3 'end of the exon2 of a pre-mRNA coding for Minx.

FIG. 2A shows the coding nucleic acid sequence in which a Th aptamer was inserted into the intron of a pre-mRNA coding for Minx.

2B shows the coding nucleic acid sequence in which a to-aptamer was inserted into the intron of a pre-mRNA coding for Minx.

FIG. 3A shows the coding nucleic acid sequence in which a Th aptamer was inserted at the 5 ′ end of the exoni of a pre-mRNA coding for Minx.

3B shows the sequence of an RNA in which a Th aptamer was inserted at the 5 'end of the exoni of a pre-mRNA coding for Minx.

4A shows the coding nucleic acid sequence which codes for a U3 pre-mRNA. At the 5 ^' end of the Exoni is an insert for a Th or To aptamer.

4B shows the coding nucleic acid sequence which codes for a U3 pre-mRNA. An insertion point for a Th or To aptamer is marked in the intron. 4C shows the coding nucleic acid sequence which codes for a U3 pre-mRNA. An insert for a Th or To aptamer is identified at the 3 'end of the Exon2.

Figure 5A shows the coding nucleic acid sequence coding for a Minx pre-mRNA. The sequence for IRE is inserted at the 3 'end of exon2.

5B shows the coding nucleic acid sequence which codes for one of the U3 pre-mRNA. The sequence for IRE is inserted at the 3 'end of exon2.

FIG. 6 shows the temporal course of the splicing of MINX and MINX-IRE pre mRNA by HeLa core extract.

MINX (lanes 1-5) and MINX-IRE (lanes 6-10) pre-mRNAs transcribed in vitro were incubated with HeLa core extract for the period shown in the figure. The samples were then separated using polyacrylamide / urea gel electrophoresis and the 32P-labeled RNA was detected using autoradiography. Multiple bands in the RNA containing IRE (lanes 6-10) can be explained by incomplete denaturation of the IRE.

Examples

1. The RNA construct:

The mRNA to be spliced consists of at least two exons that are separated by an intron. In addition to these sequences, sequences are included which are suitable for enabling specific recognition by means of RNA binding proteins or low molecular weight compounds. The mRNA is selectively bound to the matrix by coupling these binding proteins or compounds to a matrix. Alternatively, the mRNA is also coupled covalently to the matrix via 3 ^' OH groups of the ribose. Preparation of the core extracts:

2.1 Nuclear extracts from mammalian cells

For the production of nuclear extracts from mammalian cells, cell cultures with Heia cells are used. For this, the cells are centrifuged (1000 x g,

10 min.) Sedimented from the culture medium and washed with phosphate buffer. The cell sediment is then taken up in five times the volume of buffer A (10 mM HEPES, 1.5 mM MgCl ₂ , 10 mM KCI, 0.5 mM DTT, pH 7.9, 4 ° C.) and incubated for 10 minutes. The cells are sedimented again and taken up in twice the volume of buffer A. This suspension is digested with a dounce homogenizer (pestle B) (10 times the pestle is moved up and down). The cores are sedimented by centrifugation. Finally, the cores are again taken up in buffer A and centrifuged for 20 minutes at 25,000 xg. The sediment is dissolved in 3 ml buffer B (20 mM HEPES, 25% (v / v) glycerin, 0.42 M NaCl, 1, 5 mM MgCl ₂ , 0.2 mM EDTA, 0.5 mM phenylmethysisulfonyl fluoride (PMSF),

0.5 mM DTT, pH 7.9) and digested again with the Dounce homogenizer. The resulting suspension is incubated for 30 minutes on a magnetic stirrer and then centrifuged at 25,000 x g for 30 minutes. Another centrifugation at 25,000 x g (30 minutes) follows. The clear supernatant is against 50 times the volume of buffer C (20 mM HEPES, 20% (v / v) glycerol, 0.1

M KCI, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT, pH 7.9) dialyzed. The dialysate is centrifuged (25,000 x g, 20 min.) And the resulting supernatant can be stored as a core extract in liquid nitrogen (Dignam; J.D. et al. (1983) Nucleic Acid Res., 11, 1475).

2.2 Cell extracts from yeast cells

Cell extracts from yeast cells are made in a very similar manner. Yeast cells of a protease-deficient strain (BJ926, EJ101 or similar strains) are sedimented in the logarithmic growth phase by centrifugation (1500 x g, 5 min., 4 ° C.). The cells are ice-cold in two to four times the volume

Resuspended water and centrifuged again at 1,500 xg (5 min, 4 ° C). Then the cells in the simple volume of zymolyase buffer (50 mM Tris HCL, 10 mM MgCl ₂ , 1 M sorbitol, 30 mM DTT, pH 7.5) and incubated for 30 minutes at room temperature. The cells are centrifuged off (1,500 xg, 5 min., 4 ° C.), taken up in three times the volume of zymolyase buffer with 2 mg (200 U) of Zymolyase 100 T and in for 40 minutes at 30 ° C. on a shaker (50 RPM) - cubed. The spheroblasts formed are centrifuged off (1,500 xg, 5 min.,

4 ° C) and washed once in 2 ml of ice-cold zymolyase buffer. The sediment is washed with two volumes of lysis buffer (50 mM Tris HCl, 10 mM MgSO ₄ , 1 mM EDTA, 10 mM potassium acetate, 1 mM DTT, protease inhibitors, 1 mM PMSF, pH 7.5) and finally in one volume of lysis buffer added. The spheroblasts are then in a dounce homogenizer by 15 to 20 times

Moving the pestle up and down (distance 1-2 μm) lysed. The same volume of extraction buffer (lysis buffer + 0.8 M ammonium sulfate, 20% (v / v) glycerol) is added to the lysate and incubated for 15-30 minutes on an overhead shaker (4 ° C.). Then centrifuged at 100,000 x g for 90 minutes (4 ° C). The supernatant is compared to a hundred-fold volume of storage buffer (20 mM Tris HCl, 0.1 mM

EDTA, 10% (v / v) glycerin, 100 mM KCI, 1 mM DTT, Proteae Inhibitors, 1 mM PMSF, pH 7.5) dialyzed. The dialysate is centrifuged at 10,000 xg (4 ° C.) and the supernatant is stored in liquid nitrogen (Dünn, B. & Wobbe, CR (1994) Preparation of protein extracts from yeast, In: Ausubel, FM, et al. ( eds.) Current proto- cols in Molecular Biology, ^2nd Volume, John Wiley and Sons, Inc., USA pp 13.13.1. -

13.13.9).

3. In vitro splicing of constructs with IRE in a test system

By cutting out the intron from the RNA sequence, a nucleotide sequence arises at the border of the two now connected exons, which is not present in the unspliced pre-mRNA (new sequence). This sequence is used to generate complementary nucleotide sequences that selectively only bind to this new sequence. The splicing carried out is indirectly detected by covalent binding of fluorescent dyes, biotin, digoxigenin or similar molecules or by radioactive labeling. The assay is evaluated in a suitable evaluation device (ELISA reader, fluorescence measuring device, etc.). All experiments described were carried out according to standard methods as described in (Eperon,

I.C., and Krainer, A.R. (1994) Splicing of mRNA precursors in mammalian cells. In

RNA Processing, vol. I - A Practical Approach (B.D. Harnes and S.J. Higgins, eds.)

Oxford: IRL Press, pp. 57-101).

3.1. Carrying out in vitro transcription

The construct described in FIG. 5A was transcribed from the plasmid coding for it into the corresponding mRNA by means of in vitro transcription. The corresponding construct without the IRE sequence was also used in the experiments for control and later comparison.

The constructs had previously been cloned into the vector pGEM-3Zf (Pharmacia) and propagated in E. coli. The plasmids were purified using standard technologies and adjusted to the desired concentration. Before use in vitro

The transcription, the plasmids were linearized using restriction enzymes.

The reaction was carried out under the following conditions:

5 μl 5 x transcription buffer (200 mM Tris-HCl pH 7.9, 30 mM MgCl ₂ , 10 mM spermidine, 50 mM NaCI) 1 μl BSA (1 mg / ml)

1 μl RNAsin 2.5 μl DTT (100 mM) 1 μl NTP ^'s (ATP, GTP, CTP with 2.5 mM and UTP with 1.25 mM)

2 μl ³² P-UTP (3000 Ci / mM)

2 ul MINX plasmid linearized (1 mg / ml) 2.5 ul GpppG-Cap (1 mM) 2 ul SP6 polymerase ad 25 ul with H ₂ O

The transcription batch was incubated for 2 h at 37 ° C. and then cleaned using a preparative gel using standard methods. To find the marked An X-ray film was placed on the RNA for 1 minute and the band by means of an

Cut out scalpel. The gel fragment was cut and the RNA extracted from the gel overnight at 4 ° C. with elution buffer (500 mM Na acetate pH 5, 1 mM EDTA pH 8, 2.5% phenol / chloroform).

3.2 Execution of the splice reaction

7 μl core extract from HeLa cells (= 35% v / v) were mixed with 3.25 mM MgCl ₂ , 35 mM KCI, 2 mM ATP, 20 mM phosphocreatine, 1 U / μl RNAsin and 30,000 - 50,000 cpm MINX-pre- mRNA (Zillmann et al., 1988, Molecular and Cellular Biology, 8: 814) in a reaction volume of 20 μl for 0, 10, 20, 30 and 40 minutes at 30 ° C. For comparison, pre-mRNA was used which did not contain the IRE. The reactions were then ended by adding 400 μl Proteinase K buffer (100 mM Tris-HCl, pH 7.5, 12.5, mM EDTA, 150 mM NaCl, 1% SDS, 0.1 mg proteinase K) . The samples were extracted with 400 μl phenol / chloroform and the aqueous phase was precipitated with 2.5 volumes of ethanol and 1/10 volume of 3M sodium acetate (pH 5.2) at -20 ° C. The RNA was centrifuged off and washed with 70-80% ethanol. After centrifugation again, the RNA was dried.

The dried RNA was dissolved in 5 μl sample buffer (0.5 x TBE, 80% (v / v) formamide,

0.1% (w / v) xylene cyanole and 0.1% (w / v) bromophenol blue), heated for 10 minutes at 65 ° C, cooled on ice and then separated using an 8% polyacrylamide gel. The separated RNA was detected by means of autoradiography (see FIG. 6).

Lanes 1-5 of FIG. 6 show the time course (0, 10, 20, 30, 40 minutes) of the splice reaction of the MINX pre-mRNA without IRE at the 3 ^' OH end. The figure shows that after about 20 minutes a large part of the pre-mRNA has been converted into the mature mRNA. Lanes 6-10 show the same experiment with the pre-mRNA modified by IRE at the 3 ^' OH end. Here too, a clear splicing reaction can be seen after 20 minutes of incubation.

Claims

claims

1. Containing test system

(c) at least one composition containing splice components, and preferably (d) suitable detection probes, and optionally

(e) other tools.

2. Test system according to claim 1, characterized in that the splicable nucleic acid contains at least two exons which are separated by at least one intron.

3. Test system according to claim 1 or 2, characterized in that the gel-free detection system contains at least one probe.

4. Test system according to claim 3, characterized in that the probe is a nucleic acid complementary to the splicable nucleic acid, a splicable nucleic acid binding low molecular weight compound and / or a splicable nucleic acid binding peptide or protein.

5. Test system according to claim 4, characterized in that the low molecular weight

Compound is selected from theophylline, xanthine or an aminoglycoside, preferably tobramycin, and the nucleic acid-binding peptide or protein is an "iron responsive element binding protein" IBP).

6. Test system according to claim 4, characterized in that the complementary nucleic acid is complementary to at least one intron, to at least one exon and / or to at least one transition point of an exon and an intron.

7. Test system according to one of claims 3-6, characterized in that the splicable nucleic acid or the complementary nucleic acid of the gel-free detection system contains a recognition sequence for a nucleic acid-binding low-molecular compound and / or for a nucleic acid-binding peptide or protein.

8. Test system according to claim 7, characterized in that the recognition sequence for a nucleic acid-binding low-molecular compound is an aptamer sequence.

9. Test system according to one of claims 1-8, characterized in that the gel-free detection system contains a marking.

10. Test system according to claim 9, characterized in that the label is a radioactive label, a label with fluorescent dyes, with biotin, with digoxigenin and / or with antibodies.

11. Test system according to one of claims 3-8, characterized in that the splicable nucleic acid and the probe-binding nucleic acid sequence are connected to one another.

12. Test system according to claim 11, characterized in that at least two different probe-binding nucleic acid sequences are connected to a splicable nucleic acid.

13. Test system according to one of claims 1-12, characterized in that the nucleic acid is bound directly covalently or indirectly via a structural element and a binding partner to the structural element or by means of hybridization to a solid phase.

14. Test system according to claim 11, characterized in that the direct oval binding takes place via the vicinal 2 ^' , 3'-hydroxyl group of the ribose backbone of the nucleic acid.

15. Test system according to claim 13, characterized in that the

Structural element is a biotin or dicarboxylic acid linker

16. Test system according to claim 13, characterized in that the binding partner theophylline, xanthine or an aminogiycoside, preferably

Tobramycin, and / or a nucleic acid binding protein, especially IBP.

17. Test system according to one of claims 13-16, characterized in that the solid phase is selected from ceramic, metal, in particular noble metal, glass or plastic.

18. Test system according to one of claims 1-17, characterized in that at least one nucleic acid is an RNA.

19. Test system according to claim 18, characterized in that the RNA of a

Nucleic acid is derived, selected from a sequence of the formula

T CTTGGATCGG AAACCCGTCG GCCTCCGAAC GGTAAGAGCC TAGCATGTAG AACTGGTTAC CTGCAGCCCA AGCTTGCTGC ACGTCTAGGG CGCAGTAGTC CAGGGTTTCC

TTGATGATGT CATACTTATC CTGTCCCTTT TTTTTCCACA GCTCGCGGTT GAGGACAAAC TCTTCGCGGT CTTTCCAGTG GGGATCCAAG TGATACCAGC ATCGTCTTGA TGCCCTTGGC AGCACTTGGA TCC,

CACTCTTGGA TCGGAAACCC GTCGGCCTCC GAACGGTAAG AGCCTAGCAT GTAGAACTGG TTACCTGCAG CCCAAGCTTG CTGCACGTCT AGGGCGCAGT AGTCCAGGGT TTCCTTGATG ATGTCATACT TATCCTGTCC CTTTTTTTTC CACAGCTCGC GGTTGAGGAC AAACTCTTCG CGGTCTTTCC AGTGGGGATC GGCTTAGTAT AGCGAGGTTT AGCTACACTC GTGCTGAGCC GGATCC

CACTCTTGGA TCGGAAACCC GTCGGCCTCC GAACGGTAAG AGCCTAGCAT

GTAGAACTGG TTACCTGCAA AGUGAUACCA GCAÜCGUCUU GAUGCCCUÜG GCAGCACUUC TGCAGCCCAA GCTTGCTGCA CGTCTAGGGC GCAGTAGTCC

AGGGTTTCCT TGATGATGTC ATACTTATCC TGTCCCTTTT TTTTCCACAG

CTCGCGGTTG AGGACAAACT CTTCGCGGTC TTTCCAGTGG GGATCC

CCACTCTTGG ATCGGAAACC CGTCGGCCTC CGAACGGTAA GAGCCTAGCA TGTAGAACTG GTTACCTGCA GGCTTAGTAT AGCGAGGTTT AGCTACACTC

GTGCTGAGCC CTGCAGCCCA AGCTTGCTGC ACGTCTAGGG CGCAGTAGTC

CAGGGTTTCC TTGATGATGT CATACTTATC CTGTCCCTTT TTTTTCCACA

GCTCGCGGTT GAGGACAAAC TCTTCGCGGT CTTTCCAGTG GGGATCC AAGTGATACC AGCATCGTCT TGATGCCCTT GGCAGCACTT GAATTCGAGC TCGCCCACTC TTGGATCGGA AACCCGTCGG CCTCCGAACG GTAAGAGCCT AGCATGTAGA ACTGGTTACC TGCAGCCCAA GCTTGCTGCA CGTCTAGGTCCTGATCGTAGGTCCT

TTTTCCACAG CTCGCGGTTG AGGACAAACT CTTCGCGGTC TTTCCAGTGG GGATCC

GGCTTAGTAT AGCGAGGTTT AGCTACACTC GTGCTGAGCC GAATTCGAGC TCGCCCACTC TTGGATCGGA AACCCGTCGG CCTCCGAACG GTAAGAGCCT

AGCATGTAGA ACTGGTTACC TGCAGCCCAA GCTTGCTGCA CGTCTAGGGC GCAGTAGTCC AGGGTTTCCT TGATGATGTC ATACTTATCC TGTCCCTTTT TTTTCCACAG CTCGCGGTTG AGGACAAACT CTTCGCGGGTC GTTCCAGT

(Th / To) -5'-linked to a sequence of the formula CCGCGGTGGC GGCCGCTCTA GAACTAGTGG ATCCGTCGAC TGACTTCAGT ATGTAATATA CCCCAAACAT TTTACCCACA AAAAACCAGG ATTTGAAACT ATAGCATCTA AAAGTCTTAG GTACTAGAGTTTCTCAAAATTAGAGTCTCAATCTACGT

ACAGTAGGAT CATTTCTATA GGAATCGTCA CTCTTTGACT CTTCAAAAGA GCCACTGAAT CCAACTTGGT TGATGAGTCC CATAACCTTT GTACCCCAGA GTGAGAAACC GAAATTGAAT CTAAATTAGC TTGGTCCGCA ATCCTTAGCG TTCGGCCATC TATAATTTTG AATAAAAATT TTGCTTTGCC GTTGCATTTG TAGTTTTTTC CTTTGGAAGT AATTACAATA TTTTATGGCG CGATGATTCT

TGACCCATCC TATGTACTTC TTTTTTGAAG GGATAGGGCT CTATGGGTGG GTACAAATGG CAGTCTGACA AGTT,

TCCGTCGACT GACTTCAGTA TGTAATATAC CCCAAACATT TTACCCACAA

AAAACCA-3 '- (Th / To)

CCAGGATTTG AAACTATAGC ATCTAAAAGT CTTAGGTACT AGAGTTTTCA TTTCGGAGCA GGCTTTTTGA AAAATTTAAT TCAACCATTG CAGCAGCTTT TGACTAACAC ATTCTACAGT AGGATCATTT CTATAGGAAT CGTCACTCTT TGACTCTTCGGATCCATACTTCGGACCAGA TTCTCGAAGCCA

CCTTTGTACC CCAGAGTGAG AAACCGAAAT TGAATCTAAA TTAGCTTGGT CCGCAATCCT TAGCGTTCGG CCATCTATAA TTTTGAATAA AAATTTTGCT TTGCCGTTGC ATTTGTAGTT TTTTCCTTTG GAAGTAATTA CAATATTTTA TGGCGCGATG ATTCTTGACC CATCCTATGT ACTTCTTTTT TGAAGGGATA GGGCTCTATG GGTGGGTACA AATGGCAGTC TGACAAGTT,

GTCGACTGAC TTCAGTATGT AATATACCCC AAACATTTTA CCCACAAAAA ACCAGGATTT GAAACTATAG CATCTAAAAG TCTTAGGTAC TAGAGTTTTC ATTTCGGAGC AGGCTTTTTG AAAAATTTAA TTCAACCATT GCAGCAGCTT

TTGACTAACA CATTCTACAG TAGGATCATT TCTATAGGAA TCGTCACTCT TTGACTCTTC AAAAGAGCCA CTGAATCCAA CTTGGTTGAT GAGTCCCATA ACCTTTGTAC CCCAGAGTGA GAAACCGAAA TTGAATCTAA ATTAGCTTGG TCCGCAATCC TTAGCGTTCG GCCATCTATA ATTTTGAATA AAAATTTTGC TTTGCCGTTG CATTTGTAGT TTTTTCCTTT GGAAGTAATT ACAATATTTT

ATGGCGCGAT GATTCTTGAC CCATCCTATG TACTTCTTTT TTGAAGGGAT AGGGCTCTAT GGGTGGGTAC AAATGGCAGT CTGACAAGTT-3 '- (Th / Tθ) CACTCTTGGA TCGGAAACCC GTCGGCCTCC GAACGGTAAG AGCCTAGCAT GTAGAACTGG TTACCTGCAG CCCAAGCTTG CTGCACGTCT AGGGCGCAGT AGTCCAGGGT TTCCTTGATG ATGTCATACT TATCCTGTCC CTTTTTTTTC CACAGCTCGC GGTTGAGGAC AAACTCTTCG CGGTCTTTCC AGTGGGGATC GGGGATCCTG CTTCAACAGT GCTTGGACGG ATCCTCTAGA c or

TCCGTCGACT GACTTCAGTA TGTAATATAC CCCAAACATT TTACCCACAA AAAACCAGGA TTTGAAACTA TAGCATCTAA AAGTCTTAGG TACTAGAGTT

TTCATTTCGG AGCAGGCTTT TTGAAAAATT TAATTCAACC ATTGCAGCAG

CTTTTGACTA ACACATTCTA CAGTAGGATC ATTTCTATAG GAATCGTCAC

TCTTTGACTC TTCAAAAGAG CCACTGAATC CAACTTGGTT GATGAGTCCC

ATAACCTTTG TACCCCAGAG TGAGAAACCG AAATTGAATC TAAATTAGCT TGGTCCGCAA TCCTTAGCGT TCGGCCATCT ATAATTTTGA ATAAAAATTT

TGCTTTGCCG TTGCATTTGT AGTTTTTTCC TTTGGAAGTA ATTACAATAT

TTTATGGCGC GATGATTCTT GACCCATCCT ATGTACTTCT TTTTTGAAGG

GATAGGGCTC TATGGGTGGG TACAAATGGC AGTCTGACA AGTTGGGGATC

CTGCTTCAAC AGTGCTTGGA CGGATCCTCT AGAC,

where (Th / To) is a sequence selected from

AAGTGATACC AGCATCGTCT TGATGCCCTT GGCAGCACTT GAATT (Th), or

GGCTTAGTAT AGCGAGGTTT AGCTACACTC GTGCTGAGCC GAATT (To) means.

20. Test system according to one of claims 1-19, characterized in that the composition according to feature (c) contain "small nuclear ribonucleoprotein parti- des" (snRNP) components and non-snRNP components.

21. Test system according to claim 20, characterized in that the snRNP components contain U1, U2, U4, U5 and / or U6 proteins.

22. Test system according to one of claims 1-21, characterized in that the

Composition according to feature (c) is a cell extract, in particular a eukaryotic cell or cell nucleus extract.

23. Test system according to claim 22, characterized in that the cell nucleus extract is obtained from animal cells, in particular mammalian cells, or from fungi, in particular yeasts.

24. Test system according to one of claims 1-23, characterized in that the further aids according to feature (d) are selected from buffer solutions, stabilizers and / or energy equivalents, in particular ATP.

25. A method for producing a test system according to any one of claims 1-24, characterized in that at least one splicable nucleic acid according to feature (a) and at least one gel-free detection system according to feature (b) and optionally at least one composition containing splice components according to feature ( c) and, if appropriate, further aids are put together.

26. A method for finding an active substance, characterized in that

(a) one or more, identical or different immobilized nucleic acid / s with at least one splicable nucleic acid sequence in the presence of at least one substance to be examined and at least one

Composition containing splicing components and optionally other auxiliaries are incubated under suitable conditions, and

(b) the splice product that may be formed is detected using a gel-free detection system.

27. The method according to claim 26, characterized in that the active substance is a pharmaceutically active compound, a fungicide, a herbicide, a pesticide and / or an insecticide.

28. The method according to claim 27, characterized in that the pharmaceutically active substance is an antibiotic.

29. The method according to any one of claims 26-28, characterized in that the substance to be examined is selected from a naturally occurring, naturally occurring and chemically modified and / or synthetic

Substance.

30. The method according to claim 29, characterized in that the substance to be examined is used in the form of a combinatorial substance library.

31. A method for diagnosing a disease, characterized in that

(a) one or more, identical or different immobilized nucleic acid (s) are incubated with at least one splicable nucleic acid sequence in the presence of at least one composition containing splice components and optionally further auxiliaries, and

32. The method according to claim 31, characterized in that the disease is an inherited disease, a cancer and / or a viral disease.

33. The method according to claim 31 or 32, characterized in that the disease is selected from Grave's disease, spinal muscular atrophy, ß'-thalassemia, cancer related to the c-erb oncogene, hepatitis C infection and / or herpes simplex virus infection .

34. Splicable nucleic acids selected from an RNA derived from a nucleic acid selected from a sequence of the formula

T CTTGGATCGG

AAACCCGTCG GCCTCCGAAC GGTAAGAGCC TAGCATGTAG AACTGGTTAC CTGCAGCCCA AGCTTGCTGC ACGTCTAGGG CGCAGTAGTC CAGGGTTTCC TTGATGATGT CATACTTATC CTGTCCCTTT TTTTTCCACA GCTCGCGGTT GAGGACAAAC TCTTCGCGGT CTTTCCAGTG GGGATCCAAG TGATACCAGC ATCGTCTTGA TGCCCTTGGC AGCACTTGGA TCC,

CACT CTTGGATCGG AAACCCGTCG GCCTCCGAAC GGTAAGAGCC TAGCATGTAG AACTGGTTAC

CTGCAGCCCA AGCTTGCTGC ACGTCTAGGG CGCAGTAGTC CAGGGTTTCC TTGATGATGT CATACTTATC CTGTCCCTTT TTTTTCCACA GCTCGCGGTT GAGGACAAAC TCTTCGCGGT CTTTCCAGTG GGGTCGGTCTAGGAGTATCGGGT CACT CTTGGATCGG AAACCCGTCG GCCTCCGAAC GGTAAGAGCC TAGCATGTAG AACTGGTTAC CTGCAAAGTG ATACCAGCAT CGTCTTGATG CCCTTGGCAG CACTTCTGCA GCCCAAGCTT GCTGCACGTC TAGGGCGCAG TAGTCCAGGG TTTCCTTGAT GATGTCATAC TTATCCTGTC CCTTTTTTTT CCACAGCTCG CGGTTGAGGA CAAACTCTTC GCGGTCTTTC CAGTGGGGAT CC,

CCACT CTTGGATCGG AAACCCGTCG GCCTCCGAAC GGTAAGAGCC TAGCATGTAG AACTGGTTAC CTGCAGGCTT AGTATAGCGA GGTTTAGCTA CACTCGTGCT GAGCCCTGCA GCCCAAGCTT GCTGCACGTC TAGTGCCGTGT

GATGTCATAC TTATCCTGTC CCTTTTTTTT CCACAGCTCG CGGTTGAGGA CAAACTCTTC GCGGTCTTTC CAGTGGGGAT CC,

AAGT GATACCAGCA TCGTCTTGAT

GCCCTTGGCA GCACTTGAAT TCGAGCTCGC CCACTCTTGG ATCGGAAACC CGTCGGCCTC CGAACGGTAA GAGCCTAGCA TGTAGAACTG GTTACCTGCA GCCCAAGCTT GCTGCACGTC TAGGGCGCAG TAGTCCAGGG TTTCCTTGAT GATGTCATAC TTATCCTGTC CCTTTTTTTT CCACAGCTCG CGGTTGAGGA CAAACTCTTC GCGGTCTTTC CAGTGGGGAT CC,

GGCT TAGTATAGCG AGGTTTAGCT ACACTCGTGC TGAGCCGAAT TCGAGCTCGC CCACTCTTGG ATCGGAAACC CGTCGGCCTC CGAACGGTAA GAGCCTAGCA TGTAGAACTG GTTACCTGCA

GCCCAAGCTT GCTGCACGTC TAGGGCGCAG TAGTCCAGGG TTTCCTTGAT GATGTCATAC TTATCCTGTC CCTTTTTTTTT CCACAGCTCG CGGTTGAGGA CAAACTCTTC GCGGTCTTTC CAGTGGGGAT CC,

(Th / To) -5 'linked to a sequence of the formula

CCGCGGTGGC GGCCGCTCTA GAACTAGTGG ATCCGTCGAC TGACTTCAGT ATGTAATATA CCCCAAACAT TTTACCCACA AAAAACCAGG ATTTGAAACT ATAGCATCTA AAAGTCTTAG GTACTAGAGT TTTCATTTCG GAGCAGGCTT TTTGAAAAACCTTATTAGAAAACCTTA

TGACCCATCC TATGTACTTC TTTTTTGAAG GGATAGGGCT CTATGGGTGG GTACAAATGG CAGTCTGACA AGTT, TCCGTCGACT GACTTCAGTA TGTAATATAC CCCAAACATT TTACCCACAA AAAACCA-3 '- (Th / To)

CCAGGATTTG AAACTATAGC ATCTAAAAGT CTTAGGTACT AGAGTTTTCA TTTCGGAGCA GGCTTTTTGA AAAATTTAAT TCAACCATTG CAGCAGCTTT TGACTAACAC ATTCTACAGT AGGATCATTT CTATAGGAAT CGTCACTCTT

TGACTCTTCA AAAGAGCCAC TGAATCCAAC TTGGTTGATG AGTCCCATAA CCTTTGTACC CCAGAGTGAG AAACCGAAAT TGAATCTAAA TTAGCTTGGT CCGCAATCCT TAGCGTTCGG CCATCTATAA TTTTGAATAA AAATTTTGCT TTGCCGTTGC ATTTGTAGTT TTTTCCTTTG GAAGTAATTA CAATATTTTA TGGCGCGATG ATTCTTGACC CATCCTATGT ACTTCTTTTT TGAAGGGATA

GGGCTCTATG GGTGGGTACA AATGGCAGTC TGACAAGTT,

ATGGCGCGAT GATTCTTGAC CCATCCTATG TACTTCTTTT TTGAAGGGAT AGGGCTCTAT GGGTGGGTAC AAATGGCAGT CTGACAAGTT-3 '- (Th / Tθ)

CACTCTTGGA TCGGAAACCC GTCGGCCTCC GAACGGTAAG AGCCTAGCAT

GTAGAACTGG TTACCTGCAG CCCAAGCTTG CTGCACGTCT AGGGCGCAGT AGTCCAGGGT TTCCTTGATG ATGTCATACT TATCCTGTCC CTTTTTTTTC CACAGCTCGC GGTTGAGGGA AAACTCTTCG CGGTCTTGCCGTGTGGTGGTGTGGTGGTGTGGTGGT

TCCGTCGACT GACTTCAGTA TGTAATATAC CCCAAACATT TTACCCACAA AAAACCAGGA TTTGAAACTA TAGCATCTAA AAGTCTTAGG TACTAGAGTT TTCATTTCGG AGCAGGCTTT TTGAAAAATT TAATTCAACC ATTGCAGCAG CTTTTTGACTATAGATCAGACTACAT

TCTTTGACTC TTCAAAAGAG CCACTGAATC CAACTTGGTT GATGAGTCCC ATAACCTTTG TACCCCAGAG TGAGAAACCG AAATTGAATC TAAATTAGCT TGGTCCGCAA TCCTTAGCGT TCGGCCATCT ATAATTTTGA ATAAAAATTT TGCTTTGCCG TTGCATTTGT AGTTTTTTCC TTTGGAAGTA ATTACAATAT TTTATGGCGC GATGATTCTT GACCCATCCT ATGTACTTCT TTTTTGAAGG

GATAGGGCTC TATGGGTGGG TACAAATGGC AGTCTGACA AGTTGGGGATC CTGCTTCAAC AGTGCTTGGA CGGATCCTCT AGAC,

where (Th / To) is a sequence selected from

AAGTGATACC AGCATCGTCT TGATGCCCTT GGCAGCACTT GAATT (Th), or GGCTTAGTAT AGCGAGGTTT AGCTACACTC GTGCTGAGCC GAATT (To).

5. Use of a splicable nucleic acid according to claim 34 for

Manufacture of a test system.