MXPA00001978A - Oligonucleotides for identifying precursors of amidated polypeptide hormones - Google Patents

Abstract

The invention concerns novel oligonucleotides and their application as probes for identifying RNAm's coding for precursors of amidated polypeptide hormones and, thereby, identifying novel amidated polypeptide hormones. The invention also concerns oligonucleotides of the disclosed nucleotide sequence and a method for identifying precursors of amidated hormones.

Description

OLIGONUCLEOTIDES THAT ALLOW THE IDENTIFICATION OF PRECURSORS OF POLYPEPTIDIC AMID HORMONES The present invention relates to new oligonucleotides and their application as probes for the identification of the mRNAs that code for the precursor of amidated polypeptide hormones and, thus, the identification of new amidated polypeptide hormones. The invention then concerns oligonucleotides whose nucleotide sequence is described below and a method for identifying amidated hormone precursors.

The amidated polypeptide hormones are synthesized in the form of a precursor that undergoes maturation. This maturation consists of an amidation reaction.

The amidation reaction of the C-terminal end is a characteristic reaction of amidated polypeptide hormones. This reaction, which intervenes on the precursor of one or several hormones, allows the maturation of the hormone and also ensures its biostability in the physiological medium: REF. 32899 the amide group formed is less vulnerable than the free acid function. The hormone is then more resistant to carboxypeptidases, remains active longer in the cell and retains an optimal affinity with its receptor site.

Amidation has been widely described ("Peptide Amidation", Alan F. Bradbury and Derek G. S yth, TIBS 16: 112-115, March 1991 and "Functional and structural characterization of peptidyl amido glycolatelatese, the enzyme that catalyzes the second step in peptide amidation ", AG Katopodis, DS Ping, CE Smith and SW May, Biochemistry, 30 (25): 6189-6194, June 1991), its mechanism is as follows: 1. - division of the polypeptide chain precursor of the hormone by an endoprotease at the level of two basic amino acids that are arginine and / or lysine, 2. - then two divisions are produced by the carboxypeptidase leading to the extended glycine intermediate, 3. - the enzyme PAM (Peptidil-glycine-a-Amidante Monooxygenase) comprises two different enzymatic activities: in a first time, the extended glycine intermediate was converted to a-hydroxyglycine derivative, the subunit of the PAM enzyme involved is the PHM ( Peptidyl-glycine-a-hydrolilant Mono-oxygenase). The obtained derivative serves as substrate to the second sub-unit of the PAM (annotated PAL: Peptidyl-a-hydroxiglicina-a-Amidante Liasa) that fixes the amine function of the glycine on the amino acid immediately adjacent to the N-terminal side and releases the glyoxylate.

This reaction imposes the presence of a recognition site on the precursor of or of the hormones, site that always contains the sequence: glycine and two basic amino acids (arginine or lysine) (see AG Ktopodis et al., Biochemistry, 30 (25) , 6189-6194, June 1991, and cited references).

Amidated polypeptide hormones that are intended to be secreted outside the endoplasmic reticulum are known to contain a consensus sequence indicating a fortnight to thirty amino acids, this sequence is present at the N-terminal end of the polypeptide chain. It is later cut by a peptidase indicator enzyme, so that it is no longer found in the protein once secreted (see F. Cuttitta, The Anatomical Record, 236, 87-93 (1993) and references cited).

To date, the discovery of a new protein is not an easy thing. Isolation and purification of proteins can be done by various techniques: precipitation at the isoelectric point, selective extraction by certain solvents, then purification by crystallization, countercurrent distribution, adsorption chromatography, distribution or ion exchange, electrophoresis. However, these techniques involve knowledge of the properties of the protein to be isolated. On the other hand, when a pure sample of a new interesting protein is available on the therapeutic plan, there are still numerous stages before a genetically modified microorganism capable of synthesizing it is available.

The method proposed by the present invention offers the advantage, by the use of a particularity of the peptide sequence of the precursor of all amidated hormones known to date, to allow the simultaneous testing of several new hormones of this category. This search is made by the direct identification of the nucleotide sequence coding for said precursors in the cDNA libraries prepared from tissues in which the precursors of these hormones are capable of being synthesized.

The search for this method is much less compelling than the classical techniques of biochemistry mentioned above because: - may lead to isolating, following the same principle, several different precursors present in the same tissue; it makes it possible to demonstrate, in the same technical conditions, precursors corresponding to hormones that have very different biochemical and biological properties; - authorizes the concomitant identification of all peptide hormones that may be contained in the same precursor.

From this fact, this invention allows a saving of time and money not negligible in a sector in which the expenses of Research and Development represent a very important proportion of the business figures.

The present invention will also allow the pharmacological study of active substances that play a fundamental physiological role in the organism of mammals: the hormones and very particularly the amidated polypeptide neurohormones. Having first of all the cDNAs corresponding to the active substances. It will then be possible to genetically engineer the cloned vector to drive the synthesis of hormones having a therapeutic application by micro-organisms.

The subject of the invention is first of all a single-stranded oligonucleotide OX which can hybridize under mild conditions with an oligonucleotide OY of sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is deleted, Y2 represents a trinucleotide encoding Gly, Y3 and Y4 independently represent a trinucleotide encoding Arg or Lys, and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is deleted.

Nucleotide is understood as a monomeric unit of RNA or DNA that has the chemical structure of a nucleoside phosphoric ester. A nucleoside results from the binding of a purine base (purine, adenine, guanine or analogs) or a pyrimidic base (pyrimidine, cytosine, uracil or analogues) with ribose or deoxyribose. An oligonucleotide is a polymer of nucleotides that designates a priming sequence, a probe or a fragment of RNA or DNA.

The oligonucleotides cited can be obtained by synthesis, there is an automated reference method that is described in the following publications: "DNA synthesis" of S.A. Narang, Tetrahedron, 39, 3 (1983) and "Synthesis and use of synthetic oligonucleotides" by K. Itakura, J.J. Rossi and R.B. Wallace, .Annu. Rev. Biochem., 53, 323 (1984).

Preferably, OX can be hybridized under severe conditions with OY.

More preferentially, OX can be hybridized with an OY oligonucleotide of sequence Y2-Y3-Y4-Y5.

Even more preferentially, OX can be hybridized with an OY oligonucleotide of sequence Y1-Y2-Y3-Y4 or Y2-Y3-Y4.

Particularly, OX can hybridize to an OY oligonucleotide such as Y5 represents a nucleotide sequence Y6-Y7-Y8-Y9 in which Y6 represents a trinucleotide encoding Ser, Thr, or Tyr, Y7 represents a trinucleotide that codes for an amino acid any, Y8 represents a trinucleotide encoding Glu or Asp and Y9 represents a nucleotide sequence comprising from 1 to 12 nucleotides. More particularly, OX can be hybridized with an oligonucleotide OY such that Y1 and Y9 are deleted.

Most particularly, OX can hybridize with an OY oligonucleotide in which Y2 represents a trinucleotide encoding Gly, Y3 represents a trinucleotide encoding Lys, Y4 a trinucleotide encoding Arg and Y5 a sequence of 3 trinucleotides encoding Ser- Ala-Glu.

This sequence has been determined thanks to a statistical study on 27 known amidation sites and has led to define over 6 positions a given motif of amino acids: Gly-Lys-Arg-Ser-Ala-Glu.

Because of the degeneracy of the genetic code and the high number of codons corresponding to the Gly (4 codons), to the Arg (6 codons) and to the Ser (6 codons), the sequence of the oligonucleotide has been constructed with the help of two Procedures that allow to take into account this degeneration: - use in certain positions of inosine, a nucleotide whose nitrogenous base, hypoxanthine, pairs indifferently with the 4 nitrogenous bases constituting the DNA. - variation, in certain positions, of the nature of the incorporated nitrogenous base, thus generating a number of oligonucleotide combinations proportional to the number of different bases introduced.

The subject of the present invention is also an OY oligonucleotide containing from 9 to 42 nucleotides of sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence of 1 to 12 nucleotides or Y1 is deleted, Y2 represents a trinucleotide encoding Gly, Y3 and Y4 independently represent a trinucleotide encoding Arg or Lys, and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is deleted.

Preferably, the subject of the invention is an oligonucleotide OY such that Y1 is deleted or such that Y5 is deleted.

The invention is particularly characterized by an oligonucleotide OY such that 45 rq represents the nucleotide sequence Y6-Y7-Y8-Y9, in which Y6 has a trichucleotide coding for Ser, Thr or Tyr, Y7 represents a trinucleotide encoding any amino acid, Y8 represents a trinucleotide encoding Glu or Asp and Y9 represents a nucleotide sequence comprising 1 to 12 nucleotides.

The subject of the invention is more particularly an oligonucleotide OY such that Y1 and Y9 are deleted.

The subject of the invention is very particularly an oligonucleotide OY, which is characterized in that Y1 is deleted, Y2 represents a trinucleotide coding for Gly, Y3 represents a trinucleotide coding for Lys, Y4 is a trinucleotide < which encodes for Arg and Y5 a sequence of three trinucleotides encoding Ser-Ala-Glu.

The present invention also concerns a single-filament OZ oligonucleotide, which is characterized in that it comprises from 15 to 39 nucleotides and is capable of hybridizing with a consensus indicator sequence characteristic of amidated polypeptide hormones, said sequence having the formula Z1- Z2-Z3-Z4-Z5-Z6-Z7 in which Zl represents a nucleotide sequence of 1 to 12 nucleotides or Zl is deleted, Z2 and Z3 represent two trinucleotides that code for Leu, Z4 and Z5 represent two trinucleotides that code for two any amino acids, Z6 represents a trinucleotide that codes for Leu, and Z7 represents a nucleotide sequence of 1 to 12 nucleotides or Z7 is deleted.

In this invention, hormone will be understood as polypeptide hormones amidated from the endocrine system, and more particularly neurohormones.

The consensus indicator sequence is a sequence contained in the precursors of the proteins that are secreted by the cells after their maturation.

The present invention finally concerns a set of oligonucleotides OX or OZ such that it constitutes a combinatorial pool.

In the invention described, a combination of oligonucleotides synthesized by means of a model is understood to be a nucleotide sequence coding for a sequence of amino acids, some of which may be variable. From the fact of the degeneracy of the genetic code, a set of different oligonucleotides will be obtained.

Another object of the invention is a method of identifying the precursor of a peptide having an amidated C-terminal end, characterized by the following successive steps: 1. - obtaining a DNA bank; 2. - hybridization of one or more OX oligonucleotides with said DNA bank; 3. - identification of the DNA sequence of said bank that hybridizes with an OX oligonucleotide; 4. - identification in this or these sequences of one or several peptides with a possible C-terminal amidated end.

A method such as the DNA library is a cDNA library will be preferred.

Complementary DNA (cDNA) is a nucleotide chain whose sequence is complementary to that of an mRNA, the reaction that leads to single-stranded cDNA is catalyzed by reverse transcriptase. The double-stranded cDNA can be obtained by the action of the DNA polymerase, then inserted with the help of a ligase in a plasmid or a vector derived from the bacteriophage?.

A cDNA library groups the cDNAs corresponding to the cytoplasmic mRNAs extracted from a given cell. The bank is said complete when it comprises at least one bacterial clone for each initial mRNA.

Hybridization occurs when two oligonucleotides have substantially complementary nucleotide sequences, can be associated over their length by establishing hydrogen bonds between complementary bases.

A method such that oligonucleotide OX is detectable with the aid of a labeling agent, such as 3A or digoxigenin, will be particularly preferred.

The radioactive labeling agents of the most commonly used nucleotides are the elements that emit ß-rays, for example, 3H, 12C, 32P, 33P and 35S.

The labeling of the oligonucleotide is made by the addition at its 5 'end of a phosphate group contributed by the (? "32) -ATP, this reaction is catalyzed by the enzyme T4-polynucleotide kinase. The one marked by digoxigenin is immunoenzymatic, the Digoxigenin is associated with a nitrogenous base and is incor- porated to the oligonucleotide, and its presence is revealed by the use of an antibody directed against digoxigenin and coupled to an alkaline phosphate.The disclosure uses the color developed by a substrate hydrolyzed by alkaline phosphatase.

Other marking techniques can be employed: chemically modified oligonucleotides because they contain a metal-complexing agent (lanthanide complexes are frequently used), a group that contains biotin or acridine ester, a fluorescent compound (fluorescein, rhodamine, red Texas ) or another.

A method of identifying amidated polypeptide hormone precursor such that the hybridization step utilizes a combinatorial pool of oligonucleotides OX will be very particularly preferred.

Another subject of the invention is a method for identifying the precursor of a peptide having an amidated C-terminal end, comprising the following steps: 1. - obtaining a DNA bank; 2. - use of the PCR technique to amplify the fragment of interest with the help of one set of oligonucleotides OX and another set of oligonucleotides OZ; 3. - identification of the DNA sequence of said bank that hybridizes with the oligonucleotide OX and that is amplified by the PCR reaction; 4. - identification in this sequence of one or more peptides with a possible C-terminal amidated end.

A fragment of interest is understood to be the cDNA sequence coding for the precursor of one or more amidated polypeptide hormones.

The amplification reaction of DNA by PCR ("Polymerase Chain Reaction") needs a preparation of DNA denatured by heating at 95 ° C. Next, this preparation is paired to an excess of two complementary oligonucleotides in the opposite strands of the DNA, on one part and on the other of the sequence to be amplified. Each oligonucleotide then serves as bait to a DNA polymerase (extracted from thermophilic bacteria of the Thermus aquatitus: Taq polymerase type) for the copy of each of the strands of the DNA. This cycle can be repeated, automatically, by de-naturalization-successive renaturalizations.

There are numerous references that detail PCR protocols: US patents no. 4,683,192, 4,483,202, 4,800,159 and 4,965,188, "PCR Technology: Principles and Applications for DNA Amplification", H. Erlich, ed. Stockton Press, New York (1989) and "PCR Protocols: A Guide to Methods and Applications," Innis et al., Eds. Academic Press, San Diego California (1990).

Preferably, said DNA library is a cDNA library.

More preferably, said oligonucleotide OX is detectable with the aid of a labeling agent such as 3 or digoxigenin.

Particularly preferred is a method for identifying amidated polypeptide hormone precursor tql that the amplification step uses a combinatorial pool of oligonucleotides OX and another combinatorial pool of oligonucleotides OZ.

Another object of the invention is a method of identifying the precursor of a peptide having an amidated C-terminal end, comprising the following steps: 1. - obtaining a DNA bank; 2. - use of the PCR technique to amplify the fragment of interest with the help of a set of OX oligonucleotides; 3. - identification of the DNA sequence of said bank that hybridizes with the oligonucleotide OX and that is amplified by the PCR reaction; 4. - identification in this sequence of one or more peptides with a possible C-terminal amidated end.

The objective of this method is to characterize the nucleotide sequences that code for precursors that have more than one amidation site.

Preferably, said DNA library is a cDNA library.

More preferably, said oligonucleotide OX is detectable with the aid of a labeling agent such as 32P or digoxigenin.

A method of identifying amidated polypeptide hormone precursor such that the amplification step utilizes a combinatorial pool of oligonucleotides OX will be particularly preferred.

Another method proposed by the present invention for the identification of the precursor of a polypeptide having an amidated C-terminal end, is characterized by the following steps. 1. - obtaining a DNA bank; 2. - use of the PCR technique to amplify the fragment of interest with the help of an OX oligonucleotide and another single-stranded oligonucleotide capable of hybridizing under mild or severe conditions with a universal consensus sequence contained in the sequence of the plasmid vector in which they are cloned the DNA of said DNA bank, such as the T3 primers, T7, K5, SK, M13, inverse; 3. - identification of the DNA sequence of said bank that hybridizes with an OX oligonucleotide; 4. - identification in this sequence of one or several peptides with a possible C-terminal amidated end.

The universal consensus sequence is a sequence contained in the vector in which the DNA of the bank is cloned. This sequence can serve as bait for the formation of sequences. The nucleotide sequences of these primers are available from: Sambrook, J. Fritsch, E.F., Maniatis, T., "Molecular cloning, a laboratory manual", 2A Edition, 1989, Spring Harbor Laboratory Press.

The PCR reaction requires that two oligonucleotides be fixed on the cDNA cloned in a vector for amplification to take place. In the case where a single sequence of the DNA fragment to be amplified is known, a solution to prowl this problem is to use an oligonucleotide that can hybridize with a specific nucleotide sequence to the vector in which the cDNA has been cloned, such as a Universal consensus sequence.

Preferably, said DNA library is a cDNA library.

An OY oligonucleotide detectable with the aid of a labeling agent such as 32P or digoxigenin will be preferred.

An amplification step using a combinatorial pool of oligonucleotides OX will be more particularly preferred.

EXAMPLE: The method described by the invention has been validated by its application on an already isolated hormone. The selected neurohormone is cholecystokinin (CCK) which is the quantitatively most important neuromediator represented in the brain. 1. 1 . Preparation of the DNA matrix that serves the PCR reactions from a commercial Lambda bank Zapp II (Vector of the rat brain cDNA stock, Ref. 936 501) of STRATAGENE (Lafolla, U.S.A.

This Stratagene cDNA library contains the cloning of the cDNAs from rat brain cells. 1. 1.1. Release of cloned cDNAs in the form of Bluescript phagemids (Stratagene, Lajolla, USES.) .

It is done following the following protocol: it is contacted during 15 minutes at 37 ° C, 250 μl of the cDNA library in 2.108 PFU / ml, 200 μl of blue XLi bacteria (genotype: recAl endAl gyrA96 thi-1 hsdR17 supE44 relat lac [F 'proAB lacIqZ? M15 TnlO (Tetr)] c see Bullock, fernández, Short, Biotechniques, 5, 376-379 (1987) -optical density at 600 nm: OD = 2.5) and 1 μl of phage ExAssist ™ (see Hay, B., Short, J., Strategies, 5, 16-18 (1992)) at 1010 PFU / ml then the set is incubated on 50 ml of LB medium (composition: for 1 liter of sterile physiological water, mix 10 g of NaCl, 5 g of yeast extract and 10 g of Bactotriptone) for 3 hours under agitation at 37 ° C. The culture broth is centrifuged then the supernatant is inactivated by heating at 70 ° C for 20 minutes. 1. 1.2. Obtaining cDNAs in the form of a bank of double filament plasmids.

This step requires 15 minutes of incubation at 37 ° C of 100 μl of the inactivated supernatant and 200 μl of SOLR ™ bacteria (genotype: el4"(McrA") (mcrCB-hsdSMR-mrr) 171 sbcCrecB recJ uvrC umuC :: Tn5 ( Kanr) lac gyrA96 relAl thi-1 endAl? R [F 'proBlacIqZ? M15] c Su "(non-suppressor) - see Hay, B. Short, JM, strategies, 5 (1), 16-18 (1992) - DO = 1 at 600 nm) After the addition of 50 μl of ampicillin (at 100 mg / ml) and 50 ml of LB medium, the total is incubated at 37 ° C with stirring overnight. from 50 ml of culture with the QIAGEN QIAGEN Plasmid Midi protocol and columns (the QIAGEN columns contain an ion exchange resin that has positively charged diethylaminoethanol groups on its surface, which interact with the phosphates of the DNA) A DNA solution at 1.37 μg / μl was thus obtained. 1. 2. Amplification of a portion of the CCK precursor from the plasmid bank thus prepared 1. 2.1. Establishment of the sequences of two oligonucleotides necessary for the PCR reaction.

Some of these two nucleotides will contain the sequence complementary to the one that codes for the amidation site of the CCK, this site is known and has by sequence Gly-Arg-Arg-Ser-Ala-Glu. This oligonucleotide, which will be called oligo CCK amid, has a nucleotide sequence: 'CTCAGCACTGCGCCGGCC 3' The second oligonucleotide, called oligo CCK 5 ', corresponds to the consensus indicator sequence: 'GTGTGTCTGTGCGTGGTG 3' The size of the expected amplification product is 315 base pairs, it is the distance between the sequences corresponding to the two oligonucleotides on the precursor sequence of CCK. 1. 2.2. PCR reaction.

A DI dilution containing 1 μl of Goldstar Taq polymerase enzyme 5 U / μl is prepared (see Reyner, P., Pellissier, J. F., Harle, J.R., Malthiéry, Y., Biochemical and Biophysical Research Communications, 205 (1), 375-380 (1994)), 1 μl of a buffer 10 times concentrated in standard Taq polymerase and 8 μl of water.

Then 1 μl of oligo CCK 5 'is mixed at 250 ng / μl, 1 μl oligo CCK amid at 250 ng / μl, 1 μl dNTP at 10 mM each, 1 μl DNA cDNA library at 250 ng / μl , 5 μl of 10-fold concentrated buffer of the enzyme Taq polymerase, 2 μl of MgC12 at 25 mM, 1 μl of the dilution DI and 37 μl of water.

The amplification conditions are as follows: first a thermal treatment of 5 minutes at 95 ° C is carried out, then 30 cycles are renewed. Denatures are made for 45 seconds at 95 ° C, hybridization for 30 seconds at 60 ° C and elongation for 1 minute at 72 ° C. Finally, a supplementary cycle is conducted with a 10-minute rise at 72 ° C. 1. 2.3. Results The results are read by migration on agarose gel at 0.8% of 1/10 of the reaction product of PCR In the presence of 3, 8-diamino-5-ethyl-6-phenyl-phenatridinium bromide (etidium bromide), a single, intense band of size slightly larger than the 300 molecular weight marker is visualized on the gel. 1. 3. Under cloning of the PCR product in a vector that allows the formation of sequences.

The vector used is Vector pGEM T-easy (marketed by PROGEMA Corporation, Madison, U.S.A., ref A 1380 - sequence given in appendix I). The stages are the following: purification of the band corresponding to the PCR product by electroelution, - ligation overnight at 16 ° C with 1 μl of pGEM T-easy vector at 50 ng / μl, 1 μl of 10 times concentrated ligase buffer, - 3 μl of product extracted from the purified band estimated at 20 ng / μl, - complete with water up to 10 μl.

Bacteria JM 109 (genotype: el4"(McrA") recAl endAl gyrA96 thi-1 hsdR17 (r? -m? +) SupE44 relAl? Dac-proAB) [F 'traD36 proAB ladqZ? M15] -see Yanish-Perron, C, Viera, J. Messing, J., Gene, 33, 1103-199 (1985)) are made competent by a previous treatment to CaCl 2 then transformed by a thermal shock of 45 seconds at 42 ° C 1/5 of the ligation The cells are then placed in culture on the LB-ampicillin medium in a Petri dish overnight at 37 ° C.

The plasmid DNA of some recombinant clones is prepared. Then the sub-cloning is verified by the enzymatic digestion with Eco Rl. 1. 4. Sequence Formation It is carried out by the classic SANGER deoxynucleotide technique on the "Vector pGEM T-easy" vector that has incorporated the 315 base pair PCR product (prepared on a large scale by the QIAGEN tip 100). The bait used for the formation of sequences is the universal oligonucleotide T7 present on the vector plasmid pGEM T-easy. 1. 5. Results The following gross sequence is obtained: GTG TGT CTG TGC TGG GTG GTG ATG GCA GTC CTG GCA GCA GGC GCC CTG GCG CAG CCG GTC GTC CCT GTA GCT GCT GCT GG CCG ATG GAG CAG CGG GCG GAG GAG GCG CCC CGA AGG CAG CTG AGG GCT GTG CTC CGA CCG GAC AGC GAG CCC CGA GCG CGC CTG GGC GCA CTG CTA GCC CGA TAC ATC CAG CAG GTC CGC AAA GCT CCC TCT GGC CGC ATG TCC GTT CTT AAG AAC CTG CAG GGC CTG GAC CCT AGC CAC AGG ATA AGT GAC CGG GACTAC ATGGGC TGG ATG GATTTC GGCCGGCGC AGTGCT GAG The translation in amino acids of the obtained sequence ends in: VCCVV MAVLAAGALA QPWPVEAVD PMEQRAEEAP RRQLRAVLRP DSEPRARLGA UARY1QQVR KAPSGRMSVL KNLQGLDPSH RISDRDYMGW MDFGRRSAE It allows to find the nucleotide sequence of the precursor of the CCK (whose sequence has been provided by the Suiso Prot databank No. p01355).

The abbreviation for amino acids is as follows: Alanine A Leucine Argina Lysine K Aspartic acid D Methionine M Aspargin N Phenylalanine Cysteine Proline Glutamic acid E Serine Glutamine Treonine Glycine Tryptophan Histidine H Tyrosine Y Valle Isoleucine V It is noted that in relation to this date the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Having described the invention as above, it is claimed as property in the following:

Claims (25)

1. The OX single filament oligonucleotide, which is characterized in that it comprises 9 to 42 nucleotides and is capable of hybridizing under mild conditions with an OY oligonucleotide of sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence 1 to 12 nucleotides or Yl is deleted, Y2 represents a trinucleotide encoding Gly, Y3 and Y4 independently represent a trinucleotide encoding Arg or Lys, and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is deleted.

2. The oligonucleotide OX according to claim 1, which is characterized in that it comprises from 9 to 42 nucleotides and is capable of hybridizing under severe conditions with an oligonucleotide OY of sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence 1 to 12 nucleotides or Yl is deleted, Y2 represents a trinucleotide encoding Gly, Y3 and Y4 independently represent a trinucleotide encoding Arg or Lys, and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is deleted.

3. The oligonucleotide OX according to claim 1 or 2, which is characterized in that Y5 is deleted in oligonucleotide OY.

4. The oligonucleotide OX according to claim 1, 2 or 3, which is characterized in that Y5 is deleted in oligonucleotide OY.

5. The oligonucleotide OX according to claim 1, 2 or 3, which is characterized in that, in OY, Y5 represents a nucleotide sequence Y6-Y7-Y8-Y9, in which Y6 represents a trinucleotide coding for Ser, Thr, or Tyr, Y7 represents a trinucleotide encoding any amino acid, Y8 represents a trinucleotide encoding Glu or Asp and Y9 represents a nucleotide sequence comprising 1 to 12 nucleotides.

6. The oligonucleotide OX according to claim 5, which is characterized in that Yl and Y9 are deleted in oligonucleotide OY.

7. The oligonucleotide OX according to claim 6, which is characterized in that it can hybridize with said oligonucleotide OY in which Y2 represents a trinucleotide encoding Gly, Y3 represents a trinucleotide encoding Lys, Y4 a trinucleotide encoding Arg and Y5 a sequence of 3 trinucleotides that encode Ser-Ala-Glu.

8. The single filament oligonucleotide OY, which is characterized in that it comprises from 9 to 42 nucleotides of sequence Y1-Y2-Y3-Y4-Y5, in which Y1 represents a nucleotide sequence of 1 to 12 mucleotides or Y1 is deleted, Y2 represents a trinucleotide encoding Gly, Y3 and Y4 independently represent a trinucleotide encoding Arg or Lys, and Y5 represents a nucleotide sequence of 1 to 21 nucleotides or Y5 is deleted.

9. The oligonucleotide OY according to claim 8, which is characterized in that Y1 is deleted.

10. The oligonucleotide OY according to claim 8 or 9, which is characterized in that Y5 is deleted.

11. The oligonucleotide OY according to claim 8 or 9, which is characterized in that Y5 represents a nucleotide sequence Y6-Y7-Y8-Y9, in which Y6 represents a trinucleotide encoding Ser, Thr, or Tyr, Y7 represents a trinucleotide encoding for any amino acid, Y8 represents a trinucleotide encoding Glu or Asp and Y9 represents a nucleotide sequence comprising from 1 to 12 nucleotides.

12. The oligonucleotide OY according to the claim 11, which is characterized by Yl and Y9 being suppressed.

13. The oligonucleotide OY according to the claim 12, which is characterized in that Y2 represents a trinucleotide encoding Gly, Y3 represents a trinucleotide encoding Lys, Y4 a trinucleotide encoding Arg and Y5 a sequence of 3 trinucleotides encoding Ser-Ala-Glu.

14. The single-stranded oligonucleotide OZ, which is characterized in that it comprises from 15 to 39 nucleotides and is capable of hybridizing under mild or severe conditions with a consensus indicator sequence characteristic of amidated polypeptide hormones, the sequence mentioned has by formula Z1-Z2-Z3-Z4-Z5-Z6-Z7 in which Z1 represents a nucleotide sequence of 1 to 12 nucleotides or Z1 is deleted, Z2 and Z3 represent two trinucleotides which codes for Leu, Z4 and Z5 represent two trinucleotides that code for any two amino acids, Z6 represents a trinucleotide that codes for Leu, and Z7 represents a nucleotide sequence of 1 to 12 nucleotides or Z7 is deleted.

15. OX oligonucleotide assembly according to any one of claims 1 to 7 or oligonucleotide OZ according to claim 14, characterized in that it constitutes a combinatorial stock.

16. Method of identifying the precursor of a peptide having an amidated C-terminal end, which is characterized by the following successive steps: - obtaining a DNA bank; - hybridization of one or more oligonucleotides according to any one of claims 1 to 7 with said DNA bank; - identification of the DNA sequence (s) of said bank that hybridizes with an oligonucleotide according to any one of claims 1 to 7; - identification in this or these sequences of one or several peptide precursors with a possible C-terminal amidated end.

17. Method according to claim 16, characterized in that the hybridization step uses a combinatorial pool according to claim 15.

18. Method of identifying the precursor of a peptide having an amidated C-terminal end, which is characterized by the following successive steps: - obtaining a DNA bank; - use of the PCR technique to amplify the fragment of interest with the aid of a set of oligonucleotides according to any one of claims 1 to 7 and of another set of oligonucleotides according to claim 14; - identification of the DNA sequence (s) of said bank that hybridizes with an oligonucleotide according to any one of claims 1 to 7; - identification in this or these sequences of one or more peptide precursors with a possible C = terminal amidated terminal.

19. Method according to claim 18, characterized in that the amplification stage uses a combinatorial collection according to claim 15.

20. Method of identifying the precursor of a peptide having an amidated C-terminal end, which is characterized by the following successive steps: - obtaining a DNA bank; - use of the PCR technique to amplify the fragment of interest with the aid of a set of oligonucleotides according to any one of claims 1 to 7; - identification of the DNA sequence (s) of said bank that hybridizes with an oligonucleotide according to any one of claims 1 to 7; - identification in this or these sequences of one or several peptide precursors with a possible C-ter-amidated endpoint.

21. Method according to claim 20, characterized in that the amplification stage uses a combinatorial pool according to claim 15.

22. Method of identifying the precursor of a polypeptide having an amidated C-terminal extremity, which is characterized by the following steps: - obtaining a DNA bank; - use of the PCR technique to amplify the fragment of interest with the aid of an oligonucleotide according to any one of claims 1 to 7 and another single-filament oligonucleotide capable of hybridizing under mild or severe conditions with a universal consensus sequence contained in the sequence of the plasmid vector in which the cDNAs of said DNA bank are cloned, such as the primers T3, T7, KS, SK, M13, Inverse; identification of the cDNA sequence of said bank that hybridizes with an oligonucleotide according to any one of claims 1 to 7; - identification in this sequence of one or more peptide precursors with a possible amidated C-terminal end.

23. The method according to claim 22, characterized in that the amplification stage uses a combinatorial collection according to claim 15.

24. Method according to any one of claims 16 to 23, which is characterized in that the DNA library is a cDNA library.

25. Method according to any one of claims 16 to 24, which is characterized in that the single-stranded oligonucleotide is detectable with the aid of a labeling agent, such as 32P or digoxigenin.